Program

Authors: Alexandr Melnikov

Affiliations:
Charité - Universitätsmedizin Berlin

Abstract:

At the heart of any successful student-teacher interaction, regardless of whether innovative or traditional teaching methods are used, lies the simultaneous commitment of attention by both teacher and students. Effective teaching therefore depends decisively on capturing and sustaining students’ attention throughout the entire learning encounter. In my practice, deliberately forgoing or drastically reducing the use of pre-made digital presentation slides and returning to the analog whiteboard or flipchart has proven to be the single most effective strategy to achieve this. The absence of pre-made slides redirects students’ attention to what unfolds in real time – to what is said, shown, drawn, or written – enabling the teacher to guide their attention with greater precision. The board and handwriting themselves impose natural temporal and spatial constraints on the amount and rate of information that can be presented, thereby enacting a fundamental pedagogical principle: less is more. Thus, by requiring meticulous prioritization and intentional planning in advance, this shift toward developing the subject live with the audience ensures rigorous preparation on the part of the teacher. A clearly constructed central thread, the intentional use of rhetorical and stylistic devices, thoughtfully integrated demonstration experiments or technology when appropriate, and authentic passion for the subject – combined with a genuine interest in students’ learning – capture and sustain attention, making every moment of the interaction meaningful and rewarding for both teacher and students.

Authors: Prof. Jakub Otáhal, MD.,Ph.D.

Affiliations:
Department of Pathophysiology, Second Faculty of Medicine, Charles University, Prague

Abstract:

Preclinical education was built for a world in which medical knowledge was scarce, static, and accessed primarily through textbooks and lectures. Today’s students enter medicine shaped by an environment of abundant information, real-time feedback, and rapidly evolving digital tools, most notably AI systems that can generate fluent explanations, summaries, questions, and even clinical reasoning pathways. This shift forces a fundamental question: should we keep teaching preclinical subjects the same way when the technologies that shape learning, attention, and problem-solving have changed dramatically? This talk argues that the goal of preclinical teaching is not to preserve the methods of the previous century, but to design competence for clinicians who will peak in their careers 25 years from now. Books remain valuable, but they are only one medium among many. If we want students to develop durable understanding and safe decision-making, we must intentionally combine complementary approaches: simulation and scenario-based learning to create “experience” before real patients; problem-based learning to train structured thinking under uncertainty; AI-assisted tutoring to individualize feedback and practice; and immersive tools such as virtual and augmented reality to make mechanisms tangible rather than abstract. At the same time, these tools introduce new risks: illusion of competence, over-reliance on generated answers, and misinformation delivered with high confidence. Therefore, the talk proposes a framework for “preclinical education in the age of AI” focused on (1) mechanistic understanding, (2) critical appraisal and verification, (3) transparent learning processes, and (4) assessment redesign, from testing recall to testing reasoning, validation, and decision justification. The outcome is a pragmatic, future-facing blueprint: preclinical education as competence engineering, where teaching methods evolve with technology, while professional standards -rigor, responsibility, and patient safety - remain non-negotiable.

Authors: Anna Faivre, Anders M. Kristensen, Xiang Zheng, Qi Wu, Peder M. Berg, Camilla Tanderup Sjøgaard, Hanne Kidmose, Robert A. Fenton, Ina Maria Schiessl

Affiliations:
Aarhus University, Department of Biomedicine, Aarhus, Denmark

Abstract:

Acute kidney injury (AKI) is associated with high morbidity, and many patients progress toward chronic kidney disease (CKD). The mechanisms driving this failed repair remain unclear. During AKI, cellular debris accumulate as granular casts in the tubular lumen and correlate with the severity of injury, but whether they actively contribute to tubular injury is unknown. We investigated the ischemia–reperfusion injury (IRI) model in C57BL/6J male mice. Unilateral IRI was induced by 30-min renal pedicle clamping. Casts and adjacent epithelium were isolated by laser microdissection for spatial proteomics. Longitudinal intravital two-photon (2PM) imaging was used to track cast formation, as well as tubular atrophy and fibrosis development. This was done in PDGFRβCreERT2 x Salsa6F mice, which allow tracking of PDGFRβ-cells. Cast removal was attempted with osmotic diuresis (mannitol, 1 g/kg intraperitoneally twice daily for 4 days plus 20% in drinking water for 7 days). Proteomic analysis from n=5 mice revealed that casts share much of their protein content with adjacent epithelium but also contain damage-associated molecular patterns (DAMPs). Serial intravital imaging of IRI kidneys demonstrated intratubular material from day 2 and mature casts by day 7. Mannitol reduced urine osmolality as expected with this regimen. In this unilateral model, we saw no difference in GFR or albumin to creatinine ratio, but the urinary ammonium acid-base index indicates an improvement in tubular function in mannitol-treated individuals. After 6 weeks, fibrosis (assessed by Picrosirius Red staining) was 40% reduced in the mannitol-treated mice. Finally, 2PM longitudinal imaging of n=5 control and n=6 mice indicated reduced granular casts and development of tubular atrophy in the mannitol-treated group. In conclusion, casts may not only reflect injury but also exacerbate proximal tubule damage, potentially through obstruction and direct signaling on the adjacent epithelium. Cast removal with osmotic diuretics represents a feasible strategy that warrants further evaluation.  

Authors: Katharina van der Giet (1), Maria Räwer (1), Dr. Charlotte von Horn (2), Prof. Thomas Minor (2), Dr. Helmuth Lieder (3), Prof. Petra Kleinbongard (3), Prof. Lars Pape (1), Prof. Anja Büscher (1), Dr. Johannes Jägers (1)

Affiliations:
1University Hospital Essen, Children's Hospital II, Hufelandstr. 55, 45147 Essen, Germany
2 University Hospital Essen, Clinic of General, Visceral, Vascular, and Transplant Surgery, Hufelandstr. 55, 45147 Essen, Germany
3 University Hospital Essen, Institute for Pathophysiology, Hufelandstr. 55, 45147 Essen

Abstract:

For kidney transplantation histidine-tryptophan-ketoglutarate solution (HTK) is the most used organ storage solution in Europe. Tryptophan is a central metabolite for various metabolic pathways, such as the NAD+-Synthesis and indole- and kynurenine pathway, which play crucial roles in the energy metabolism and in immunomodulation. Kynurenine in particular is used in clinical settings as a marker for infectious and inflammatory processes. Especially in kidney transplantation, the tryptophan pathway is linked to the occurrence of acute kidney rejection. Therefore, alterations in the tryptophan metabolism may impact kidney function and graft survival after static cold storage. Rat (n=5) and pig (n=5) kidneys were rinsed with cold HTK immediately after retrieval and stored at 4 °C for 6 hours (rats) and 24 hours (pigs). Afterwards rat kidneys were reperfused, using an ex-vivo normothermic kidney perfusion setup, for 2 hours. Samples of cortex and medulla were collected before and after cold storage and after reperfusion. To investigate the effect of cold storage, tryptophan and its metabolites HK-2 cells were stored overnight at 4 °C or 37 °C with media containing 2 mM tryptophan, kynurenine, or without supplementation. We applied qPCR, Western Blot analysis and immunohistochemistry to investigate the expression of key enzymes of the different pathways (IDO1, TDO2, ACMSD, KYAT1, QPRT and TPH) We observed a significant but reversible change in tryptophan metabolism in cold-stored kidneys. IDO1 and TDO2 (kynurenine pathway) showed species-specific expression differences between pigs and rats. Upregulation of KMO, QPRT, and ACMSD in pigs (acetyl-CoA synthesis) may help to maintain renal energy supply during cold storage. Increased TPH expression (serotonin synthesis) in the medulla aligns with findings that normothermic hypoxia enhances serotonin synthesis in murine hepatocytes. Reduced IDO1 or TDO2 expression together with increased downstream enzyme activity lower nephrotoxic kynurenine, potentially protecting transplanted organs.

Authors: Malotka L (1), Kulow V A (1), Erdogan C (1), Labes R (1), Fähling M (1)

Affiliations:
Institute of Translational Physiology, Charité – Universitätsmedizin Berlin

Abstract:

Aim Acute kidney injury (AKI) affects approximately 20 % of hospitalized patients and more than 50 % of those in intensive care. Independent of etiology, renal cellular injury is commonly associated with regional hypoxia. As neither early diagnostic markers nor causal therapies are currently available, we investigated whether pharmacological modulation of oxygen–hemoglobin binding affinity influences renal oxygen-related responses in health and under conditions of acute kidney injury. Methods Efaproxiral (alias RSR13; 300 mg/kg body weight, i.p.), a synthetic allosteric modulator of oxygen-hemoglobin binding, was administered to healthy DBA2/J mice and to mice with rhabdomyolysis-induced acute kidney injury (RIAKI). Blood and organ samples were collected 2, 6, 12, and 24 hours after RSR13 treatment. Oxygen dissociation curves were assessed by tonometry. Potential direct cellular effects of Efaproxiral were examined in human embryonic kidney cells in vitro. Renal gene expression was analyzed by next-generation sequencing and validated by qPCR. Tissue injury and protein localization were assessed by immunohistochemistry and immunofluorescence. Results Efaproxiral induced a rightward shift of the oxygen dissociation curve (up to +7 mmHg) with an apparent half-life of approximately 4 hours. Efaproxiral treatment alone did not induce histological kidney injury. Notably, Efaproxiral administration was associated with increased expression of HIF 1 target genes in healthy kidneys in vivo and further enhanced HIF-associated gene expression in RIAKI. These effects were not reproduced in renal cells in vitro. In contrast to the kidney, other organs, including the heart, showed transcriptional signatures consistent with improved tissue oxygenation. Conclusion Pharmacological reduction of oxygen-hemoglobin binding affinity elicits organ-specific oxygen-related responses in vivo. While several organs exhibit signatures compatible with improved oxygen availability, the kidney displays a distinct transcriptional response characterized by activation of hypoxia-associated pathways. These findings point toward unique renal determinants of oxygen handling that modulate the physiological consequences of altered oxygen-hemoglobin affinity.

Authors: Motalli A (1), Borrelli M (1), Papini N (2), Tringali C (2), Rampichini S (1), Esposito F (1)

Affiliations:
1. Department of Biomedical Sciences for Health - Università degli Studi di Milano, Milan, Italy
2. Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Segrate, Milan, Italy

Abstract:

Introduction Conventional cigarette smoking (CS) is the leading preventable cause of death worldwide. CS impairs cardiovascular, respiratory and muscle function, thereby altering exercise responses. Nowadays, electronic nicotine products are increasingly used as safer alternatives. Yet, evidence suggests early cardiovascular effects comparable to CS with also cardiopulmonary impairments at rest. Given the heterogeneity of these effects coming from over 7000 different devices, we focused on a specific heated tobacco product (HTPs), widely spread in Europe, especially in young individuals. Aim We investigated whether chronic HTPs use alters cardiorespiratory and metabolic kinetics in response to moderate-intensity square-wave exercise in young, physically active individuals. Methods Four HTPs users (age: 22.8±2.2 years; stature: 1.73±0.08 m; body mass: 67±9.9 kg; 14±11sticks/day; 5±2 years HTPs exposure) and 4 non-smokers (CTRL; age: 22.0±0.8 years; stature: 1.70±0.09 m; body mass: 75.0±17.2 kg) underwent spirometry and carbon monoxide diffusing capacity (DlCO) assessment. On two different days, participants performed an incremental step test and four 6-min square-wave bouts at 50% of peak power output to assess expiratory ventilation (V'E), oxygen uptake (V'O2) and heart rate (fH) kinetics during both ON and OFF phases. Breath-by-breath data were averaged and fitted with a mono-exponential model to derive amplitude (AMP) and time constant (𝜏). Results No group differences were observed in pulmonary function or diffusing capacity, nor cardiorespiratory and metabolic responses at peak exercise. During square-wave exercise, HTPs users showed slower ON-transient kinetics for V'E (𝜏: 82.0±17.1 vs 43.7±2.9 s for HTPs and CTRL, respectively; p=0.004) and V'O2 (𝜏: 49.9±11.3 vs 28.4±11.9 s for HTPs and CTRL, respectively; p=0.040). Conclusion Despite similar pulmonary function and peak responses, chronic HTPs use was associated to a slower ventilatory and metabolic response to moderate-intensity exercise even in young, physically active individuals, which is consistent with early cardiopulmonary alterations previously described in HTPs users at rest.

Authors: Körber J (1), Gensch T (1), Fahlke C (1)

Affiliations:
1. Institute of Biological Information Processing, Molekular- und Zellphysiologie (IBI-1), Forschungszentrum Jülich, 52428, Jülich, Germany

Abstract:

Disbalanced neuronal chloride homeostasis can hamper brain function by promoting hyperexcitability. Additionally, increased intracellular [Cl^-] and corresponding hyperosmolarity might trigger cerebral edema in ischemic stroke. Despite this physiological significance, reports of absolute [Cl^-]_int in neurons are rare, in part due to the technical hurdles in assessing [Cl-]int directly. In this study we investigate neuronal [Cl^-]_int under physiological conditions in organotypic cortical brain slices by 2-photon fluorescence lifetime imaging microscopy of the Cl- sensitive dye MQAE. Under resting conditions, we obtained [Cl^-]_int values of 15 ± 4 mM (n =26 slices). Neuronal chloride concentrations are under control of the potassium-chloride-cotransporter 2 (KCC2), with inhibition of KCC2 resulting in an increased average of 21 ± 7 mM (n= 10 slices). Chemical ischemia; i.e. ATP deprivation by inhibition of both glycolysis and respiratory chain increased [Cl^-]_int to 60 ± 22 mM (n= 9 slices) after 10 minutes. Application of bumetanide, an inhibitor of the sodium-potassium-chloride-cotransporter 1 (NKCC1) attenuates this increase to 31 ± 8 mM (n = 7 slices), identifying NKCC1 as a major entry path for Cl^- into neurons in ischemia. Application of DIDS - a nonspecific anion exchange inhibitor also inhibits ischemic Cl^- uptake with [Cl^-]_int = 27 ± 16 mM (n=6 slices) after 10 minutes chemical ischemia. The combination of bumetanide and DIDS potentiates the effect of the individual compounds, resulting in a [Cl^-]_int of 21 ± 4 mM (n=4 slices) after 10 minutes chemical ischemia. Our findings are in agreement with previous studies on volume changes during ischemia/oxygen-glucose deprivation in neurons and highlight the central role of Cl^- in neuronal osmoregulation, providing potential targets for novel treatment options in ischemic stroke.

Authors: Eschholz L S (1), Wiegert J S (1), Dieter A (1)

Affiliations:
1. Department of Neurophysiology, Medical Faculty Mannheim, MCTN, University of Heidelberg, Mannheim, Germany

Abstract:

Almost all brain functions are controlled by a small set of transmitter molecules termed neuromodulators. Disbalanced neuromodulation can lead to altered, pathological brain states. However, despite their importance, it remains poorly understood how the different neuromodulatory systems interact dynamically to control brain function. Recent advances in the genetic targeting of neuromodulatory neurons and the emergence of fluorescent indicators sensitive to specific neuromodulators finally allow to address this gap of knowledge. Here, we investigate the interaction between noradrenergic neurons of the locus coeruleus(LC-NE) and dopaminergic neurons of the ventral tegmental area (VTA-DA). By simultaneously recording calcium activity from LC-NE and VTA-DA neurons in awake mice, we found that these two neuromodulatory systems are differentially coupled to both locomotion and pupil-linked arousal. Es expected, LC-NE activity positively predicts locomotion onset and pupil dilation. In contrast, VTA-DA activity is negatively coupled to these events. Temporal dynamics of neuromodulatory activity suggest that LC-NE activity precedes and suppresses VTA-DA activity. These findings were corroborated by anatomical and functional evidence for norepinephrine release from LC neurons within the VTA. Finally, exposure of the mouse to a natural stressor as well as optogenetic activation of LC-NE neurons demonstrate the causal relationship of LC-NE activation on VTA-DA suppression. In conclusion, our findings suggest a negative coupling between these two catecholaminergic systems, which is governed by the LC-NE system.

Authors: Nadja Sharkov (1), Tina Sackmann (1), Nikolas A. Stevens (1), Janina Kupke (2,3), Andreas Draguhn (1,4), Ana M.M. Oliveira (2,4,5), Martin Both (1,4)

Affiliations:
1. Institute of Physiology and Pathophysiology, Medical Faculty, Heidelberg University, Germany
2. Department of Neurobiology, Heidelberg University, Germany
3. Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Vrije Universiteit Amsterdam, 1081 HV, Netherlands
4. Interdisciplinary Center for Neurosciences (IZN), Heidelberg University, 69120 Heidelberg, Germany
5. Department of Molecular and Cellular Cognition Research, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, 68159 Mannheim, Germany

Abstract:

Hippocampal pyramidal cells are involved in spatial coding and memory formation which requires the reliable integration of multiple synaptic inputs within single neurons. According to current understanding, this takes place at the soma which is directly connected with the axon, where supra-threshold inputs trigger action potentials. However, in about 50% of hippocampal CA1 pyramidal neurons the axon emerges from a basal dendrite (AcD, ‘axon-carrying dendrite’). This particular dendrite is largely independent from somatic signal integration and can efficiently convert excitatory inputs into action potentials (APs). We therefore hypothesize that AcD cells are more active during the formation and consolidation of memories. To test this hypothesis, we trained mice on a spatial memory task. Active neurons are expected to express immediate early genes (e.g. cFos), and can be identified by ex vivo staining. cFos expression of cells were analyzed at different time points of the training process and cell were classified into AcD and canonical cells. The number of cells expressing cFos was higher in dorsal compared to medio-ventral portions of the hippocampus. AcD cells and canonical neurons showed different learning-related time courses of cFos. While the number of cFos expressing cells in the dorsal hippocampus decreased within canonical cells during the learning process, it increased in AcD cells. Interestingly, the proportion of AcD cells in medio-ventral CA1 decreased during the learning protocol, indicating structural plasticity of axon initial segment location. Our findings indicate distinct roles of AcD and nonAcD cells during formation and consolidation of spatial memories in the hippocampus.

Authors: Damato M (1), Aboulouard S (2), La Pesa V (3), Gentile F (3), Lunghi G (4), Perrone M (1), Pranzo B (1), Chiricozzi E (4), Fournier I (2), Romano A (3,5,6), Quattrini A (3), Salzet M (2), Maffia M(1)

Affiliations:
1. Dept of Experimental Medicine, Univ. of Salento, Lecce, Italy
2. Universitè de Lille, Inserm, CHU Lille, U1192, Laboratoire Protéomique, Réponse Inflammatoire et Spectrométrie de Masse (PRISM), F-59000 Lille, France
3. Neuropathology Unit, Institute of Experimental Neurology, Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy.
4. Department of Medical Biotechnology and Translational Medicine, University of Milano, Segrate, Milano, Italy.
5. Department of Life Sciences, Health and Health Professions, Link Campus University, Rome, Italy
6. CNR NANOTEC – Institute of Nanotechnology, Campus Ecotekne, Lecce, Italy.

The author did not consent to the publication of the abstract.

Authors: Wagner C (1,2)

Affiliations:
(1) Department of Physiology, Universität Zürich
(2) Editor-in-Chief, Pflügers Archiv of Physiology - European Journal of Physiology

Abstract:

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Authors: Alena E. Shulutkova (1), Thomas Gensch (1)

Affiliations:
1. Institute of Biological Information Processing, Department of Molecular and Cellular Physiology (IBI-1) – Forschungszentrum Jülich

Abstract:

Ion concentrations of the lysosomal lumen are scarcely investigated, but crucial for understanding cellular processes such as ion homeostasis or membrane potential in lysosomes. We are developing a potassium biosensor for application in lysosomes of living cells. The largest challenge – next to effective targeting to the lysosome – is the low lysosomal pH (4.5-5). Our design is based on native E. coli potassium-binding protein (Kbp) and a pair of fluorescent proteins leading to Förster Resonance Energy Transfer (FRET), where potassium binding causes a FRET change. It will allow to determine lysosomal potassium concentrations with both lifetime and ratiometric fluorescence intensity change. We determined the pH dependence of potassium binding of isolated Kbp and tested various combinations of fluorescent proteins with high pH stability and good spectral overlap to create a biosensor suitable for the low lysosomal pH. The best candidate was characterized in vitro and in vivo (expressed in HEK293T cells).

Authors: Lara Heinrich (1), Anastasia Pyanova (1), Rudolf Schubert (1)

Affiliations:
1. Physiology, Institute of Theoretical Medicine, Faculty of Medicine, University of Augsburg, Augsburg, Germany

Abstract:

Motivation: Organ perfusion is regulated by arterial tone, which is determined by the membrane potential of vascular smooth muscle cells. Potassium (K+) channels are key modulators of this membrane potential. Notably two-pore domain K+ (K2P) channels play roles in negative feedback regulation of vasoconstriction and pH-induced vascular tone. While well studied in the pulmonary circulation, their function in the systemic circulation remains largely unexplored. We hypothesized that K2P channels, particularly TASK-1 or TREK-1 channels, contribute to tone regulation in the systemic circulation. Methods: Intact segments of a rat systemic artery (Arteria saphena) were studied ex vivo using wire myography. For TREK-1 channels, the selective blocker spadin and the opener ML335 were explored. TASK-1 channel function was examined using the selective blocker ML365. Due to their small conductance, we aimed to unmask them by blocking major potassium conductances using iberiotoxin (BKCa channels) and DPO-1 (Kv1.5 channels). Results: We did not detect any relevant effect of spadin alone or in the presence of ML335, either in the absence or in the presence of IBTX+DPO-1. ML335 reduced methoxamine-induced contractions, indicating an unspecific effect at the applied concentration. We did not detect any relevant effect of ML365 in the absence of IBTX+DPO-1 but increased vascular tone in the presence of IBTX+DPO-1, with ML365 being more potent than the previously studied TASK-1 blocker AVE1231, but having a non-specific effect at higher concentrations. AVE1231 has been shown earlier to induce comparable effects in pulmonary arteries, whereas corresponding data for ML365 in the pulmonary circulation are currently lacking. Conclusion: Our data suggest that in A. saphena TREK-1 channels do not contribute to basal or methoxamine-induced tone. In contrast, TASK-1 channels contribute to negative feedback on vasoconstriction when dominating potassium conductances are suppressed, indicating a functional role for TASK-1 channels in the regulation of systemic arterial tone.

Authors: Zizhuo Lu (1), Sönke Cordeiro (1), Han Sun (2), Thomas Baukrowitz (1)

Affiliations:
1. Institute of Physiology, Kiel University, Kiel, Germany
2. Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany

Abstract:

TMEM175 is an ion channel localized to endosomal and lysosomal membranes. It is primarily characterized as a K⁺ channel but also exhibits proton permeability, thereby contributing to lysosomal pH regulation. Proper lysosomal pH homeostasis is essential for hydrolase activity and efficient protein degradation. Loss-of-function variants of TMEM175 have been linked to Parkinson’s disease, likely through impaired lysosomal proteolysis and the consequent accumulation of toxic proteins in dopaminergic neurons of the substantia nigra. In contrast, gain-of-function TMEM175 variants have been reported to exert neuroprotective effects. Collectively, these findings suggest that pharmacological activation of TMEM175 represents a promising therapeutic strategy for Parkinson’s disease. A diverse range of compounds has been reported to activate TMEM175; however, the molecular mechanisms underlying TMEM175 activation remain poorly understood. A recently published study elucidated the cryo-EM structure of human TMEM175 and proposed docking models for small molecules activating TMEM175 channels. Notably, our own molecular dockings suggested an alternative binding site for DCPIB based, highlighting unresolved structural questions regarding small molecule–TMEM175 interactions. Building on these structural and pharmacological studies, the present work aims to define the binding sites of the activators DCPIB and TUG-891 on TMEM175 and to elucidate the structural mechanisms underlying TMEM175 activation. Site-directed mutagenesis combined with patch-clamp electrophysiology is employed to functionally interrogate ligand–channel interactions. These observations should be applied to other activators we have identified. Identification of key residues involved in drug binding will provide a foundation for the rational optimization of TMEM175-targeted therapeutics.

Authors: Júlia Castro-Marsal, Franziska M. Schneider, Marc Behrendt, Raissa Enzeroth, Olaf Pinkenburg, Johannes Oberwinkler

Affiliations:
Institut für Physiologie und Pathophysiologie, Philipps-Universität Marburg, Marburg, Germany

Abstract:

In the dendritic tips of ON-bipolar cells, TRPM1 is found in a multiprotein complex together with mGluR6. In the absence of light glutamate is released from photoreceptors, activating mGluR6, which leads to the inhibition of TRPM1 channels and to ON-bipolar cell hyperpolarization. Given the difficulty of studying TRPM1 channels in heterologous systems, the details of this negative regulation are still unclear. TRPM3 channels, which are closely related to TRPM1, are inhibited by binding to Gβγ proteins, and the interaction site has been mapped in TRPM3. Interestingly, this short (10 aa) N-terminal Gβγ-interacting sequence is also found in TRPM1. Using HEK293T cells for heterologous expression, we investigated TRPM1 inhibition produced by GPCR activation with whole-cell patch-clamping. We specifically tested the hypothesis that Gβγ-mediated inhibition of TRPM1 occurs by Gβγ-binding to the same binding site sequence as in TRPM3. Testing different Gi-coupled receptors, we found a larger inhibition when co-expressing TRPM1 with the morphine-activated μOR than with the glutamate-activated mGluR6. We therefore used μOR for further experiments. The non-hydrolyzable GDP analog GDPβS deactivates G-proteins. Applying this substance intracellularly reduced TRPM1 inhibition, indicating a dependence on the G-protein activity. Furthermore, TRPM1 inhibition was abolished by the additional overexpression of a Gβγ-binding peptide, demonstrating that Gβγ subunits are responsible for TRPM1 inhibition. TRPM1 inhibition was not reduced during the intracellular application of AMP-PCP, a non-hydrolyzable ATP analogue which inhibits several enzymes potentially implicated in a Gi-regulated pathway, such as PKA or adenylate cyclase. This indicates that these downstream enzymes are not involved in TRPM1 inhibition. Importantly, a mutant TRPM1 without the Gβγ-interacting sequence did not present any inhibition after μOR or mGluR6 activation. In conclusion, our results suggest that TRPM1 channels are inhibited by direct interaction of Gβγ proteins on the same Gβγ-interacting sequence as has been identified in TRPM3.

Authors: Joel A. M. Porto (1,2), Bassam G. Haddad (2), Jan-Philipp Machtens (1,2), Christoph Fahlke (1)

Affiliations:
(1) Molecular and Cellular Physiology (IBI-1), Forschungszentrum Jülich, Jülich, Germany
(2) Institut für Neurophysiologie, Medizinische Hochschule Hannover, Hannover, Germany

Abstract:

Amino acids are essential not only as protein building blocks, but also in metabolism, cell signaling, and cellular homeostasis. The SLC1 family is important for amino acid uptake and distribution. ASCT2 (SLC1A5) is a sodium-dependent exchanger that transports neutral amino acids such as alanine, serine, and glutamine and contributes to cancer progression, making it a potential therapeutic target. While sodium’s role in ASCT2 activity is well established, the impact of potassium is less understood. Some studies suggest potassium may interfere with ligand binding and transport, though the mechanism is unclear. To investigate ion selectivity and transport, we performed molecular dynamics (MD) simulations combined with Markov state modeling (MSM) and umbrella sampling to characterize ion binding, conformational dynamics, and Hairpin 2 (HP2) gate energetics during substrate translocation. Kinetic analyses of putative binding sites revealed a higher thermodynamic affinity for sodium compared to potassium. KDs and binding ΔGs revealed limited Na⁺/K⁺ selectivity without ligand, whereas in the presence of serine, ΔG became highly favorable for Na⁺ and less favorable for K⁺, indicating ligand-dependent ion selectivity. Umbrella sampling simulations of the HP2 gate indicated a more stable conformational landscape with both sodium and ligand compared to sodium alone, highlighting the role of ligand in stabilizing transport-relevant conformations. MSM revealed that sodium, in the presence of serine, supports formation of a stable binding configuration necessary for transport, whereas potassium disrupts this process by destabilizing the binding site and preventing proper HP2 closure. To evaluate ASCT2 function, whole-cell patch-clamp electrophysiology was performed under varying extracellular ionic conditions, using multiple transported ligands and defined intracellular substrate conditions. Electrophysiology data showed a strong dependence on sodium, with a notable reduction in current when potassium was present. These findings clarify ASCT2’s ion selectivity and interaction mechanisms, demonstrating that ligand presence modulates sodium preference and stabilizes transport-relevant conformations.

Authors: Dominik Lenz-Schwab (1), Annalisa Questino (1), Muhammad Younus (1), Dominik Oliver (1)

Affiliations:
(1) Philipps-Universität Marburg, Institut für Physiologie & Pathophysiologie

Abstract:

The Solute Carrier Family 26 (SLC26) comprises 10 homologous anion transporters with key functions in essentially all transport organs. They share a [7+7]-inverted-repeat topology and dimerize, mainly along the intracellular STAS domain. It is established that SLC26A4 (pendrin) mediates an electroneutral exchange of anion such as Cl⁻, I- and HCO₃⁻, putatively by an elevator-movement of the transport vs the scaffold domain as shown for the closely related protein prestin (SLC26A5, Kuwabara et al. 2023). Recent cryo-EM structures of mixed inward-open and outward-open states (Liu et al. 2023) raised the question, whether the movement of the two protomers’ transport domains is cooperative or not. Mutations in the anion binding site in the center of trans-membrane domain (TM) 10 were performed to change and assess behaviour of both anion binding and transport. Mammalian cell lines were either transfected with wildtype (wt) or mutant pendrin alone or co-transfected aiming towards constructing heterodimers. Exchange of HCO3- against Cl- was measured by monitoring the fluorescence intensity of the pH-dependent dye pHrodo after transfection of HEK293T cells. Whole-cell patch-clamp experiments were performed on CHO cells to measure transport currents. While transfection of several mutants changed transport mode (R409S, R409C, A406E) and/or intensity (R409S, R409C, R409H, A406E), co-transfection of either of those mutants with wt rescued the HCO3- transport closely to the wt level, with a minor reduction in exchange kinetics, though. This can, however, easily be explained by a combined transport action of homomeric wt proteins and heterodimers with a functional wt subunit acting independently while the mutated subunit would still act as an elevator but without transport capacity. Despite the initial hopes, a movement-locked mutation could not yet be identified, therefore leaving the research question still open.

Authors: Sure F (1), Kißler A (1), Schreiber F (1), Korbmacher C (1), Ilyaskin AV (1)

Affiliations:
1. Institute of Cellular and Molecular Physiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany

Abstract:

The epithelial sodium channel (ENaC) is a heterotrimeric ion channel that typically assembles in either an αβγ- or δβγ-configuration. A unique feature of ENaC is its activation by proteolytic removal of inhibitory tracts from extracellular GRIP (gating relief of inhibition by proteolysis) domains in the channels' α- and γ-subunits. Although cryo-EM structures indicate that β- and δ-subunits also contain structurally similar GRIP domains, their functional relevance for proteolytic ENaC activation remains unclear. To address this, we combined molecular dynamics (MD) simulations with two-electrode voltage-clamp recordings in Xenopus laevis oocytes expressing human αβγ- or δβγ-ENaC. MD simulations revealed that inhibitory tracts of α- and γ-ENaC predominantly interact with four conserved residues in their respective GRIP domains (αF226/W251/H255/Y447 and γF204/W229/H233/Y425). Importantly, alanine substitutions of these critical residues significantly reduced the inhibitory effect of synthetic peptides that mimic the endogenous inhibitory tracts of α- or γ-ENaC. In contrast, the introduction of artificial proteolytic cleavage sites within β-ENaC to remove the corresponding region from the β-GRIP domain did not result in channel activation. δβγ-ENaC was activated by chymotrypsin, which cleaved both δ- and γ-subunits. However, chymotrypsin failed to stimulate δβγ-ENaC when dissociation of the γ-inhibitory peptide was prevented by covalently tethering it to its binding site with a newly introduced disulfide bond. This indicates that proteolytic activation of δβγ-ENaC is mediated primarily by cleavage of the γ-subunit rather than the δ-subunit. In summary, despite structural similarity, β- and δ-GRIP domains do not appear to contribute to proteolytic ENaC activation, which critically depends on a conserved set of aromatic residues in the α- and γ-GRIP domains.

Authors: Rott C (1), Merza O (1), Cordeiro S (1), Hu M (1)

Affiliations:
1. Institute of Physiology, Kiel University (CAU)

Abstract:

The endoskeleton of the sea urchin larva forms at an early embryonic stage and is composed of CaCO₃ produced by primary mesenchyme cells (PMCs). The biomineralization takes place in vesicles after endocytosis of sea water like fluid of the primary body cavity. In these vesicles, tight regulation of the ionic composition is critical for mineralization. Therefore, identification and characterization of vesicular ion channels is important to understand the underlying regulatory mechanisms. In mammals the transmembrane protein 175 (TMEM175) has been identified as lysosomal K⁺ and proton channel. Here, we show that TMEM175 is almost exclusively expressed in PMCs of the sea urchin Strongylocentrotus purpuratus, suggesting that it may be a critical factor in the larval skeletal development by regulating the vesicular pH and/or K⁺ concentration. This is supported by gene expression data showing strong upregulation of the sea urchin TMEM175 (spTMEM175) at 56 hours post fertilization, a time point within the critical phase of skeletogenesis, highlighting its potential in regulating vesicular ion composition during mineralization. To characterize the properties of spTMEM175, the gene was cloned and heterologously expressed in Xenopus laevis oocytes. Two-electrode voltage clamp analyses indicated that spTMEM175 activation depends on extracellular ion composition, particularly elevated magnesium concentrations and pH, resembling seawater conditions. These findings support the potential role for spTMEM175 as a pH- or ion-sensitive regulator. Future studies will aim to elucidate the ion selectivity and subcellular localization of spTMEM175 in greater detail. A deep mechanistic understanding of calcification processes enables improved explanation and prediction of how environmental changes affect marine calcifying systems in both past and future oceans.

Authors: Ömer Kocdölü(1), Birgit Begemann(1) , Jan-Philipp Machtens(1), Christian Wahl-Schott(2), Martin Fischer(1)

Affiliations:
1. Hannover Medical School, Institute of Neurophysiology.
2. Ludwig-Maximillian-University Munich, Biomedical Center (BMC), Institute for Cardiovascular Physiology and Pathophysiology.

Abstract:

Two-pore channels (TPC1 and TPC2) are cation-selective ion channels of the endo-lysosomal system. Deletion or inhibition of TPCs disrupts endo-lysosomal trafficking and ion homeostasis, contributing to various physiological functions. Mouse TPC1 (mTPC1) is activated by ligands (PI(3,5)P₂ and NAADP) and simultaneous membrane depolarization, which results in a rearrangement of a voltage-sensing domain and subsequent opening of a hydrophobic gate formed by bundle crossing of S6 helices. Major constriction sites are leucines at positions 317 and 688 and phenylalanine at position 321. Here, we substituted these amino acids by alanine (triple mutation L317A/L321A/F688A). After transient expression of wild-type (WT) and mutated mTPC1 in HEK293T cells, we investigated electrophysiological properties of mTPC1 using the endo-lysosomal patch-clamp technique. Even though confocal microscopy displayed lower expression of the mutant channel on enlarged vesicles (vs. WT), whole-lysosome recordings revealed significantly larger currents for the mutated mTPC1. Both channels showed time-dependent activation upon depolarization and deactivation upon hyperpolarization with comparable low relative open probabilities at negative potentials. In contrast, single-channel recordings obtained in the lysosome-attached mode demonstrated voltage-dependent gating only for the WT channel. The hydrophobic gate mutation rendered the channel largely voltage-independent with a high absolute open probability. Occasional closures putatively depended on episodic ligand release. We conclude that the hydrophobic gate mutation constitutively opened the pore of mTPC1, explaining the large currents obtained from whole lysosomes. Large current amplitudes in combination with a small vesicle lumen, however, altered ion gradients through ion depletion or accumulation in the whole-lysosome configuration, leading to an apparent voltage-dependent current decease at negative potentials. Despite this challenge of endo-lysosomal recordings, we confirm here, based on single-channel recordings from lysosomal mTPC1, the functional relevance of important constriction sites in the hydrophobic gate, as previously predicted exclusively from structural data.

Authors: Kühs S (1), Stawarski M (1), Kuspiel S (1), Kläser E (1), Gründer S (1)

Affiliations:
Institute of Physiology, RWTH Aachen University, Aachen D-52074

The author did not consent to the publication of the abstract.

Authors: Sven Schütte (1), Florian Schick (1), Emilia De Martino (1), Hannah Schmitz (1), Katharina Zander (1), Eva Corvest (3), Verena Wilmes (3), Moritz Linder (5), Aytuğ K Kiper (1,2), Silke Kauferstein (3,4), Martin K-H Schäfer (6), Susanne Rinné (1) & Niels Decher (1)

Affiliations:
1. Institute of Physiology and Pathophysiology, Vegetative Physiology, University of Marburg, Marburg, Germany.
2. Institute of Physiology, University Medicine Greifswald, Greifswald, Germany.
3. Centre for Sudden Cardiac Death and Institute of Legal Medicine, University Hospital Frankfurt, Goethe-University, Frankfurt/Main, Germany.
4. DZHK (German Centre for Cardiovascular Research), Partner Site Rhein-Main, Frankfurt, Germany.
5. Institute of Biomedical Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
6. Institute of Anatomy and Cell Biology, University of Marburg, Marburg, Germany.

Abstract:

Two-pore domain potassium channels (K_2P) are crucial for the regulation of the membrane potential in both excitable and non-excitable cells. As they are modulated by various physiological stimuli, they play an important physiological role in different tissues throughout the human body. Over the last couple of decades, increasing evidence has emerged that K_2P channels are not only limited to homomer formation, but also form functional heterodimers with altered channel characteristics. This increases the functional and pharmacological heterogeneity within this channel family. In the current study, we describe the formation of a novel functional heterodimer consisting of TREK-1 and TASK-4. Utilizing two-electrode voltage-clamp experiments (TEVC) and inside-out macropatch-clamp recordings, as well as ELISA-based HA-assays for quantifying surface expression, we characterized the heteromerization of these two K_2P channels. The TREK-1/TASK-4 heteromeric channels display an altered regulation by physiological stimuli, a unique pharmacology and G-protein-coupled receptor-mediated modulation. Therefore, the formation of functional TREK-1/TASK-4 heterodimers with their distinct pharmacological profile and altered responses to different physiological stimuli must be considered in studies utilizing native tissues, in which both channels are expressed, as well as in the development of drugs that selectively target homo- or heterodimeric K_2P channels.

Authors: Emilia De Martino (1), Susanne Rinné (1), Stefanie Scheiper-Welling (2), Sven Dittmann (4), Tina Jenewein (2), Katharina Herpertz (1), Silke Kauferstein (2,3), Eric Schulze-Bahr (4), Niels Decher (1)

Affiliations:
1. Institute for Physiology and Pathophysiology, Vegetative Physiology, University of Marburg, Marburg, Germany
2. Centre for Sudden Cardiac Death and Institute of Legal Medicine, University Hospital Frankfurt, Goethe‐University, Frankfurt/Main, Germany
3. DZHK (German Centre for Cardiovascular Research), Partner Site Rhein‐Main, Frankfurt, Germany
4. Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases, University Hospital Münster, Münster, Germany

Abstract:

The syntrophin family includes three dystrophin-associated intracellular proteins: α1 (SNTA1), β1 (SNTB1), and β2 (SNTB2). While β-syntrophins are found in a greater variety of tissues, α1-syntrophin is primarily expressed in skeletal muscle. As part of the dystrophin protein complex these modular adaptor proteins can recruit and anchor various signaling proteins. According previous studies, syntrophins interact with the voltage-gated sodium channel Na_v1.5 (SCN5A) within a multi-protein complex, affecting channel expression and function. Genetic variants in SCN5A and SNTA1 were previously linked to Sudden Infant Death Syndrome (SIDS) and Sudden Cardiac Death (SCD). Using whole-exome sequencing (WES), we identified a heterozygous variant in the SNTB1 gene, in a family affected by SCD. In voltage-clamp experiments co-expression of Na_v1.5 with SNTB1 reduced the sodium current amplitudes while this effect was absent for the SNTB1 variant, resulting in increased channel activity. Neither SNTB1 nor its genetic variants altered Na_v1.5 gating properties. Therefore, the observed changes in current densities were likely due to altered surface expression of Na_v1.5. As a consequence of the results, we subsequently screened a large cohort of young patients deceased from SIDS or SCD for putative disease-causing SNTB1 variants. Here we identified three rare SNTB1 variants with negative pathogenicity predictions. Functional studies revealed that these variants similarly induced a significant impairment of SNTB1-mediated current reduction of Na_v1.5, leading to increased sodium currents. Overall, our data suggest that the dysregulation of the voltage-activated Na_v1.5 channel by SNTB1 variants is a novel arrhythmia mechanism and a potential putative cause of SIDS and SCD.

Authors: Kuwabara MF (1,2), Berger TK (1), Machtens JP (2), Oliver D (1)

Affiliations:
1. Department of Neurophysiology, Institute of Physiology and Pathophysiology, Philipps University Marburg
2. Institute of Neurophysiology, Hannover Medical School

Abstract:

Voltage-sensitive membrane proteins sense membrane potential, converting voltage changes into downstream electrical, chemical, or mechanical signals. Classical electrophysiological techniques such as patch-clamp and two-electrode voltage clamp provide high-resolution readouts of gating-charge movement and ionic currents, but offer limited direct information about the underlying protein conformational changes. Voltage-clamp fluorometry (VCF) bridges this gap by recording fluorescence changes from an environmentally sensitive fluorescent probe while controlling membrane voltage. Conventional VCF typically labels genetically introduced extracellular cysteines with thiol-reactive dyes. It is often performed in Xenopus laevis oocytes as a heterologous expression system and labels introduced cysteines by applying dyes from the extracellular side. This restricts labeling to extracellularly accessible sites and limits applicability to proteins that can be robustly expressed and functionally characterized in oocytes. Here we are working to implement ANAP-based VCF in cultured human cells. ANAP, a fluorescent non-canonical amino acid (3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid), can be genetically encoded at selected positions that tolerate incorporation, enabling site-specific fluorescence reporting from extracellular, membrane-embedded, or intracellular sites. This approach was pioneered for voltage-gated channels in Xenopus laevis oocytes by Kalstrup and Blunck (2013) and has recently been implemented in human cells (Suárez-Delgado et al., 2023). Our goal is to apply this technique to a range of voltage-sensitive membrane proteins and relate conformational changes to function. This effort should facilitate the accessibility of VCF, broaden the range of questions that can be addressed, and support functional studies across protein families under physiologically relevant cellular conditions.

Authors: Emelie Dehmer, Júlia Castro-Marsal, Doris Newel, Olaf Pinkenburg, Johannes Oberwinkler

Affiliations:
Institut für Physiologie und Pathophysiologie, Philipps-Universität Marburg, Marburg, Germany

The author did not consent to the publication of the abstract.

Authors: Héctor Noguera Hurtado (1), Luca Matteo Todesca (2), Christina Kick (3), Martina Düfer (3), Bernhard Wünsch (3), Ildiko Szabo (2), Albrecht Schwab (1)

Affiliations:
1. University Hospital Münster, Institute of Physiologie II, Münster, Germany
2. University of Padova, Department of Biology, Padova, Italy
3. University of Münster, Institute for Pharmaceutical and Medicinal Chemistry, Münster, Germany

Abstract:

Mitochondria play a crucial role in cancer development by regulating cellular metabolism, redox balance, and apoptotic signaling. Tumor cells rely on mitochondrial function to sustain proliferation and survival. Ion channels in the inner mitochondrial membrane control ion homeostasis and are essential for mitochondrial activity. Among these, the mitochondrial K_Ca 3.1 channel (mitoK_Ca 3.1) impacts mitochondrial bioenergetics and cell survival. In this project, we aim to determine how the mitoK_Ca3.1 channel contributes to cancer progression by targeting it with new TPP⁺-conjugated drugs. To inhibit mito_KCa3.1, we used two new senicapoc derivatives (mito-sen and mito-rev-sen) that are targeted to mitochondria by conjugation with tri-phenyl-phosphonium⁺ (TPP⁺). We validated these compounds by measuring mitochondrial membrane potential (Δψmito) in lung and breast cancer cell lines. Blocking K⁺ influx through mito_KCa3.1 hyperpolarized Δψmito. Since mitochondrial function influences energy and signaling pathways supporting cancer progression, we examined whether inhibiting mito_KCa3.1 affects cell migration. After treating lung cancer cells with mito-sen for 24h, migration was reduced. In addition, channel blockade increased reactive oxygen species, disrupted mitochondrial respiration, and upregulated endoplasmic reticulum stress markers, leading to cell death in both single-cell and spheroid tumor models. Moreover, we observed that blocking mito_KCa3.1 caused a loss of mitochondrial cristae and swelling of mitochondria. Notably, the effects of the novel blocker mito-sen were achieved at a 30-fold lower concentration than those of the original inhibitor senicapoc. Based on these results, we conclude that the mito_KCa3.1 channel plays a role in regulating mitochondrial function and cancer-related cell behaviors, including migration and survival. Targeting mito_KCa3.1 disturbs mitochondrial ion balance and affects downstream metabolic and stress pathways, supporting its potential as a mitochondrial target for controlling tumor cell viability.

Authors: Donati C (1), Cazzola J (1), Talpo F (1), Faravelli G (1), Maramai S (2), Saletti M (2), Giuliani G (2), Paolino M (2), Cappelli A (2), Anzini M (2), Sommi P (3), Trucco A (1), Biella G R (1,4), Spaiardi P (1,4)

Affiliations:
1. Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, Pavia, Italy
2. Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy
3. Department of Molecular Medicine, Human Physiology Unit, University of Pavia, Pavia, Italy
4. Istituto Nazionale di Fisica Nucleare, Sezione di Pavia, Pavia, Italy

Abstract:

Excitotoxicity has been identified as a hallmark in different neurodegenerative conditions. This pathogenic process induces toxic effects due to dysregulated glutamate signaling, which can ultimately lead to neuronal function loss and cell death. Excitotoxicity is targeted by Riluzole (Rilutek®), a benzothiazole derivative and the first clinically approved drug for the treatment of amyotrophic lateral sclerosis (ALS), which showed to slow down disease progression and prolong patients’ survival. Riluzole has been proven to exert a neuroprotective role by a multimodal mechanism of action, by modulating voltage-dependent sodium and potassium channels and reducing glutamatergic neurotransmission, thereby controlling overall neuronal excitability. However, Riluzole displays a concentration-dependent mechanism of action, and its efficacy is often reduced in more advanced stages of the disease. To address the pharmacological limitations of Riluzole, novel derivatives such as VA945 and VA942 were synthesized in the laboratory of Prof. Anzini at the University of Siena. These compounds are currently being electrophysiologically explored by our lab at the University of Pavia. Having successfully characterized the modulatory effects of VA945 on voltage-gated sodium and potassium currents across a wide concentrations range (5-50-100 μM), we subsequently proceeded to investigate its parent compound, VA942. We have begun analyzing the effect of VA942 on channel conductance, kinetics and voltage-dependence. Our goal is to determine whether VA942 displays a neuroprotective profile similar to that of Riluzole, while potentially offering an improved pharmacological profile. Our findings will clarify if VA942 is a promising candidate for further preclinical analysis in disease-relevant models, with potential for translational and clinical applications on ALS patients.

Authors: A. Heuer (1), D. Ciechanska (1), S. Schimmelpfennig (1), S. Barbakadze (2), A. Schwab (1)

Affiliations:
1. Institut for Physiology II, Robert-Koch-Str. 27b, 48149 Münster, Germany
2. European Institute for Molecular Imaging, Röntgenstraße 16, 48149 Münster, Germany

Abstract:

KCa3.1 is a calcium-activated potassium channel that regulates membrane potential and cell migration in various cell types, including immune cells.[1] Neutrophils are key mediators of innate immune defense and important components of the tumor microenvironment. In non–small cell lung cancer (NSCLC), the neutrophil-to-lymphocyte ratio (NLR) has been identified as a prognostic factor for tumor progression and clinical outcome. [2] In this study, we investigated the effects of pharmacological inhibition of KCa3.1 in neutrophils using senicapoc, a selective KCa3.1 inhibitor. Live-cell imaging was applied to analyze neutrophil chemotaxis and membrane potential upon senicapoc treatment. By confocal microscopy, we examined the NLR in senicapoc-treated lung tumors. We established a protocol enabling the comparison of the NLR between tumor core and the peripheral 250 µm of a tumor node. Our data demonstrate differential distributions of neutrophils and CD8+ T cells in these regions. Based on the findings, we evaluated whether KCa3.1 channel inhibition impacts neutrophil tumor infiltration by impairing chemotaxis. In chemotaxis experiments, we observed reduced migratory capabilities of neutrophils after senicapoc-treatment. This approach expands the knowledge of the KCa3.1 channel function in neutrophils. Moreover, it reveals the impact of senicapoc treatment on the immune profile in NSCLC. [1] Schwab A, Fabian A et al., Physiol Rev, 2012, 92(4), 1865-913 [2] Mitchell KG, Lee Y et al., J Immunother Cancer, 2025, 13(6):e011458

Authors: Johannes Jung (1), Martina Düfer (2), Albrecht Schwab (1), Héctor Noguera Hurtado (1)

Affiliations:
1. Institute for Physiology II, University of Münster
2. Institute for Pharmaceutical and Medical Chemistry, University of Münster

Abstract:

Ca²⁺-activated K⁺ channels (K_Ca3.1 and K_Ca1.1) have emerged as important regulators of cancer cell behavior. In breast cancer for example, increased expression and activity of K_Ca1.1 channels contribute to tumor aggressiveness and poor prognosis, including enhanced proliferation, migration, invasion, and resistance to cell death. The K_Ca1.1 channel is overexpressed in human lung cancer tissue as well; however, its role remains largely unexplored. In this project, we aim to define the (patho)physiological role of the plasma membrane and mitochondrial K_Ca1.1 channel in different non-small cell lung cancer (NSCLC) cell lines. The mitochondrial K_Ca1.1 channel is of particular interest as it regulates the mitochondrial membrane potential and redox balance, and thereby links channel activity to metabolic adaptation and survival under stress conditions. We used A549-3R and PC9 cells, human lung adenocarcinoma cell lines, for our experiments and confirmed the channel expression in both cell lines. Channel expression increases in PC9 cells after 24h when K_Ca1.1 channels are blocked with paxilline. As reactive oxygen species are important signaling molecules for cancer progression, we measured the intracellular oxidant activity by using DCFH-DA fluorescent dye. In both cell lines, paxilline, which blocks both plasma membrane and mitochondrial K_Ca1.1 channels, increased oxidative stress. This was not the case when only the plasma membrane K_Ca1.1 channel was blocked with iberiotoxin. To confirm the supposed effect of paxilline on mitochondrial K_Ca1.1 channels, we tested whether the mitochondrial metabolism was also affected after 24h of paxilline treatment in A549-3R cells. For this purpose, the oxygen consumption rate was measured and we could observe an overall decrease in mitochondrial respiration. In summary, our results show that K_Ca1.1 channels, mostly the one expressed in the inner mitochondrial membrane, is relevant for lung cancer cells.

Authors: Leon-Patrick Sprenger (1), Ömer Kocdölü (1), Birgit Begemann (1), Jan-Philipp Machtens (1), Christian Wahl-Schott (2), Martin Fischer (1)

Affiliations:
1. Hannover Medical School, Institute of Neurophysiology
2. Ludwig-Maximillian-University Munich, Biomedical Center (BMC), Institute for Cardiovascular Physiology and Pathophysiology

Abstract:

Two-pore channels (TPC1 and TPC2) are cation-selective channels activated by ligand-binding of either PI(3,5)P₂ or NAADP. Their endo-lysosomal localization incorporates TPC function into a dynamic subcellular signaling network, which results in manifold physiological implications. Even though their pseudo-tetrameric assembly places them firmly in the voltage-gated ion channel superfamily, only TPC1 exhibits canonical voltage-dependent gating, whereas TPC2 has been considered voltage-insensitive. TPC2 pores are constricted by a cytosolic hydrophobic gate (HG) and luminal selectivity filter (SF). Structural studies primarily attributed ligand-dependent gating to the HG, while the SF has been described as a static determinant of Na⁺ selectivity. In contrast, recent molecular dynamics simulations hint at an active functional role by demonstrating conformational SF flexibility, which could possibly be the origin of TPC2’s agonist-dependent selectivity. We thus set out to investigate voltage-dependent modulation of hTPC2 currents and examine its connection to the SF using voltage-clamp recordings after heterologous channel expression in HEK293 cells, a Goldman–Hodgkin–Katz equation-based current simulation approach and site-directed mutagenesis. Whole-lysosomal recordings of WT hTPC2 exhibited large current amplitudes accompanied with shifts in reversal potential following prolonged voltage pulses, consistent with luminal Na⁺ accumulation or depletion. Incorporating altered ion concentrations into the GHK current equation demonstrated that simulation of observed current trajectories additionally required voltage-dependent pore permeability. In contrast, mutating SF residues abolished voltage-dependence of permeability, with current relaxations explicable exclusively by changes in luminal Na⁺ concentration. Examining hTPC2 redirected to the plasma membrane (PM) for examination in excised patches and non-stationary noise analysis confirmed voltage-dependent currents and variance distribution while avoiding shifts in luminal ion concentration. Additionally exchanging the SF residues in the PM mutant abolished these attributes. Together, these findings identify the hTPC2 selectivity filter as an active voltage-dependent modulator of Na⁺ permeability, supporting its role beyond static ion selectivity.

Authors: Weronika Wilczak (1), Albrecht Schwab (1), Zoltan Pethö (1)

Affiliations:
(1) University of Münster, Institut of Physiology II, Münster, Germany

Abstract:

Pancreatic ductal adenocarcinoma (PDAC) is driven by oncogenic KRAS mutations in the majority of patients and shows limited response to current therapies. Direct inhibition of KRAS^G12D has recently emerged as a promising strategy in PDAC therapy. However, its efficacy is limited, suggesting that additional targets are needed. K⁺ channels are frequently dysregulated in PDAC and contribute to malignant phenotypes including proliferation, migration, and survival. Therefore, we investigated whether combining KRAS inhibition with modulation of K⁺ channel activity could improve treatment response. We established a 3D multicellular spheroid platform composed of PDAC cancer cells, pancreatic stellate cells, and endothelial cells embedded in a complex extracellular matrix to better mimic the tumor microenvironment. To address the limited efficacy of KRAS inhibition, we adopted a drug repurposing approach and screened a panel of clinically tested K⁺ channel modulators, including both inhibitors and activators. Using our model, we confirmed that the KRAS^G12D inhibitor MRTX1133 selectively reduces viability of KRAS^G12D-mutated cancer cells, while sparing non-tumorous cells. Viability assays revealed that several K⁺ channel modulators enhance the response of KRAS-mutant spheroids to MRTX1133. In addition, combined modulation of K⁺ channels (e.g. with linopirdine) and KRAS inhibition reduced cancer cell migration. Overall, our data suggest that modulation of K⁺ channel activity can influence the response of PDAC spheroids to KRAS inhibition and support further investigation of ion channel–based combination strategies in PDAC.

Authors: Klinger L (1), Scholl U (1), Stölting G (1)

Affiliations:
1. Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Center of Functional Genomics, Berlin, Germany

Abstract:

The voltage-dependent calcium channel Ca_V3.2 is encoded by the gene CACNA1H and has a significant influence on aldosterone synthesis. When the channel opens upon depolarization, the intracellular calcium concentration increases, activating various calcium-dependent cell processes. This includes the synthesis of aldosterone, a steroid hormone produced by the adrenal gland, which regulates blood pressure. Mutations in CACNA1H often lead to an increased channel function, also known as a gain-of-function. They result in an increased calcium influx, increased aldosterone synthesis and high blood pressure, also known as primary hyperaldosteronism. The aim was to investigate the functional properties of the CACNA1H^R428W and CACNA1H^I1430T variants using electrophysiological methods. Stably transfected Flp-In T-REx 293 cell line of both variants were generated and whole-cell patch clamp recordings were performed under standardized conditions. Comparative measurements were performed with cells expressing the wildtype channel, focusing on the steady state voltage-dependent activation and inactivation, but also on the kinetics of both calcium channels. Both CACNA1H^R428W and CACNA1H^I1430T exhibited functional differences, suggesting a gain-of-function. Particularly striking was the shift in activation and inactivation towards the resting membrane potential, making the channels presumably easier to activate. The window current describes a voltage range in which activation and inactivation overlap, allowing a sustained calcium influx. Since the range for CACNA1H^R428W also shifted to more negative potentials and is closer to the resting membrane potential, the cellular excitability may be increased, as even small changes in the potential are sufficient to open the channel. In addition to the shift of activation and inactivation and therefore the window current of the Ca_V3.2 I1430T channel to more negative potentials, a significantly slower opening and closing rate was observed, suggesting that the calcium influx lasts for a significantly longer period of time, leading to a permanently elevated intracellular calcium concentration.

Authors: Paul J. Wagner (1), Fang Zheng (1), Thomas Seidel (2), Efthymios Oikonomou (1), Johannes Broichhagen (3), Stefan Düsterhöft (4), Christian Alzheimer (1), Tobias Huth (1)

Affiliations:
1. Friedrich-Alexander-Universität Erlangen-Nürnberg, Institut für Physiologie und Pathophysiologie
2. Friedrich-Alexander-Universität Erlangen-Nürnberg, Institut für Zelluläre und Molekulare Physiologie
3. Leibniz Forschungsverband, Forschungsinstitut für Molekulare Pharmakologie, Berlin
4. RWTH Aachen, Institut für Molekulare Pharmakologie

Abstract:

Currents mediated by voltage-gated sodium channels (NaVs) are divided into transient, resurgent, and persistent current entities. Dysregulation of any of these subtypes can cause severe illnesses, such as epilepsy or neuropathic pain syndromes. Therefore, understanding the mechanisms and gating behaviors underlying these currents is crucial for developing precision therapies. Specific intracellular peptides have been identified as the mediators of resurgent sodium currents; however, studying these currents without compromising cellular integrity has proven difficult. To address these challenges, our study focused on an innovative method to modulate sodium channel activity without compromising the integrity of the cells. Using a novel construct consisting of a cell-penetrating peptide (CPP) and a fibroblast growth factor homologous factor (FGF) fragment, we induced pronounced resurgent and persistent currents in heterologous systems by adding the peptide to the culture medium of the cells. This peptide is remarkably stable against cellular proteases and exhibits effects in HEK cells, cardiac slices and neurons for over 48 h, without notable cytotoxicity. This opens the door to developing synthetic peptides capable of penetrating cells and specifically targeting NaV channels. These peptides serve as innovative tools for manipulating sodium currents to better understand their biophysics and for potential therapeutic applications.

Authors: Franziska Schreiber, Florian Sure, Christoph Korbmacher, Alexandr V. Ilyaskin

Affiliations:
Institute of Cellular and Molecular Physiology, Friedrich-Alexander-Universität Erlangen Nürnberg, Erlangen, Germany

The author did not consent to the publication of the abstract.

Authors: Maria Antonietta Coppola (1), Margherita Festa (1), Alice Giusto (1), Elena Angeli (2), Abraham Tettey-Matey (1), Irene Mazza (1), Elena Gatta (2), Raffaella Barbieri (1), Alessandra Picollo (1), Paola Gavazzo (1), Cristiana Picco (1), Francesca Sbrana (1), Michael Pusch (1)

Affiliations:
1. Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Italy
2. DIFI Lab, Dipartimento di Fisica, Università di Genova, Italy

Abstract:

Variants in CLCN3 and CLCN4, encoding the neuronal 2Cl-/H+ exchangers ClC-3 and ClC-4, are linked to various neurodevelopmental disorders. Both ClC-3 and ClC-4 exist as dimers, either as homodimers or heterodimers in regulating activities in endosomes. But their molecular mechanisms and role in underlying disease-associated variants remain elusive. In particular, most ClC-3 and ClC-4 variants exhibit wild-type (WT) like phenotypes. This suggests the involvement of an unidentified accessory protein in the functional regulation. In this study, we identified TMEM9 Domain Family Member B (TMEM9B), a type I transmembrane protein of unknown function, to strongly interact with the neuronal endosomal ClC-3 and ClC-4 transporters. Using Xenopus laevis oocytes (Two-Electrode Voltage-Clamp) and HEK293 cells (Whole-cell Patch-Clamp), we characterized the resulting ionic currents to determine the impact of this potential interaction. Results indicate a strong inhibitory effect of one of the TMEM9B on ClC-3 and ClC-4 transporter activity in both oocytes and HEK cells. For ClC-3, kinetic modulation analysis using patch-clamp recordings revealed significant alterations in the kinetic properties of ClC-3 currents, providing strong evidence for a direct physical or functional interaction with the TMEM9B. Further, strong evidence for direct interaction was detected using Förster Resonance Energy Transfer (FRET), exploiting fluorescence lifetime microscopy-based (FLIM-FRET) techniques between TMEM9B and ClC-3 and ClC-4, but hardly any FRET with controls ClC-1 or ClC-7. These findings suggest that the TMEM9B acts as a potent modulator of ClC-3 and ClC-4 function, with its inhibitory effects on only endosomal but not lysosomal CLC-7. Ongoing investigations aim to elucidate how TMEM9B modifies the electrophysiological properties of disease-causing ClC-3 and ClC-4 variants, potentially uncovering the missing link in the molecular pathogenesis of these neuronal disorders.

Authors: Søndenaa, K.Q.(1), Espedal, H.(2), Matre, I.T.(2,3), Skogstrand, T.(2), Maya, M.B.S.(2), Spriet, E.(2), Leh, S.(3,4), Nordanger, B.(2), Karlsen T.V.(2), Tenstad, O.(2)

Affiliations:
1. University of Bergen - Medical school
2. University of Bergen - Institute of Biomedicine
3. Haukeland University Hospital
4. University of Bergen - Clinical Institute II

Abstract:

Introduction & aims Chronic kidney disease affects a large portion of the global population. Preclinical research on mice has relied on unreliable practices such as blood- and urine sampling. Our aim has been to develop a new method using PET-CT imaging, as well as provide insight into the long-term mechanisms behind uninephrectomy. Methods n=5 mice were subjected to a unilateral nephrectomy. n=5 mice were subjected to a SHAM operation. Normal chow and tap water ad libitum. 18F-coupled cytochrome C was injected into sevofluorane anesthesised mice. A CT-scan for anatomical reference was overlaid with a PET-scan. Data were analysed using InterView Fusion version 3.01.021.0000. To obtain tracer signal, we use geographical spheres extending from the central abdominal aorta just proximal to the iliac branching, as well as autosegmentation in the kidneys. GFR is obtained by dividing kidney activity by the area under the curve found in the aortic measurements. Kidneys were harvested following unaliving 63 weeks post uninephrectomy. Kidney pathology were analysed using several methods: Periodic Acidic Schiff and Sirius Red staining for histopathological analysis, electron microscopy for basement membrane damage, and measurement of albumine-creatinine ratio. Results Uninephrectomized mice compensate with a rapid growth of the remaining kidney. Volume growth driven compensation in GFR is lasting. There was no signs of glomerular hyperfiltration. Pathological analyses are largely non significant across groups, supporting our results on GFR. Podocyte fusion was significantly elevated in uninephrectomized mice. This is supported by litterature as an early marker of kidney damage. PET-CT data were comparable between two different observers.

Authors: Tanja Liesenberg (1), Allein Plain (1), Vanessa Mikicz (1), Anja Süß (1), Katrin Pollok (1), Eva Wacker (1), Ben Davies (2), Detlef Böckenhauer (3), Markus Reichold (1), Ralph Witzgall (4), Katja Dettmer-Wilde (5), Robert Kleta (6), Felix Knauf (7), Richard Warth (1)

Affiliations:
1. Medical Cell Biology, University of Regensburg
2. The Francis Crick Institute, London, UK
3. UZ Leuven, Belgium
4. Molecular and Cellular Anatomy, University of Regensburg
5. Institute of Functional Genomics, BioPark Regensburg
6. Department of Nephrology, University College London, UK
7. Mayo Clinic, Rochester, USA

The author did not consent to the publication of the abstract.

Authors: (1)Yipeng Chen, (2)Minze Xu,(3)Karin M. Kirschner

Affiliations:
1.Charité - Universitätsmedizin Berlin Institut für Translationale Physiologie
2.Charité - Universitätsmedizin Berlin Institut für Translationale Physiologie
3.Charité - Universitätsmedizin Berlin Institut für Translationale Physiologie

Abstract:

Polyamines are emerging as important regulators of kidney injury, potentially exerting protective effects through modulation of the renal microvasculature. In pathological conditions such as ischemia–reperfusion injury, vascular dysfunction impairs vasoconstriction and vasodilation, leading to sustained tissue hypoxia, inflammation, fibrosis, and progressive loss of renal function. Restoration of microvascular perfusion is therefore considered a key strategy for improving long-term renal outcomes. Polyamines—including putrescine, spermidine, and spermine—are small positively charged molecules involved in gene regulation, cell growth, and ion channel modulation. They are known to interact with potassium (K⁺) channels, particularly inwardly rectifying potassium (Kir) channels, by binding within the channel pore. Potassium channels such as Kir2.x and calcium-activated potassium (KCa) channels play central roles in setting membrane potential, regulating vascular smooth muscle cell contractility, and controlling vascular tone. These properties suggest that polyamines could directly influence vascular reactivity. To test this hypothesis, we used an ex vivo vascular perfusion system to examine freshly isolated afferent and efferent arterioles from wild-type mouse kidneys. Vasoconstriction was first induced using a dose-response protocol with angiotensin II. Various polyamines were then applied to evaluate their direct effects on vessel diameter and vascular reactivity. Preliminary findings indicate that spermidine modulates renal arteriole tone and produces vasodilation under the experimental conditions. This provides the first functional evidence that individual polyamines can directly alter renal microvascular behavior. Ongoing studies are expanding the analysis to additional polyamines, including putrescine and spermine, to determine whether these vascular effects are compound-specific. Together, these results support the hypothesis that the renoprotective effects of polyamines are mediated, at least in part, through regulation of renal microvascular function, laying the groundwork for future studies on the underlying ion channel and signaling mechanisms.

Authors: Minze Xu (1), Yipeng Chen (1), Pontus Persson (1), Karin Kirschner (1)

Affiliations:
1. Institute of Translational Physiology, Charité – Universitätsmedizin Berlin

Abstract:

Background Acute kidney injury (AKI) is frequently accompanied by persistent impairment of renal microcirculation following ischemia–reperfusion injury (IRI). In particular, sustained constriction of the descending vasa recta contributes to the renal medullary “no-reflow” phenomenon. Pericytes are key regulators of vasa recta tone, yet the inflammatory mechanisms that sensitize pericytes to vasoconstrictors after IRI remain incompletely understood. Complement activation, especially signaling via the anaphylatoxin C5a, has emerged as a potential mediator of microvascular dysfunction. Methods To assess the direct impact of C5a on renal microvascular reactivity, we established an ex vivo perfusion model using isolated descending vasa recta from murine kidney slices. Vessels were perfused under isobaric conditions and continuously imaged to quantify real-time changes in luminal diameter. Angiotensin II (Ang II) dose–response curves (10⁻¹²–10⁻⁶ M) were performed under five experimental conditions: baseline control, post-treatment with the C5aR1 antagonist Avacopan, C5a + Avacopan co-treatment, C5a pre-treatment followed by Avacopan rescue, and C5a pre-treatment time control. This design allowed discrimination between preventive and reversal effects of C5aR1 inhibition. Results Pre-treatment with C5a significantly increased the sensitivity of vasa recta to Ang II-induced vasoconstriction, lowering the threshold concentration required to induce measurable diameter reduction. Co-administration of Avacopan completely normalized Ang II responsiveness to control levels, demonstrating effective blockade of C5a–C5aR1 signaling. Notably, Avacopan applied after Ang II-induced preconstriction partially reversed vasoconstriction in both C5a-treated and untreated vessels, indicating a direct vasoregulatory effect beyond prevention of inflammatory sensitization. Conclusion These findings provide direct functional evidence that C5a enhances pericyte-dependent vasoconstriction in renal microvessels and identify C5aR1 inhibition as an effective strategy to both prevent and reverse microvascular dysfunction. The established perfusion platform forms the basis for ongoing studies combining kidney slice electrophysiology, in vivo IRI models, and molecular profiling to link complement-driven pericyte dysfunction with hypoxia, fibrosis, and AKI–CKD transition.

Authors: Sophia Vorreuther, Laura Opitz, Schirin Czajkowski, Shobika Karuppusamy, Moritz Pernecker, Bayram Edemir

Affiliations:
Universität Witten/Herdecke, Fakultät für Gesundheit, Department Humanmedizin, Lehrstuhl für Physiologie und Pathophysiologie

Abstract:

To generate a concentrated urine, the kidneys create an osmotic gradient between renal cortex and inner medulla. This hypertonic environment presents a stress factor. Kidney cells have developed mechanisms to adapt and prevent resulting cellular damage. Under hyperosmotic conditions specific gene expression patterns are induced to protect the cells. Besides genes with known osmoprotective functions, hundreds of other genes are deregulated with so far unknown cell functions. FXYD2 is one of the genes which is highly upregulated. It encodes for the γ-subunit of the Na+-K+-ATPase and is present in most nephron segments. It has regulatory functions in modulating Na+-K+-ATPase. This suggests that FXYD2 plays a vital role in the adaptation to osmotic stress. With this study we want to denote the expression pattern to help understand the function of FXYD2 in kidney cells. We used the CRISPR/Cas9 method to functionally suppress the expression of FXYD2 in the renal cell line of mouse principal cells. After validation of functional knockout, we performed a proliferation assay using live cell imaging and analyzed changes in gene expression profile by next generation sequencing to characterize the cellular phenotype in cells that were cultivated at 300 (control) or 600 mOsmol/kg. We further used gene ontology classification for functional analysis. Analysis showed no major differences in cellular proliferation. However, loss of FXYD2 function affected gene expression, with 3420 genes down- and 3604 upregulated at 300 mOsmol/kg, and 2541 down- and 2149 upregulated at 600 mOsmol/kg. Functional annotation showed that loss of function leads to deregulation of genes that are associated with various aspects of nucleic acid metabolism. These results indicate a novel finding regarding the physiological function of FXYD2. Further research is needed to investigate how exactly FXYD2 modulates the changes in gene expression.

Authors: J. Janz (1), A. Lieven (1), T. Tertel (2), K. Meyer-Schwesinger (4), K. Lahme (4), U. Brockmeyer (4), A. Büscher (1), L. Pape (1), J. Jägers (1)

Affiliations:
1. University Hospital Essen, Children's Hospital II, Hufelandstr. 55, 45147 Essen
2. University Hospital Essen, Transfusion Medicine, Hufelandstr. 55, 45147 Essen
3. Institute of Cellular and Integrative Physiology, Center for Experimental Medicine, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
4. University Hospital Essen, Department of Neurology, Hufelandstr. 55, 45147 Essen

Abstract:

Introduction Extracellular vesicles (EVs) are nanoparticles that play an important role in cell-cell communication by carrying RNA, DNA and proteins through extracellular fluids. Under pathological conditions, adjustment of the quantity of released EVs and their cargo was reported. The proximal tubular epithelial cells (PTEC) are strictly aerobic, but start to perform glycolysis during kidney disease. Whether podocytote derived EVs (pEVs) are taken up by PTECs and may influence this metabolic switch and thus the progression of kidney disease is yet unknown. Methods To investigate pEVs uptake by the PTECs, an mT/mG mouse model that expresses ACTB-eGFP under the NPHS2 promoter was used to label pEVs. Kidneys were fixated in 4%PFA and sliced into 50 µm sections using a vibratome. pEV-uptake was than observed with confocal imaging of the kidney sections without further processing. To investigate the effect of EVs we harvested the EVs from human conditionally immortalized podocytes, in which we mimicked different glomerular diseases by applying different stressors: cold stress (6h/4°C/SCS) followed by warm reperfusion (24h/37°C/Rep) and high-glucose (48h/300mg/dL Glu). EVs were purified by dual centrifugation (900xG, 3000xG) and size-exclusion-chromatography. EVs were characterized using Cryo-transmission electron microcopy and quantified by nanoparticle tracking analysis (NTA). PTECS were treated with the obtained pEVs for 16h. Changes in the metabolism and redox status were analysed using resazurin-reduction-assay, seahorse metabolic flux analysis, fluorescence lifetime imaging, qPCR and Western Blot. Results GFP-labeled pEVs are taken up by PTECs in vivo, suggesting podocyte-to-nephron communication. Surprisingly, in cell culture, PTECs treated with EVs from healthy control podocytes showed a significantly lower capability to reduce resazurin after 16 hours of incubation. qPCR revealed fundamental alterations in glucose metabolism. Westernblot analysis revealed response to oxidative stress. Outlook Metabolic labelling with proteogenic 13C6-lysine will be used to investigate which proteins enter the HK2-cells that can facilitate these metabolic changes.

Authors: Saadé C (1,2), Schaeffner E (1), Aigner A (3), Eckardt KU (2), Knauf F (2,4)

Affiliations:
1- Institute of Public Health, Charité – Universitätsmedizin Berlin
2- Department of Nephrology and Medical Intensive Care, Charité – Universitätsmedizin Berlin
3- Institute of Biometry and Clinical Epidemiology, Charité – Universitätsmedizin Berlin
4- Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN

Abstract:

BACKGROUND AND HYPOTHESIS: Sulfate is an essential anion for cellular function. The kidneys play an important role in regulating circulating sulfate levels, via two transporters encoded by SLC13A1 and SLC26A1. Damaging variants in these genes lead to lower sulfate levels. Higher circulating sulfate levels are observed in advanced chronic kidney disease (CKD), possibly associated with adverse health outcomes. However, there are limited data on sulfate levels in patients with less advanced CKD. Using data from the observational German Chronic Kidney Disease study, we evaluated the distribution of plasma sulfate levels in patients with mild to severe CKD and in carriers of qualifying damaging variants (QVs) in SLC26A1 and SLC13A1. We also investigated the relationship of plasma sulfate levels with common clinical and biochemical parameters. METHODS: Data from 5144 patients aged 18-74 years with an estimated glomerular filtartion rate (eGFR) of 30-<60ml/min x 1.73m² or eGFR ≥60 and overt proteinuria at recruitment were evaluated. Semi-quantitative plasma sulfate levels were measured using non-targeted mass spectrometry. Associations of plasma sulfate with clinical and biochemical parameters were examined visually using scatterplots with LOESS smoothing and Spearman correlations. Linear regression models quantified differences in plasma sulfate across CKD etiologies and in carriers of SLC26A1 and SLC13A1 QVs. RESULTS: Plasma sulfate was strongly correlated with urea (rho = +0.78 [95% CI: 0.77; 0.80]) and eGFR (rho = -0.70[95%CI: -0.72;-0.68]). Plasma sulfate increased exponentially with decreasing eGFR. Levels were similar across leading causes of CKD and other comorbidities. Plasma sulfate levels were 19% and 8% lower in carriers of QVs in SLC13A1 and SLC26A1, respectively, independent of eGFR and albuminuria. CONCLUSION: Among CKD patients, we found that plasma sulfate increases progressively with the decline of kidney function, regardless of the cause of renal disease. Carriers of QVs in SLC13A1 and SLC26A1 had lower plasma sulfate levels.

Authors: Erdogan C (1), Xu M (1), Lichtenberger FB (1), Fei Lingyan (2), Schröter Bonk JS (1), Patzak A (1), Khedkar PH (1)

Affiliations:
1 Charite - Universitätsmedizin Berlin, Institute of Translational Physiology
2 Sun Yat-Sen University, Department of Nephrology, Center of Kidney and Urology, the Seventh Affiliated Hospital, Shenzhen, China

Abstract:

Background Diabetic nephropathy is a major cause of chronic kidney disease, and although SGLT2 inhibitors preserve glomerular filtration rate (GFR), the underlying mechanisms remain elusive. We hypothesized that SGLT2 inhibition improves glomerular microvascular function by normalizing vasoactive responses in afferent and efferent arterioles and thereby helps prevent diabetic hyperfiltration, which leads to chronic kidney disease in the long term. Methods db/db and db/- control mice were randomized into four groups (two genotypes, with or without SGLT2 inhibitor treatment) and followed for 8 weeks. Serum glucose, body weight and kidney histology were assessed. Microperfusion of isolated afferent and efferent arterioles was used to quantify vasodilatory responses to acetylcholine and the NO donor S-Nitroso-N-acetylpenicillamine (SNAP). Results Serum glucose was markedly elevated in untreated db/db mice and was significantly reduced by SGLT2 inhibition, confirming effective glucose lowering. However, body weights were significantly higher in diabetic mice, but within the genotypes body weights were comparable. Afferent arterioles from untreated db/db mice showed increased acetylcholine‑induced vasodilation versus untreated controls, an effect blunted by SGLT2 inhibition. In contrast, efferent arterioles from untreated db/db mice displayed impaired vasodilation after NO-synthase inhibition and a reduced response to SNAP, both improved by SGLT2 inhibition. Kidney histology revealed no relevant structural differences among the groups during the 8‑week period. Conclusion SGLT2 inhibition reduces hyperglycemia and partly reverses dysregulated vasoactivity of glomerular arterioles in db/db mice, particularly sGC‑dependent responses in efferent vessels. These findings support a microvascular mechanism by which SGLT2 inhibitors help maintain GFR in diabetic nephropathy and may contribute to protection from hyperfiltration.

Authors: Julia Naujox (1), Juliana Roeder (1), Sergio Esquivel-Ruiz (2), Fabian Schumacher (3), Burkhard Kleuser (3), Wolfgang M. Kuebler (1,4-7), Laura Michalick (1,4)

Affiliations:
1. Institute of Physiology, Charité-Universitätsmedizin Berlin, corporate member of the Freie Universität Berlin and Humboldt Universität zu Berlin, Berlin, Germany
2. Departamento de Farmacología de la Universidad de Granada
3. Institute of Pharmacy, Pharmacology and Toxicology, Freie Universität Berlin, Berlin, Germany
4. DZHK (German Centre for Cardiovascular Research), partner site Berlin, Berlin, Germany
5. DZL (German Centre for Lung Research), Berlin, Germany
6. Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, Ontario, Canada
7. Departments of Surgery and Physiology, University of Toronto, Toronto, Ontario, Canada

Abstract:

Introduction Ventilator-induced kidney injury (VIKI) is a frequent complication in patients receiving mechanical ventilation (MV), yet mechanisms underlying this adverse lung-kidney crosstalk remain largely unclear. Overdistension of the pulmonary endothelium activates mechanosensitive signalling pathways that enhance the release of extracellular vesicles (EVs) into the circulation, potentially delivering injurious cargo to the glomerular endothelium. We thus hypothesized that EVs released from overventilated lungs exhibit an increased potential to injure the integrity of the glomerular barrier. Methods & Results In vivo, mice (n = 17) were subjected to 2 h MV with either an injurious high tidal volume (HV_T = 20 ml/kg BW) or a protective volume (LV_T = 7 ml/kg BW). Plasma EV quantification by nanoparticle tracking analysis (NTA) showed a significant increase in circulating EVs in the HV_T group compared to LV_T. Renal assessment after 2h of HV_T-ventilation revealed kidney dysfunction evident as elevated plasma urea and creatinine levels, enlarged glomerular tuft area, immune cell infiltration, and increased podocyte effacement. Similarly, in vitro mechanical stretch (18% elongation) of pulmonary microvascular endothelial cells (HPMECs) increased EV release (100-120 nm) relative to static cultured HPMECs (6.0×10⁷± 3.8×10⁷ [mean ± SD] vs. 11.2×10⁷± 4.6×10⁷ EVs). To assess the pathogenic potential of EVs released from mechanically stretched HPMECs on renal cells, we performed ECIS-based barrier measurements in human renal glomerular endothelial cells (HRGECs) treated with equal amounts of EVs from static or stretched HPMECs. EVs from stretched, but not static cultured pulmonary endothelial cells disrupted barrier function in HRGECs. Discussion MV triggers the release of pathogenic EVs from lung endothelial cells, which in turn can disseminate via the systemic circulation and injure the glomerular barrier. Endothelial-derived EVs may serve as potential biomarkers and therapeutic targets for monitoring and mitigating VIKI.

Authors: Alina Lampert (1-3), Xueyu Li (1,2), Lisa Seipold-Zimmermann (3), Julia Reichelt (1,2), Desiree Loreth (1,2), Helga Vitzthum (1), Lukas Blume (1,2), Lena-Marie Reimers (1,2), David Rosenbaum (3), Simonas Gaudatis (3), Karen Lahme (1,2), Sarah Frömbling (1,2), Christopher Kosub (1,2), Stefanie Zielinski (1,2), Thorsten Wiech (4), Tobias N. Meyer (5,6), Paul Saftig (3), Catherine Meyer-Schwesinger (1,2)

Affiliations:
1 Institute of Cellular and Integrative Physiology, Center for Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
2 Hamburg Center of Kidney Health, Hamburg, Germany
3 Institute of Biochemistry, Christian-Albrechts-University Kiel, 24118 Kiel, Germany
4 Institute of Pathology, Neuropathology Section, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
5 Nephrology, Asklepios Klinikum Barmbek, 22307 Hamburg, Germany
6 Faculty of Medicine, Semmelweis University Budapest, Asklepios Campus Hamburg, 22307 Hamburg, Germany

Abstract:

Background The autoimmune kidney diseases membranous nephropathy (MN) and minimal change disease (MCD) are the leading cause of nephrotic syndrome in adults and children, respectively. Nephrotic syndrome is characterized by massive proteinuria resulting from disruption of the glomerular filtration barrier (GFB) due to podocyte damage, accompanied by hypoalbuminemia, edema, and hyperlipidemia. Podocytes form the slit diaphragm, a key component of the GFB, through the transmembrane proteins nephrin and neph1 on their foot processes and are targeted by autoantibodies in disease. We recently showed that the sheddase A Disintegrin And Metalloproteinase 10 (ADAM10) cleaves the podocyte surface proteins recognized by auto-antibodies in MN. Recently, auto-antibodies directed against nephrin have been identified in MCD. Methods and Results Using proximal ligation assays and super-resolution confocal imaging, we visualized a distinct colocalization pattern of ADAM10 and nephrin along the membrane in healthy human biopsies, which became noticeably pronounced along the membrane and aggregated in the podocyte cytosol in MCD patients. Co-immunoprecipitation of ADAM10 and nephrin in pig glomeruli confirmed their physical interaction. Isolated pig glomeruli and HEK cells were used for functional in vitro analyses. Pharmacologic inhibition and genetic deletion of ADAM10 resulted in a stabilization of full‑length nephrin, together with a reduction or shift in extracellular N‑terminal shedding prod-ucts and intracellular C‑terminal nephrin fragments, as shown by western blot analysis. These changes were not observed for neph1. C-terminal nephrin fragments were stabilized upon pharmacologic inhibition and genetic deletion of γ-secretases. Conclusion Our findings demonstrate a homeostatic nephrin shedding by ADAM10 and subsequent processing of the intracellular C-terminal shedding fragments by regulated intramembrane proteolysis (RIP). Potential effects of the small proteolytic products e.g. in intracellular signalling, remain to be elucidated. Altered ADAM10–nephrin colocalization in MCD biopsies suggests a disease‑related modification in their interaction, whose role in MCD pathophysiology requires clarification in vivo.

Authors: Witte KE (1), Spallek M (1), Lee WK (1)

Affiliations:
1. Physiology and Pathophysiology of Cells and Membranes, Medical School OWL, Bielefeld University

Abstract:

Exposure to cadmium (Cd), is associated with numerous human diseases. Driven by ongoing industrialization and its global consequences, the impact of Cd on the human body remains a subject of continuous relevance in pathophysiological research. In particular, contaminated soil, water and resulting food, are the main sources of intestinal Cd uptake. Within the human body, Cd is partially excreted but accumulates in the kidneys over time, where it contributes to nephrotoxicity and cancer development. In the renal proximal tubule, endocytic and exocytic processes play a crucial role due to the intensive turnover of macromolecules and ions. The endo-lysosomal cation channel mucolipin-1 (TRPML1) is known as a key regulator of exocytosis. Nevertheless, the influence of Cd on TRPML1-mediated lysosomal exocytosis remains poorly understood. To elucidate potential Cd-triggered effects on renal exocytosis, human proximal convoluted tubule (HPCT) cells were employed as an in vitro model. Live-cell-microscopy of Cd-treated HPCTs revealed more scattered lysosomes and significantly increased lysosomal distance from the nucleus compared with untreated controls. TRPML1 was subsequently identified as a key candidate, possibly mediating Cd-induced lysosomal alterations, and its overexpression (OE) was established by transient transfection. In Cd‑treated TRPML1‑OE, immunofluorescent analysis demonstrated a similar but more rapid increase in vesicular distance from the nucleus. Furthermore, co-expression of TRPML1 and lysosome‑associated membrane glycoprotein 1 (LAMP1) on the plasma membrane (PM) was significantly elevated following Cd exposure. This effect showed distinct time dependence, peaking after 5min of Cd treatment and diminishing by 60min. Complementary biotinylation assay depicted higher TRPML1/LAMP1 abundance on PM, supporting a Cd‑triggered enhancement of lysosomal exocytosis. Collectively, these results suggest a transient upregulation of lysosomal exocytosis as an early adaptive mechanism in the renal response to Cd stress. We hypothesize that the Cd-triggered TRPML1 activation drives this process, which might represent a crucial role in the progression of Cd‑induced nephrotoxicity.

Authors: Anders M. Kristensen, Francesco De Luca, Isabela Binotti de Araujo, Hao Yuan, Hanne Kidmose, Anna Faivre, Duncan Sutherland, Lin Lin, and Ina M. Schiessl

Affiliations:
Department of Biomedicine, Aarhus University, Aarhus, Denmark

Abstract:

Diabetic nephropathy is the leading cause of end-stage kidney disease globally and is associated with microvascular complications including abnormal angiogenesis and vascular remodeling. Here, we combined serial intravital multiphoton microscopy over 14 days with single-cell RNA sequencing (scRNAseq) of endothelial cells (ECs) to investigate structural, functional, and molecular aspects of vascular remodeling in the diabetic kidney. Using STZ-induced diabetic transgenic mice with tdTomato expression in ECs (Cdh5CreERT2) we visualized dynamic changes in vivo (n = 11 mice) and isolated endothelial cells for RNA sequencing (n = 8 mice). STZ treatment resulted in hyperglycemia (23 vs. 9 mmol/L, p < 0.001 at week 15) with increased albumin–creatinine ratio (63 vs. 26 mg/g, p = 0.015), but no change in GFR (p =0.51). In vivo imaging revealed reduced capillary blood velocity (0.44 vs. 0.68 um/ms, p = 0.005) in diabetic mice. Interestingly, longitudinal imaging of capillaries revealed that some capillaries fluctuated between perfused and non-perfused states. These stop-flow events were associated with tubular atrophy, with the risk of an event occurring next to an atrophic tubule being 47% vs. only 6% next to healthy tubules. scRNAseq of capillary ECs revealed an upregulation of markers associated with endothelial activation and adhesion, including Vcam1, as well as thrombosis related genes such as Gas6 and Serpine2, indicating locally micro thrombosis as a course for stop-flow events. Compared to controls, diabetic kidneys displayed areas of increased vascular permeability visualized by extravasated albumin, closely linked to atrophic tubules, 91% of which were within such regions. Intriguingly, EC-lineage cells were disassociated from the capillary bed in these areas, alongside interstitial collagen deposition (5-fold increase, p = 0.016), indicating endothelial-to-mesenchymal transition (EndMT) By combining serial imaging with scRNAseq, we link vascular remodeling to transcriptional changes in endothelial cells, highlighting its contribution to vascular dysfunction and fibrosis in diabetic nephropathy.

Authors: Yuanyuyue Huang, Sarah Mittmann, Anna Seebohm, Nina Himmerkus, Catarina Quintanova, Markus Bleich

Affiliations:
Institute of Physiology - CAU

Abstract:

Research Question: Claudin-7 is present in both, the tight junction and at basolateral side of mouse renal collecting duct. Higashi (2023) suggested that EpCAM–claudin-7 complexes in MDCK cell culture become exposed to apical membrane-anchored serine proteinases at tight-junction break sites. EpCAM can be cleaved by these proteinases, resulting in the release of claudin-7 to repair tight-junction damage. We hypothesize that a comparable mechanism is present in the collecting duct. To test this, we investigated the localization and expression of claudin-7 and EpCAM in the collecting duct and conducted initial experiments on proteolytic cleavage. Methods: Freshly isolated renal segments from male and female C57BL/6 mice were examined by immunofluorescence and Western blot analysis. Results: Western blot analysis revealed clear protein expression of claudin-7 and EpCAM in the thick ascending limb of the loop of Henle, the distal convoluted tubule, and the collecting duct. Female mice additionally showed expression in the final straight segment of the proximal tubule. Immunofluorescence demonstrated the presence of claudin-7 and EpCAM in the TJs and in the basolateral membrane along the entire corticomedullary axis of the collecting duct, with decreasing TJ localization and partial colocalization. In the basolateral membranes, EpCAM staining was preferentially observed in intercalated cells, whereas claudin-7 was predominantly detected in principal cells. Upon trypsin treatment, the 43 kDa EpCAM band disappeared (with the 34 kDa band preserved), and EpCAM localization within the TJ was reduced. Conclusion: Both claudin-7 and the cell adhesion molecule EpCAM are present throughout the distal nephron and are localized in both, basolateral membranes and tight junctions in the native collecting duct. EpCAM can be processed by proteolytic cleavage. Future studies will investigate the effects of this proteolytic regulation on tight junction repair and tubular function in more detail.

Authors: Odochi O. Chukwu (1), Albert E. Okorocha (2), Anthony C. U. Ezimahb (2).

Affiliations:
1. Department of Physiology, Faculty of Basic Medical Sciences, College of Medical Sciences, David Umahi Federal University of Health Sciences, Uburu, Ebonyi State, Nigeria.

2. Department of Physiology, Faculty of Basic Medical Sciences, College of Medical Sciences, Alex Ekwueme Federal University, Ndufu-Alike, Abakaliki, Ebonyi, State, Nigeria.

Abstract:

Background: Prenatal environments shape reproductive development via endocrine and tissue-specific pathways. Gestational chronic unpredictable stress (CUS) elevates fetal glucocorticoid exposure, potentially impairing reproductive maturation. Moringa oleifera leaf extract (MoLE), rich in bioactive phytochemicals, may modulate oxidative and hormonal stress, but its interaction with glucocorticoid signaling and impact on offspring reproductive outcomes remain unclear. This study combines in vivo developmental programming with in silico glucocorticoid receptor (GR) docking to examine MoLE’s influence on GR-mediated stress responses and reproductive integrity in female offspring. Methods: Twenty-five pregnant Wistar rats were randomly assigned to control, MoLE-only (low/high dose), or CUS + MoLE (low/high dose) groups. CUS involved unpredictable stressors during gestation (restraint, light/dark cycle disruption, and cage tilting). Offspring were assessed at puberty for body weight and length, age at vaginal opening, serum estradiol, reproductive organ weights, and ovarian/uterine histology. Lesions were semi-quantitatively scored (0–3) by blinded observers and analyzed using one-way ANOVA. MoLE phytochemicals were identified via GC–MS, and 41 compounds were docked to human GR using AutoDock, with mifepristone as reference, to predict binding affinity and interaction profiles. Results: Gestational CUS delayed puberty and altered reproductive organ morphology, including ovarian cystic changes, and uterine epithelial loss. Low-dose MoLE co-exposure partially exacerbated histological lesions, whereas high-dose MoLE conferred moderate protection. Estradiol levels were reduced in MoLE-only and CUS + MoLE groups, with dose-dependent changes in uterine weight. Docking analysis identified 1-propanol, 3,3′-oxybis- and dodecanoic acid, methyl ester as strongest GR-binding phytochemicals, demonstrating favorable binding energies relative to mifepristone and stable interactions within GR ligand-binding domain, suggesting potential selective glucocorticoid receptor modulator (SGRM) activity. Conclusion: Gestational CUS disrupts reproductive endocrine function and tissue integrity, while MoLE modulates these effects dose-dependently. GR-binding phytochemicals may underlie MoLE’s partial protective effects, highlighting their potential as SGRMs and utility of integrating molecular docking with developmental physiology.

Authors: (1)C. Münter, (1)A. Simon, (1,2)D. Wenzel

Affiliations:
(1) Department of Systems Physiology, Medical Faculty, Ruhr-University Bochum, Germany
(2) Institute of Physiology I, Life&Brain Center, Medical Faculty, University of Bonn

Abstract:

Chronic obstructive airway diseases represent a leading cause of mortality worldwide. Asthmatic airway constriction, inflammation and enhanced mucus production are well known characteristics of this disease entity. We have previously shown that the endocannabinoid anandamide (AEA) plays a crucial role in acute airway tone regulation via its metabolization by FAAH. Here, we want to analyze the role of FAAH in the development of asthma bronchiale. Faah-/- and WT C57BL/6J mice were subjected to the acute ovalbumin (OVA) mouse model. Mice were sensitized by i.p. injection of OVA and Alum followed by aerosol challenge with OVA. Airway resistance was determined by Flexivent measurements, the immune cells of the bronchoalveolar lavage fluid (BALF) were quantified, and histologic staining was performed to investigate inflammatory cell infiltration, goblet cell metaplasia as well as collagen deposition. Our experiments showed that airway hyperresponsiveness develops in OVA WT animals but not in Faah-/- mice. Interestingly, BALF cell counts were similarly enhanced in OVA Faah-/- and WT animals. In contrast, the number of goblet cells was significantly decreased in OVA Faah-/- mice compared to OVA WT. Collagen deposition in OVA Faah -/- mice showed a slight reduction compared to OVA WT animals. Our results show that FAAH deficiency may alter the pathophysiology of acute asthma in the mouse. However, further experiments are required to investigate the underlying mechanisms.

Authors: Rahal L (1), Ghouini A (2), Ouled Kouider M (3), Kheloui Y (3), Alihallasa S (3)

Affiliations:
1. Departement of clinical physiology and functional explorations, Faculty of Medicine of Bechar- TAHRI MOHAMMED Bechar University/Public Hospital Establishment of Blida- Algeria
2. Departement of Physiology, Faculty of Medicine of Blida- Blida 01 University- Algeria
3. Department of Pulmonology, Faculty of Medicine of Blida, Blida 01 University/ Public Hospital Establishment of Blida- Algeria

Abstract:

Introduction. Chronic obstructive pulmonary disease (COPD) is characterized not only by airflow limitation but also by systemic manifestations, including skeletal muscle dysfunction and sarcopenia, which contribute to disease severity and poor outcomes. Peripheral muscle mass is closely linked to exercise tolerance, metabolic efficiency, and inflammatory burden. Mid-upper arm muscle circumference (MUAMC) is a simple anthropometric surrogate of skeletal muscle mass and may reflect underlying physiological vulnerability to exacerbations. This study investigated the relationship between MUAMC and exacerbation frequency in patients with COPD. Materials and Methods. In this multicenter, cross-sectional study, 105 patients with COPD across different GOLD stages were recruited from Algerian hospitals. Anthropometric assessments included body mass index (BMI), triceps skinfold thickness (TSF), mid-upper arm circumference (MUAC), and MUAMC. Patients were classified as frequent exacerbators (≥2 exacerbations/year) or infrequent exacerbators (<2 exacerbations/year). Statistical analyses were conducted using SPSS 27, including correlation and receiver operating characteristic (ROC) analyses. Results. Participants had a mean age of 68.2 ± 9.9 years, and 38.1% were frequent exacerbators. Mean BMI was 22.1 ± 4.3 kg/m². Mean TSF, MUAC, and MUAMC were 7.2 ± 3.4 mm, 24.6 ± 3.1 cm, and 22.3 ± 2.5 cm, respectively. Frequent exacerbators exhibited significantly lower MUAC and MUAMC values (p < 0.001). Exacerbation frequency was inversely correlated with MUAC (r = −0.466; p < 0.001) and MUAMC (r = −0.427; p < 0.001). ROC analysis demonstrated good discriminative performance of MUAMC for identifying frequent exacerbators (AUC = 0.763; 95% CI: 0.671–0.855). Discussion and Conclusion. Reduced MUAMC reflects peripheral skeletal muscle depletion, a key physiological component of systemic COPD involvement. Its strong association with exacerbation frequency suggests that MUAMC captures clinically relevant muscle-related vulnerability. As a low-cost and easily applicable measure, MUAMC may be integrated into routine physiological assessment to identify high-risk patients and support targeted preventive and rehabilitative interventions.

Authors: Yilmaz Arda Ates (1), Niklas Meyer (2), Johannes Vierock (2), J. Simon Wiegert (1)

Affiliations:
1. Department of Neurophysiology, Medical Faculty Mannheim, MCTN, University of Heidelberg, Mannheim, Germany.
2. Subcellular Optogenetics Lab, Charite Universitätmedizin, Berlin, Germany.

Abstract:

By introducing light-sensitive ion channels, pumps, enzymes, or other proteins into genetically identified target cells, optogenetics allows precise manipulation of electrical and biochemical activity in these specific cells with light (Deisseroth et al., 2011). Recently, we developed BiPOLES (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing), which is a two-channel fusion protein composed of blue light sensitive anion channel GtACR2 and redlight sensitive cation channel Chrimson (Vierock et al. 2021). BiPOLES offers: (1) efficient blue-light silencing and redlight spiking of neurons; (2) bidirectional control of single neurons with two-photon holographic light; (3) dual-color control of two neuron populations using a second blue-light-sensitive ChR without interference, across various light intensities; (4) precise optical adjustment of membrane voltage between chloride and cation reversal potentials; (5) bidirectional manipulation of neuronal activity in various invertebrate and vertebrate models, such as worms, fruit flies, mice, and ferrets. Here, we present a strategy to spectrally invert BiPOLES. InverseBiPOLES (iBiPOLES) is composed of the bluelight-sensitive cation channel Chrome2S for excitation and the red-shifted anion channel raACR for inhibition. Preliminary findings in rat hippocampal organotypic tissue cultures (OTCs) suggest that iBiPOLES is a promising tool to bidirectionally control neuronal activity orthogonal to BiPOLES. Neuronal spiking was driven with blue light, and neuronal silencing was achieved with red light. iBiPOLES can provide various opportunities when combined with the original BiPOLES or other optogenetic tools to investigate the precise function of multiple neuronal populations in the same brain region.

Authors: M. Langer (1), M. Hadamitzky (2), J. Fandrey (1), T. Leu (1)

Affiliations:
1. University Duisburg-Essen, Institute of Physiology, Essen, Germany
2. University Duisburg-Essen, Institute of Medical Psychology, Essen, Germany

Abstract:

Metformin has been reported to exert anti-proliferative effects across multiple cancer types, including glioblastoma, the most common malignant primary brain tumor. These effects are mainly mediated through inhibition of mTORC1 signaling, which in turn reduces the expression of hypoxia-inducible factors (HIF), central regulators of cellular responses to low oxygen availability. While increased HIF signaling is generally associated with unfavorable clinical outcomes in glioblastoma, recent findings suggest that HIF activation may also support antitumor immune mechanisms. However, the specific involvement of HIF-2α in metformin-induced effects on glioblastoma cells, as well as its potential association with primary cilia, specialized sensory structures implicated in cellular signaling, remains unclear. To address this question, two glioblastoma cell lines (RG2 and U87) will be exposed to metformin alone or in combination with either a HIF stabilizer or a HIF-2α inhibitor. Alterations in gene expression will be quantified by qPCR, and corresponding protein changes will be assessed by Western blot analysis, with a focus on HIF-dependent pathways. Cellular viability and migratory behavior will be examined using LDH-based cytotoxicity assays and scratch assays, respectively. In addition, primary cilia number and morphology will be analyzed by immunofluorescence microscopy. Overall, this project seeks to clarify how modulation of hypoxia-inducible factor signaling influences the cellular response of glioblastoma cells to metformin treatment.

Authors: Anaïs Leuze(1), Aurelia C. Nowak(2), Nora M. Biermann(3), Franziska Dengler(1)

Affiliations:
1. Institute of Animal Science, University of Hohenheim, Stuttgart, Germany
2. Institute of Physiology and Pathophysiology, Vetmeduni Vienna, Vienna, Austria;
3. Clinic for Horses, Vetmeduni Vienna, Vienna, Austria

Abstract:

Free fecal liquid (FFL) is a frequent problem in domestic horses, associated with conditions like enteritis and recurrent colic. Despite its high incidence, effective long-term treatment options are limited. To investigate underlying pathomechanisms, we analyzed fecal metabolomic profiles of affected individuals compared to healthy controls. Fecal samples were collected from N=8 horses (3 geldings and 5 mares, aged 16±7 years) with FFL and N=8 healthy controls (CON, 4 geldings and 4 mares, aged 15±7 years) every 8 weeks for one year. The FFL group was subdivided into an active (aFFL, n=10) and a quiescent phase (qFFL, n=38). Samples were stored at -80°C and analyzed using a mass spectrometry-based approach (MxP® Quant 500 assay, Biocrates, Innsbruck, Austria). Data analysis was performed using Metaboanalyst (version 6.0) and statistical comparisons were done using a Mann-Whitney test (GraphPadPrism 10.6.1) assuming statistical significance at p<0.05. Metabolomic profiling revealed a clear clustering among the groups. Compared to aFFL, concentrations of cysteine (Cys), γ-amino butyric acid (GABA) and proline betaine (PB) were significantly higher in the CON group. qFFL also showed significantly higher levels of these metabolites than aFFL, while there was no significant difference between CON and qFFL. The gradual increase of Cys and PB from aFFL via qFFL to CON parallels clinical recovery of the fecal texture. GABA concentrations tended to be higher in qFFL than in CON (p=0.0645). These findings imply a role of Cys, PB and GABA in FFL pathophysiology. Similar changes have also been reported in human patients with inflammatory bowel disease (IBD), suggesting similar pathomechanisms in human IBD and equine FFL. However, the three metabolites are associated with diverging effects, ranging from potentially mucosa-protective, anti-inflammatory (PB, GABA) to prosecretory (GABA) effects and may even be a sign of dysbiosis (GABA, Cys). Therefore, their exact role in FFL pathophysiology requires further investigation.

Authors: Sarumathi E, Velkumary S, Pooja Sethi, Nivedita Nanda, JB Prasad

Affiliations:
1. Department Of Physiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, INDIA
2. Department Of Physiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, INDIA
3. Department Of Radiation Oncology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, INDIA
4. Department Of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, INDIA
5. Department Of Biostatistics, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, INDIA

Abstract:

A comparative study to assess the cardiac autonomic functions before and three months after neck radiation therapy in Head and Neck Cancer patients. Background: Neck radiotherapy is crucial in managing head and neck cancer. However, irradiation can affect the baroreceptor and the afferent nerves coursing through it, leading to baroreceptor failure, which remains an unaddressed issue. Screening for baroreceptor failure is essential for assessing CVD risk in these patients. Objective: This study evaluated the cardiac autonomic functions and biomarkers before and three months after neck irradiation therapy in head and neck cancer patients. Methodology: A total of 32 advanced head and neck cancer (HNC) patients were recruited for the study. Cardiac autonomic functions were assessed non-invasively before and three months after neck radiation therapy (RT). The parameters measured were resting short-term heart rate variability (sHRV), baroreflex sensitivity (BRS), and cardiac autonomic reactivity tests, which include heart rate response to standing, deep breathing (E: I), Valsalva ratio, and isometric handgrip test. Serum biomarkers (Neuropeptide Y, Xanthine oxidase, Acetylcholine esterase, and Endothelial Nitric oxide Synthase) were estimated using the ELISA method. Result: In short-term analysis, the HF power ms2 ( p= 0.03) and the cardiac autonomic reactivity tests E: I ratio ( p=0.006) and VR ratio ( p=0.017) were significantly increased in post-RT-HNC patients, indicating better cardiac parasympathetic tone, the biomarker measurement revealed a significant increase in serum Xanthine oxidase level( p=0.034) and decrease in Endothelial Nitric-oxide Synthase( P =0.002) indicating ongoing oxidative stress and endothelial dysfunction, potentially elevating cardiovascular disease (CVD) risk. CONCLUSION: Three months post RT-HNC patients revealed increased cardiac vagal activity. However, persistent oxidative stress may have reduced nitric oxide bioavailability, potentially increasing their future CVD risk.

Authors: Dahbi Hafsa(1), El Hangouche Abdelkader Jalil(1,2)

Affiliations:
1.Departement of Physiology laboratory of Faculty of Medicine and Pharmacy of Tangier, Abdelmalek Essaâdi University
2.New England University of Tangier

Abstract:

The response of heart rate to changes in body position is an essential aspect of cardiovascular regulation and is particularly relevant for Postural Orthostatic Tachycardia Syndrome (POTS). The objective of this study was to evaluate heart rate in response to postural variations in healthy young adults, using two distinct methods: an active test and a tilt test. A cross-sectional study was conducted at the Physiology Laboratory of the Faculty of Medicine and Pharmacy of Tangier, 18 participants (13 males and 5 females) were included in the study. Heart rate was continuously measured and the data were analyzed using SPSS software. The results of the active test showed a significant increase in heart rate, in contrast, the tilt test demonstrated a progressive increase in heart rate with postural changes. These findings suggest that heart rate in healthy young adults responds significantly to postural changes, during the active test and the tilt test. Highlighting the importance of using different assessment methods for better understanding of orthostatic disorders. Keywords: heart rate, POTS, postural changes, active test, tilt test, young adults.

Authors: Hayriye Çakır Atabek (1), Talha Uysal (1)

Affiliations:
1. Eskişehir Technical University, Faculty of Sport Sciences, Department of Coach Training in Sports

Abstract:

Background: The ‘post-activation performance enhancement” (PAPE) is one of the main objectives of warm-up protocols, apart from the physiological benefits derived from the rise of body temperature, including enhanced neuromuscular and cardiometabolic responses. The main mechanism related to potentiation has been suggested to be the phosphorylation of the regulatory light chain of myosin, a muscle memory mechanism which increases the sensitivity to Ca2+, thus favouring transient increments in peak force and rate of force development. Aim: This study aimed to examine the effects of PAPE on jumping performance and the association its’ effects with strength. Methods: Thirty physically-active young (age 18.96±1.8 years, height 169.8±9.5 cm, body weight 64.92±12.4 kg) volunteered in this study. First, they warmed up by pedalling on a bicycle ergometer at 65-70 rpm for 7-8 minutes. Then, they performed 3 single-leg counter movement jumps (CMJ) with both legs. Thus, the pre-test measurements were done, and the dominant leg was determined. Following, participants performed five maximum voluntary isometric contractions (MVIC) on an isokinetic dynamometer at a 135-degree knee angle. Maximum and average values were recorded for extensors and flexors. Following 8-minute of active-recovery after the MVIC, the participants performed three more single-leg CMJ with the dominant leg, the highest score was used for analyses. The data were analysed with dependent student-t test, the relationship between muscle strength and jump height was evaluated with Pearson Correlation. P was set at <0.05. Results: The post-test single-leg CMJ height was significantly higher than the pre-test value (18.69±5.38 cm vs. 17.48±4.62 cm; p<0.001). The single-leg CMJ performance was significantly strongly correlated with flexors’ muscle peak and mean strength (r(28)>.610; p<0.001). The extensors’ muscle peak and mean strength values were also corelated but at the medium level (r(28)>.394; p<0.05). Conclusion: Single MVIC used as PAPE significantly affect the jumping performance in physically-active young.

Authors: Daniel Prince Torregrosa (1), Lucía Morán Lazcano (1),Begonya Vicedo Jover (2)

Affiliations:
1 - Predepartmental Nursing Unit, Faculty of Health Sciences, Jaume I University, Castellón, Spai

2 - Department of Biology, Biochemistry, and Natural Sciences, Jaume I University, Castellón, Spain.

Abstract:

In accordance with the widely known 'five-second rule', an experimental study was conducted to evaluate the scientific validity of this popular belief. Since childhood, people are warned about the unhygienic nature of food that falls on the floor; however, it is commonly implied that food remains safe to consume if retrieved within five seconds. This study aimed to simulate the action of dropping and collecting food from the ground, allowing contact with different floor surfaces for varying time intervals, and to analyze the resulting microbiological contamination. Methods: Samples were collected using solid culture medium (nutrient agar) from two different surfaces: the floor of a microbiology laboratory (controlled environment) and a general corridor (public environment). The culture medium was placed in contact with each surface for 1 and 4 seconds. After exposure, samples were incubated for 48 hours at 27 °C. Following incubation, the total number of Colony Forming Units (CFUs) was counted, and morphological variability among colonies was assessed. Results: The results demonstrated a higher microbiological load in samples exposed for longer periods and in those taken from the public floor. Notably, detectable contamination was already present after just one second of contact. Additionally, morphologically distinct colonies were observed between samples from the laboratory and public environments, indicating differences in microbial composition. Conclusion: These findings indicate that the 'five-second rule' is not scientifically supported. Contamination occurs immediately upon contact with a surface, regardless of whether the environment is considered clean or public. Although differences in microbial morphology were observed between surfaces, no exposure time could be considered safe, thus disproving the notion that food dropped briefly on the floor remains uncontaminated.

Authors: Fabian Spahiu*(1), Moritz Lampkemeyer(1), Max Hagemann(1), Michelle Ottlik(1), Lars C. Helbig(1), Eric J. Stöhr(1)

Affiliations:
1COR-HELIX, Institute of Sport Science, Leibniz University, Hannover

Abstract:

Background: Cardiometabolic thresholds during incremental exercise emerge from the integrated interaction of cardiac contraction mechanics, central hemodynamics, peripheral blood flow and oxygen extraction. Peak oxygen uptake (V̇O₂peak) is a key marker of training status, yet it is insufficiently understood how aerobic fitness modulates the timing of cardiac and hemodynamic limitations during incremental aerobic exercise. Methods: Participants (n = 18, 26 years ± 3.12, VO2 Peak = 3362.31 ± 561.95 ml/min) performed an incremental exercise test to exhaustion with simultaneous spiroergometry and high-frame-rate echocardiography. Plateau timing (PT) was determined for left-ventricular deformation parameters (PT–twist, PT–Ap.Rot), volumetric function (PT–SV, PT–EF), and peripheral hemodynamics (PT–SVR), expressed as percentage of maximal heart rate. Peak oxygen uptake was analyzed as relative (ml·kg⁻¹·min⁻¹) and absolute (ml·min⁻¹) values. Associations were assessed using Pearson or Spearman correlation analyses. Results: Relative V̇O₂peak was significantly associated with the timing of cardiac deformation plateaus. Higher relative V̇O₂peak was related to later PT–twist and PT–Ap.Rot, indicating a delayed onset of deformation plateauing in individuals with superior aerobic fitness. For volumetric parameters, a moderate but non-significant inverse association was observed between relative V̇O₂peak and PT–SV, whereas PT–EF showed a significant negative association with relative V̇O₂peak. No significant association was found between relative V̇O₂peak and PT–SVR. Higher absolute V̇O₂peak was linked to greater EDV@rest and SV@rest, as well as higher Q@peak, SV@peak, EDV@peak, and systolic blood pressure@peak exercise. Conclusion: Cardiac deformation plateau timing is modulated by aerobic fitness, whereas volumetric plateau timing shows no significant modulation and tends toward an inverse association with V̇O₂peak. Higher V̇O₂peak is associated with greater peak SV, EDV, Q, and systolic blood pressure, indicating that training status primarily affects myocardial deformation and maximal cardiovascular performance rather than volumetric threshold behavior.

Authors: Ozdede M.R.

Affiliations:
Necmettin Erbakan University, Konya, Turkiye

Abstract:

Background: Hepatokines are liver-derived secreted factors, defined operationally here as genes encoding predominantly hepatocyte-origin secreted proteins/peptides with potential systemic effects. In hepatocellular carcinoma (HCC), hepatocyte identity/differentiation can be variably attenuated, which tends to reduce liver-function–related transcriptomic signatures; consequently, canonical hepatokines may appear reduced in tumor tissue. This motivates an axis-aware readout in which tumor biology and liver-function–related phenotypes are evaluated jointly rather than interpreted in isolation. Methods: Data were accessed from the NCI Genomic Data Commons (GDC) using project TCGA-LIHC (hg38), data category Transcriptome Profiling, data type Gene Expression Quantification, and workflow STAR - Counts, and processed in R using TCGAbiolinks. Differential expression was performed with DESeq2 comparing Tumor (n=371) versus Normal (n=50), and variance-stabilized transformation (VST) was used for visualization. A curated 20-gene hepatokine panel was evaluated; 18/20 genes were detected in the expression matrix. For within-tumor analyses (tumor samples only), three z-mean signature scores were computed (hepatocyte identity, fibrosis/inflammation, proliferation) and used in multivariable linear models relating each hepatokine to these axes. Overall survival (OS) was tested in tumor samples with OS information (n=370) using Cox proportional hazards models (unadjusted; and adjusted for the three axis scores). P-values were controlled using Benjamini–Hochberg FDR. Results: Hepatokines exhibited heterogeneous tumor–normal expression shifts, with multiple genes decreased in tumors (e.g., HAMP, ANGPTL6) and a subset increased (e.g., PCSK9, FGF21). Within tumors, VST expression of LECT2, RBP4, and PCSK9 showed positive trends with the hepatocyte identity score. In unadjusted OS models, LECT2 (HR=0.878, FDR=0.00197), RBP4 (HR=0.867, FDR=0.00213), and IGF1 (HR=0.854, FDR=0.0404) met BH-FDR<0.05; after axis adjustment, no hepatokine met BH-FDR<0.05. Conclusion: Hepatokine expression differences co-vary with hepatocyte identity and other biological axes in TCGA-LIHC, and OS associations observed in unadjusted models do not persist after axis adjustment, consistent with axis-linked variation rather than independent prognostic signals in this dataset.

Authors: Fabrizio Di Giovanni (1), Giulio Musotto (2), G. Gambino (1), and Giuseppe Giglia (1) (3)

Affiliations:
(1) Department of Biomedicine Neuroscience and Advanced Diagnostics, Section of Human Physiology, School of Medicine, University of Palermo, 90127 Palermo, Italy.
(2) Bioengineering Unit, Ri.MED Foundation, Palermo, Italy
(3) Euro-Mediterranean Institute of Science and Technology (IEMEST), 90139 Palermo, Italy.

Abstract:

According to grounded cognition theory, language is closely linked to sensorimotor system. Human visuospatial processing distinguishes between peripersonal (PPS) and extrapersonal space (EPS), whose boundary can be modified by tool use. Intriguingly, most languages encode space dichotomously (near vs. far) and spatial language use can also be influenced by a tool. The present study investigates the relation between visuospatial perception and spatial semantics using a lexical decision task in a virtual reality (VR) environment. Fourteen healthy participants were presented with pairs consisting of a real word (Near or Far semantics) and a pseudoword, displayed either in PPS (60 cm) or EPS (120 cm). Participants read aloud and pointed to the meaningful word and reaction times (RTs) were collected. Two conditions were tested. In the No Tool condition, participants pointed using their arms only. In the Tool condition, they held a virtual stick long enough to reach EPS. The task was repeated after inhibitory cathodal transcranial direct-current stimulation (tDCS) of the right posterior parietal cortex (rPPC), a region involved in visuospatial and spatial semantic processing. Baseline results showed a congruency effect between semantic content and spatial location. In the No Tool condition, RTs were faster when Near and Far semantics were presented at congruent distances. In the Tool condition, Near semantics showed decreased RTs and Far semantic increased RTs in EPS, consistent with tool-induced remapping of EPS into PPS. TDCS selectively disrupted these effects. In the No Tool condition, rPPC inhibition abolished the congruency effect for Near semantics in PPS. In the Tool condition, tDCS interfered with tool-induced remapping, with reduced RTs for Far Semantics and increased RTs for Near Semantics when presented in EPS. These findings support a close coupling between spatial semantics and visuospatial perception, and demonstrate that both tool use and rPPC interference can modulate spatial language processing.

Authors: Kucner A (1), Gałdyszyńska (1), Drobnik J (1)

Affiliations:
1.Laboratory of Connective Tissue Metabolism, Department of Pathophysiology, Chair of General and Experimental Pathology, Medical University of Lodz

Abstract:

Elevation of interleukin-6 (IL-6) within the heart exerts proinflammatory and profibrotic effects. Regulation of its’ release and synthesis is dependent on physical forces. Forces in the cellular environment are transmitted by collagen and then submitted to collagen binding α2β1 integrin and its’ intracellular messengers Src kinase but not focal adhesion kinase. Inhibition of the α2β1 integrin or silencing of α2 integrin gene by siRNA decrease IL-6 secretion (Gałdyszyńska, 2020). The study is aimed at explanation whether IL-6 release remains under control of α1β1 integrin and to check involvement of p38 kinase, in transmission of the information. The experiment was conducted on an immortalized human cardiac fibroblasts. Cells were cultured on collagen-coated plates. During the experiment, the cells were treated with obtustatin (10^-6 -10^-8 M) to inhibit α1β1 integrin. SB203586 was used as the inhibitor of p38 kinase (10^-5 - 10^-7 M). The following parameters were then evaluated: intracellular and extracellular IL-6 levels by ELISA, IL-6 gene expression by q-PCR and cell proliferation by BrdU. Inhibition of integrin α1β1 increased both intracellular and extracellular levels of IL-6 comparing to control at each of the concentration tested. Treatment of cells with obtustatin increased IL-6 gene expression after 1 and 4 days of treatment. Obtustatin did not modify fibroblasts proliferation. Inhibition of p38 kinase with its’ inhibitor increased IL-6 levels in cardiac fibroblasts. The results show that IL-6 release remains under control of α1β1 integrin and p38 kinase, the element of this integrin signaling pathway. The effect is dependent on augmentation of IL-6 gene expression – the early step of IL-6 synthesis. Moreover, influence of α1β1 integrin on IL-6 release is opposite to effect described for α2β1 integrin inhibition. In conclusion IL-6 dependent of inflammatory and fibrotic processes regulation within the heart are reliant on opposable effects exerted by integrins α1β1 and α2β1.

Authors: Borrelli M (1), Motalli A (1), Toninelli N (1), Rampichini S (1), Esposito F (1)

Affiliations:
1. Department of Biomedical Sciences for Health, Università degli Studi di Milano, Italy

Abstract:

BACKGROUND: The harmful effects of cigarette smoking (CS) on cardiovascular and pulmonary health are well documented; however, its influence on the cardiorespiratory and metabolic response to exercise performed until exhaustion is not yet fully elucidated. PURPOSE: Evaluate the CS effects on exercise tolerance by assessing the cardiorespiratory and metabolic response to an exhausting moderate-intensity sinusoidal exercise in young, physically active smokers without known disease. METHODS: After pulmonary function assessment, 8 young physically active male smokers (SM; 22.1±2.2 years; 79.9±4.4 kg; maximum pulmonary oxygen uptake, V’O2max 3385±341 ml·min⁻¹) and 8 age-matched non-smokers (CTRL; 22.6±1.4 years; 74.3±7.9 kg; V’O2max 3623±302 ml·min⁻¹) performed a sinusoidal cycling exercise to exhaustion. The protocol was defined by a midpoint (MP) set 50 W below the lactate threshold (LT), an amplitude (AMP) of 50 W, and a period of 4 minutes. For each sinusoidal cycle, MP, AMP, and the time delay (tD) between mechanical power output and expiratory ventilation (V’E), V’O2, and heart rate (fH) were quantified. Blood lactate concentration ([La⁻]) was collected. RESULTS: Despite similar static lung volumes, SM exhibited lower maximal voluntary ventilation (-9%; P=0.011), lower LT (199±26 vs 219±28 W; P=0.001) and shorter time to exhaustion (−45%; P=0.001) compared to CTRL. No between-group differences were observed in MP, AMP, or tD for any cardiorespiratory variable, nor in [La⁻]. In SM, V’O2 MP remained steady, whereas CTRL showed an increase in the last cycle compared to the first cycle (P=0.001). In both groups, fH MP (P<0.01) and V’E MP (P<0.05) progressively increased during the test. CONCLUSIONS: Despite the absence of evident pulmonary disease and regular engagement in physical activity, SM displayed reduced exercise tolerance. The preserved cardiorespiratory and metabolic responses suggest that this limitation is unlikely attributable to cardiorespiratory dysfunction, but may reflect peripheral impairments, related to CS-induced oxidative stress and altered muscular oxygen utilization.

Authors: Anton Fürniss (1), Andreas Unger (1), Franziska Koser (1), Lena Wildschütz (1), Johanna Freundt (1), Nicolas Dammeier (2), Nils Voigt (2), Wolfgang A Linke (1)

Affiliations:
1. Institute of Physiology II, University of Münster, Münster, Germany.
2. Institute of Pharmacology and Toxicology, University Medical Center Göttingen (UMG), Göttingen, Germany.

The author did not consent to the publication of the abstract.

Authors: Rima Sarkar(1), Arnab Nayak(1), Theresia Kraft(1), Mamta Amrute-Nayak(1)

Affiliations:
1. Institute of Molecular and Cell Physiology, Hannover Medical School, 30625, Hannover, Germany.

The author did not consent to the publication of the abstract.

Authors: Khadka P (1),Mishal J (2)

Affiliations:
Department of Clinical Physiology and Yogic Sciences, Madan Bhandari Academy of Health Sciences,Nepal

Abstract:

ABSTRACT Introduction: Yoga is an ancient practice originating from India that has been potentially used to improve physical, mental, and spiritual health. The long-term effects of the practice of yoga are evident; however, the immediate impact of yoga is not very well established, particularly among the population with high academic stress, like medical students. Objective: To investigates the physiological and psychological impacts of a 12-week yoga and meditation program on undergraduate medical students at Madan Bhandari Academy of Health Sciences. Methods: By employing cross-sectional analytical design, the target population of 80 students were grouped into the yoga group and the control group. The pre-test and post test data of yoga group on the key health indicators, including the pulse rate, systolic and diastolic blood pressure on the student’s health profile, were taken before and after the student underwent the intervention. The Sahaja yoga technique was applied in the intervention. Result: The results revealed significant reductions in heart rate (-15.53%) and blood pressure (Systolic blood pressure by -6.12% and Diastolic blood pressure by -13.25%) in the yoga group compared to the control group (-4.43% HR, +1.84% Systolic blood pressure, and -2.13% Diastolic blood pressure, all p < 0.001), alongside improvements in perceived stress and anxiety levels. Conclusion: The study concludes that a 12-week yoga and meditation program significantly improved both physiological parameters and psychological well-being in undergraduate medical students.

Authors: Beatrice Mantuano (1 ), C.Rubeo (1) , C. Penna (1) , T. Angelone( 2 ), M. Bertinaria (3 ), P.Pagliaro (1)

Affiliations:
1 Department of Clinical and Biological Sciences, University of Turin.
2 Cellular and Molecular Cardiovascular Pathophysiology Laboratory, Department of Biology, E. and E.S. (DiBEST), University of Calabria.
3 Department of Drug Science and Technology, University of Turin.

Abstract:

Cellular senescence plays a critical role in the development of cardiovascular diseases. Oxidative stress is a major driver of senescence, and hydrogen peroxide (H 2 O 2 ) is commonly used to mimic this condition in experimental models. In cardiac cells, excessive oxidative stress induces structural and functional alterations characteristic of myocardial aging. In this study, we investigated whether INF195, a novel and selective inhibitor of the NLRP3 inflammasome, could attenuate oxidative stress-induced senescence in H9c2 cardiomyoblasts. To establish optimal conditions for senescence induction, H9c2 cells were exposed to increasing concentrations of H 2 O 2 (100–400 µM) for 2 or 4 hours, followed by culture in DMEM +2% FBS for 72h. Based on the best results, senescence was reliably induced by treatment with 100 µM H 2 O 2 for 4 hours. INF195 (10–20 µM) was applied either prior to H 2 O 2 exposure or following oxidative stress induction to evaluate its preventive and restorative effects. Cellular senescence was assessed using a senescence-associated colorimetric β-galactosidase assay and western blot technique, while NLRP3 expression and IL-1β levels were quantified by ELISA kit. The data demonstrated that H 2 O 2 exposure significantly increased Sa-β-gal activity, NLRP3 expression, and IL-1β production compared with control. The results confirming the induction of oxidative stress driven senescence and inflammasome activation. Pre-treatment with INF195 markedly reduced all senescence markers, restoring NLRP3 and IL-1β levels close to control values. In contrast, post-treatment with INF195 resulted in only a partial attenuation of inflammasome activation, indicating limited reversal of senescent phenotype. In conclusion, these findings demonstrate that inhibition of the NLRP3 inflammasome by INF195 effectively attenuates oxidative stress–induced senescence in H9c2, particularly when administered prior to senescence induction. This study supports the potential of inflammasome-targeting strategies as approaches to modulate cardiac aging and reduce cardiovascular dysfunction.

Authors: Runhe Qin1, Alexandr Melnikov1, Wolfgang M Kuebler1-5

Affiliations:
1. Institute of Physiology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität Zu Berlin, Berlin, Germany;
2. German Centre for Cardiovascular Research (DZHK), partner site Berlin, Berlin Germany;
3. German Centre for Lung Research (DZL), associated partner site Berlin, Berlin, Germany;
4. Keenan Research Centre, St. Michael´s Hospital, Toronto, ON, Canada;
5. Departments of Physiology and Surgery, University of Toronto, Toronto, ON, Canada.

Abstract:

Objective:Primary cilia (PC) are critical sensory organelles that coordinate mechanical and biochemical signalling in vascular cells and are essential for maintaining pulmonary vascular homeostasis. In previous work, we had found PC in lung vascular cells to be significantly shortened and the proportion of ciliated cells to be reduced in pulmonary arterial hypertension (PAH), suggesting a potential link between ciliary dysfunction and pulmonary vascular remodeling. Subsequent work showed that this loss of PC drives PAH remodeling processes. Aurora kinase A (AURKA) is a well-established regulator of ciliary disassembly for cell cycle progression.Hence, this study aimed to test whether AURKA contributes to PC loss and PAH-associated cellular phenotype in lung vascular cells in response to TGF-β,a central driver of lung vascular remodeling in PAH. Method: Human pulmonary arterial smooth muscle cells (hPASMCs) and human pulmonary arterial endothelial cells (hPAECs) were stimulated with TGF-β (10 ng/ml). PC abundance and length were assessed by immunofluorescence microscopy following staining for acetylated α-tubulin and ARL13B. Cell migratory capacity was quantified in wound-healing assays. Protein expression of AURKA, cilia-associated proteins, TGF-β downstream signalling molecules,and the hypertrophy- and mesenchymal-associated marker α-smooth muscle actin (α-SMA) were analysed by Western blot. Results: TGF-β stimulation increased AURKA activity in both hPASMCs and hPAECs and was associated with a reduction in the proportion of ciliated cells and shortening of PC. AURKA inhibition using MK-5108(10 ng/ml) increased cilia length and ciliated cell frequency compared to TGF-β alone. Immunoblots revealed that MK-5108 attenuated TGF-β-induced α-SMA upregulation in hPAECs and hPASMCs by 30–40%, indicating suppression of both mesenchymal transition and hypertrophic responses. Similarly, MK-5108 reduced cell proliferation and migration in response to TGF-β. Conclusion: These findings identify AURKA activation as a novel pathogenic mechanism in PAH, promoting lung vascular cell deciliation and remodeling. Pharmacological inhibition of AURKA may represent a novel therapeutic strategy for PAH.

Authors: García-Blázquez A (1), Vicente M (1), Martínez-Sielva A (1), Salgado-Almario J (1), Domingo B (1), Llopis J (1)

Affiliations:
1. Physiology and Cell Dynamics Group, Instituto de Biomedicina de la Universidad de Castilla-La Mancha, Facultad de Medicina de Albacete, Universidad de Castilla-La Mancha, and Instituto de Investigación Sanitaria de Castilla-La Mancha (IDISCAM), C/Almansa 14, 02006 Albacete, Spain

Abstract:

The phospholamban arginine 9 to cysteine (R9C) mutation induces a cardiomyopathy in humans, with ventricular dilation and premature death. The phospholamban R9C is a loss of function mutant unable to inhibit SERCA2a in heterozygosis. In a previous study, we generated a transgenic zebrafish line overexpressing plnb^R9C in the heart, Tg-plnb^R9C, and we showed that 3 days post-fertilization (dpf) larvae had a hyperdynamic heart with increased cardiac output. In turn, 9 dpf larvae showed systolic dysfunction associated with reduced inotropism and ventricular dilation, which seemed to normalize the cardiac output by the Frank-Starling mechanism, compensating for the loss of contractility. In the present work, we studied cardiac function and bioenergetics of the Tg-plnb^R9C zebrafish from the larval stage to adulthood. We hypothesize that PlnbR9C leads to a progressive reduction of inotropism (stroke volume) and cardiac energy efficiency. Cardiac function was assessed by fluorescence imaging in larvae and echocardiography in adults. ATP levels were measured with a transgenic line expressing the mitochondrial ATP biosensor mitATeam1.03, and gene expression profile was analyzed through RNAseq. We observed that the Tg-plnb^R9C zebrafish had altered cardiac function, with ventricular dilation and reduced contractility from 14 dpf larvae to adulthood at 10 months post-fertilization (mpf), while cardiac output remained preserved. In parallel, mitochondrial ATP levels were consistently increased across all ages, unveiling an increase in energy production. In line with these results, the transcriptomic analysis also revealed enrichment in pathways associated with mitochondrial function and ATP synthesis. However, the relationship between ATP levels and stroke area showed a decrease in cardiac efficiency in Tg-plnb^R9C zebrafish. Together, these results suggest that plnb^R9C mutation causes a cardiac dysfunction in which the cardiac output is compensated by an increase in mitochondrial ATP production, leading to reduced cardiac efficiency.

Authors: Bujnová K (1), Barta A (1), Stanko P (2), Čačanyiová S (1), Cebová M (1,2)

Affiliations:
1. Institute of Normal and Pathological Physiology, Centre of Experimental Medicine, Slovak Academy of Sciences, Bratislava, Slovakia
2. Institute of Pathophysiology, Medical Faculty, Comenius University, Bratislava, Slovakia

Abstract:

Myocardial infarction (MI), the most severe manifestation of ischemic heart disease, results from an abrupt interruption of coronary blood flow, leading to cardiac muscle necrosis. The subsequent inflammatory response is essential for clearance of necrotic tissue; however, dysregulated inflammation may promote adverse cardiac remodeling characterized by fibrosis, ventricular dilation, and impaired contractility, ultimately contributing to heart failure. Although reperfusion is necessary to limit ischemic damage, it may paradoxically exacerbate injury through excessive production of reactive oxygen species and activation of inflammatory pathways. High mobility group box-1 (HMGB-1) protein is a key trigger of post-infarction inflammation. Upon release from necrotic cardiomyocytes, HMGB-1 acts as an alarmin, inducing pro-inflammatory cytokine production and initiating the inflammatory cascade. The aim of this study was to inhibit HMGB-1 using glycyrrhizin, a natural compound derived from licorice, following experimentally induced MI under hypertensive conditions. MI was induced in 12-week-old spontaneously hypertensive rats by reversible ligation of the left anterior descending coronary artery. Glycyrrhizin (10 mg/kg) was administered intravenously 20 minutes after occlusion, immediately prior to reperfusion. Animals were euthanized 28 days after surgery. Blood pressure was measured non-invasively, cardiac function was assessed by echocardiography, total nitric oxide synthase (NOS) activity was determined by L-arginine to L-citrulline conversion, and vascular reactivity was evaluated using serotonin-induced contraction in isolated aortic rings. Glycyrrhizin significantly improved left ventricular systolic function without affecting blood pressure. MI reduced ejection fraction and fractional shortening, whereas glycyrrhizin restored both parameters to control levels. Diastolic function was not significantly affected. MI increased myocardial NOS activity and impaired aortic contractility, both of which were attenuated by glycyrrhizin. In conclusion, HMGB-1 inhibition by glycyrrhizin improved cardiac systolic function, normalized NOS activity, and restored vascular reactivity, indicating its potential therapeutic benefit in MI under hypertensive conditions. This work was supported by APVV-22-0271 and VEGA 2/0131/24.

Authors: Lina Elshareif (1), Theresia Kraft (1), Bogdan Iorga (1)

Affiliations:
(1) Institute of Molecular and Cell Physiology, Hannover Medical School, Hannover, Germany

The author did not consent to the publication of the abstract.

Authors: Fesseler L (1), Heinz V (1, 2), Pilz N (3), Bothe TL (4)

Affiliations:
1. Charité – Universitätsmedizin Berlin, Institute of Physiology, Center for Space Medicine and Extreme Environments Berlin
2. Charité – Universitätsmedizin Berlin, Institute of Integrative NeuroAnatomy, Center for Space Medicine and Extreme Environments Germany
3. Department of Cardiology and Angiology, Hannover Medical School, Hannover, Germany
4. Sydney School of Health Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, Australia

Abstract:

Objective: High-intensity exercise induces systemic acidosis, which contributes to muscular performance decline. Controlled pre-exercise hyperventilation has been proposed as a non-invasive intervention to induce respiratory alkalosis and counteract acidosis-related acute fatigue. In this study, we hypothesized that a real-world-applicable pre-exercise hyperventilation protocol can improve incline sprint performance. Methods: This randomised, single-centre, controlled crossover study included 36 recreational athletes aged 18 to 40 years. Each participant completed a series of three inclined treadmill interval sprint sessions on two separate days: one with pre-exercise hyperventilation and one without. Respiratory parameters were measured via spirometry, and post-sprint capillary blood gas analysis was performed. Results: Pre-exercise hyperventilation resulted in higher mean CO2 elimination during hyperventilation (32.8 ± 5.1 vs. 21.5 ± 2.5 mL/kg, p < 0.001) and lower mean end-tidal CO₂ partial pressure (17 ± 2.5 mmHg vs. 30 ± 2.7 mmHg, p < 0.001) compared to control condition. Mean cumulative altitude gain did not differ between pre-exercise hyperventilation (83.2 ± 27.6 m) and control condition (84.3 ± 26.0 m, p = 0.555). Differences in CO2 elimination did not correlate with differences in performance (p = 0.590). We found no alterations in post-sprint capillary blood lactate and pH between both conditions (p > 0.05 for all sprints cumulative and separately). Participants subjectively assessed the breathing protocol as unpleasant, and felt it negatively influenced their performance. Conclusion: The easily translatable pre-exercise hyperventilation protocol investigated in this study did not improve performance in repeated high-intensity incline sprint intervals. Therefore, we discourage its application in real-world applications

Authors: Johannes P.-H. Seiler (1), Jonas Elpelt (2), Giuseppe Cazzetta (1), Takahiro Noda (1), Aida Ghobadi (1), Matthias Kaschube (2), Simon Rumpel (1)

Affiliations:
1. Institute of Physiology, Focus Program Translational Neurosciences, University Medical Center of the Johannes Gutenberg University Mainz, Germany
2. Frankfurt Institute of Advanced Studies, Frankfurt am Main, Germany

Abstract:

Our brain is thought to encode sensory information, such as incoming sound stimuli, in form of representational maps, reflecting relations between different sensory inputs. While the structure of a representational map effectively captures different features of sensory stimuli, it remains unclear how this structure affects perceptual decisions and associations. Here, we address this issue, studying representational maps in humans and mice. We apply a translational discrimination task and a human scaling task to assess the representational similarities between a broad set of pulsed sounds, using statistical modeling to estimate individual representational maps. We find that naïve and task-based maps in humans and mice share key structures, suggesting a stable representational architecture conserved across species. Moreover, we find that task-naïve maps predict how well humans discriminate sounds, how quickly they learn, and how likely they form creative associations. We combine these behavioral estimates of representational maps with large-scale two-photon calcium imaging in the mouse auditory cortex, observing that perceptual similarities between sounds are predicted by global structures of stimulus-evoked activity in the cortex. Taken together, our findings indicate that the representational architecture of individual provides a general and evolutionary conserved underpinning of perception and creative thought.

Authors: Urone G (1), Scordino M (1), Frinchi M (1), Giuffrè C (1), Montalbano A (2), Barreca M (2), Barraja P (2), Raimondi M R (2), Bivacqua R (2), Malhaire F (3), Goudet C (3), Mudò G (1), Spanò V (2), Di Liberto V (1)

Affiliations:
1. Department of Biomedicine, Neuroscience and Advanced Diagnostics, University of Palermo, Palermo, Italy
2. Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Palermo, Italy
5. IGF, Université de Montpellier, CNRS, INSERM, Montpellier, France

Abstract:

Parkinson's Disease (PD) is a neurodegenerative disorder marked by the progressive loss of dopaminergic neurons in the nigrostriatal circuit. Currently, available pharmacological treatments offer only symptomatic relief with adverse effects. Within this framework, indirect evidence has indicated that activating metabotropic Glutamate Receptor 3 (mGlu3) may exert neuroprotective effects in animal models of PD, but selective agonists for this receptor were lacking until now. Recently, a groundbreaking development has emerged in the form of a new Agonist/Positive Allosteric Modulator (Ago/PAM) that, although showing equal affinity for mGlu2 and mGlu3, displays greater efficacy at mGlu3 receptors in recombinant cells. Our study shows for the first time that the novel mGlu2/3 Ago/PAM protects the human neuroblastoma cell line SH-SY5Y against cell death induced by exposure to the dopaminergic toxin 6-hydroxydopamine (6-OHDA) through the modulation of key signaling pathways, such as mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3K)-AKT pathways. Moreover, in vivo experiments revealed that administration of the original mGlu2/3 Ago/PAM upregulates the expression of crucial neurotrophins, such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), while influencing MAPK/ERK and PI3K-AKT cascades in the brain of healthy mice. Finally, our results provide new insights into mGlu3 specific function and suggest the therapeutic potential of the new mGlu2/3 Ago/PAM in the management of PD.

Authors: Iva Marinova, Noa Lipstein

Affiliations:
1. Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)

Abstract:

Synaptic vesicle (SV) cycling ensures the reliable transmission of information across neuronal networks. Although the core molecular machinery mediating vesicle fusion and retrieval is well characterized, the modulatory mechanisms that fine-tune vesicle turnover remain poorly understood. Members of the Tumor Protein D52 family (TPD52 and TPD54) have been implicated in calcium-dependent vesicular trafficking in non-neuronal cells. Proteomic studies have identified them in glutamatergic terminals, suggesting potential involvement in SV dynamics. The specific contribution of TPD52/54 to synaptic transmission remains unknown.

Authors: Francesca Giammello (1), Erica Cecilia Priori (1), Francesca Dalle Sasse (1), Daniela Ratto (1), Elisa Roda (2), Federico Brandalise (3), Paola Rossi (1)

Affiliations:
1. Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Pavia 27100, Italy.
2. Laboratory of Clinical & Experimental Toxicology, Pavia Poison Centre, National Toxicology Information Centre, Toxicology Unit, Istituti Clinici Scientifici Maugeri IRCCS Pavia, 27100 Pavia, Italy.
3. Department of Biomedical Sciences, Division of Neuroscience and Clinical Pharmacology, University of Cagliari, Cittadella Universitaria di Monserrato, Monserrato 09042, Italy.

The author did not consent to the publication of the abstract.

Authors: Isis Gil-Miravet (1); Ángel Núñez-Molina (2); Mónica Navarro-Sánchez (1); Esther Castillo-Gómez (1); Francisco E Olucha-Bordonau (1)

Affiliations:
1. Departamento de Medicina, Facultad de Ciencias de la Salud, Universitat Jaume I, Castellón de la Plana, Spain.
2. Departamento de Anatomía, Histología y Neurociencia, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain.

Abstract:

Theta oscillations in the medial temporal lobe are fundamental for spatial navigation and memory formation and are classically driven by the medial septum (MS). The nucleus incertus (NI), a brainstem structure rich in relaxin-3 (RLN3) neurons, has been proposed as an upstream modulator of hippocampal and entorhinal theta, but the anatomical and physiological pathways underlying this influence are not fully understood. Using dual retrograde tracing combined with RLN3 immunolabeling, we mapped the ascending projections of NI neurons to the MS and medial temporal lobe in rats. We found that the majority of NI projections target the MS rather than directly innervating entorhinal or hippocampal structures, and that RLN3-expressing neurons show higher collateralization and a relative bias toward medial temporal lobe targets. In parallel, in vivo electrophysiological recordings revealed that electrical stimulation of the NI reliably induced theta-band activity in both the MS and medial entorhinal cortex. Critically, inactivation of the MS or pharmacological blockade of RXFP3 receptors within the MS abolished NI-evoked entorhinal theta. Together, these data demonstrate that the MS functions as a central physiological relay through which NI and RLN3 signaling synchronize medial temporal lobe networks. This brainstem–septal–entorhinal axis provides a mechanistic framework for understanding how arousal and neuromodulatory systems regulate large-scale oscillatory dynamics in cognitive circuits.

Authors: Marton Molnar(1), Alexander Dieter(1), J. Simon Wiegert(1)

Affiliations:
1. Department of Neurophysiology, Medical Faculty Mannheim, University of Heidelberg, Mannheim Center for Translational Neuroscience (MCTN)

Abstract:

The Locus Coeruleus (LC) is a compact nucleus located in the brainstem and represents the principal source of norepinephrine (NE) in the brain. Due to its reciprocal connectivity with almost all brain structures ranging from the brainstem to distant cortical areas such as the medial prefrontal cortex (mPFC) and the dorsal hippocampus (dHPC), it is thought to act as a major broadcasting hub for NE, which is involved in a large number of brain functions ranging from novelty detection to arousal to stress responses. Several theories have been proposed to explain how the LC-NE system regulates behavior, primarily based on differences in its neuronal firing patterns (tonic, phasic) or modulation of sensory gain. However, a precise anatomical, physiological, and molecular characterization of projection-specific LC neurons is still lacking. In this project, we investigate potential spatial modularity within the LC, which may manifest as specialized sub-populations of LC neurons with specific projection targets and differential neuromodulator release profiles (e.g., NE versus dopamine [DA]). In this way, the LC may shape neuronal activity in a target-specific manner. We combine recently developed sensors for neuromodulators with optogenetic stimulation of LC neurons and fiber photometry to differentiate NE and DA release in different target regions. Moreover, we apply retrograde tracing approaches to obtain a detailed picture of the specific projection profile of the LC.

Authors: Meyer K V (1,2,3), Miller P (1), Schmucker J (1), Bahnemann J (2,4)*, Meissner A (1,3,4,5)*

Affiliations:
1. University of Augsburg, Institute of Theoretical Medicine, Division of Physiology & Vascular Biology, Augsburg, Germany
2. University of Augsburg, Institute of Physics, Augsburg, Germany
3. Lund University, Department of Experimental Medical Science, Faculty of Medicine, Lund, Sweden
4. University of Augsburg, Centre for Advanced Analytics and Predictive Sciences (CAAPS), Augsburg, Germany
5. Lund University, Wallenberg Centre for Molecular Medicine, Lund, Sweden

Abstract:

Stroke is a leading cause of long-term disability worldwide. Most strokes are ischemic, arising from an acute reduction in cerebral blood flow due to vascular occlusion. Limited knowledge of neurovascular unit (NVU) alterations in stroke hinders effective treatment development. 3D microfluidic culture systems offer a promising in vitro approach to better understand NVU alterations by replicating physiological conditions while reducing animal use. The current study develops a microfluidic "stroke-on-a-chip" device that mimics the NVU and enables studying its response to ischemia by precisely controlling perfusion conditions and allowing for cell-specific sampling or manipulation. The system enables microscopic live-cell imaging as well as recovery of the 3D cell cultures to maximize analysis options. A functional assessment of ischemia-induced NVU alterations can be achieved by integrating non-invasive chemical optical sensors for O2- and pH-measurements and electrodes for impedance measurements to enable assessment of barrier integrity. The cultivation device consists of two 3D-printed parts that form the media channels and chambers for culturing hydrogel-embedded cells. Channels and chambers are separated by a permeable membrane which prevents hydrogel detachment and doubles as substrate for endothelial cells. The system therefore allows for the cultivation of endothelial cells exposed to shear stress in close contact with other important NVU cells, such as astrocytes, embedded in a hydrogel that their native extracellular microenvironment. A transparent bottom plate allows for good microscopic imaging and screws and gaskets are used to reversibly seal the device to allow recovery of the cells after an experiment. Ongoing studies include long-term reliability testing with mouse brain endothelial cells and hydrogel-embedded mouse astrocytes. The high level of customization enabled by additive manufacturing allows the microfluidic culture system to be readily adapted to diverse experimental requirements. Consequently, it holds significant potential as a versatile platform for 3D cell culture applications.

Authors: Büttner T (1), Wang X (2), Krishnacoumar B (1), Papadamakis A (2), Cicek S (1), Schild Y (1), Winning S (1), Littwitz-Salomon E (2), Dittmer U (2), Bayer W (2), Fandrey J (1), Malyshkina A (1)

Affiliations:
1 Institute of Physiology, University of Duisburg-Essen, University Hospital Essen, Essen, Germany
2 Institute for Virology, University of Duisburg-Essen, University Hospital Essen, Essen, Germany

Abstract:

Hypoxia is a hallmark of solid tumors and represents a major barrier for effective cancer therapies, including oncolytic virotherapy. While adenoviruses are widely studied as oncolytic agents, the impact of hypoxic tumor microenvironments on viral infection and spatial spread remains incompletely understood. In this study, we investigated how oxygen availability influences adenovirus infection in two-dimensional (2D) cultures and three-dimensional (3D) tumor spheroids. We first confirmed hypoxia responsiveness in commonly used adenovirus research cell lines (HEK293A and A549) as well as in KP4 pancreatic cancer cells, demonstrating hypoxia-induced HIF-1α stabilization. Hypoxia markedly reduced adenoviral protein production in 2D cultures across all cell lines. To model oxygen gradients found in solid tumors, we established stable KP4 spheroids using a hanging-drop approach supplemented with methylcellulose. Spatial analysis of HAdV5_GFP-infected spheroids revealed that productive infection was largely restricted to the well-oxygenated outer rim when infection occurred during spheroid formation and hypoxia development. In contrast, infection performed under normoxic conditions prior to spheroid formation resulted in a more uniform distribution of infected cells throughout the spheroid. Together, these findings demonstrate that hypoxia not only suppresses adenoviral replication but also shapes the spatial pattern of infection in 3D tumor models, highlighting the importance of incorporating hypoxia-relevant 3D systems in preclinical evaluation of oncolytic adenoviruses.

Authors: Evgeniya Kochina (1), Friederike Stumpff (2), Dorothee Günzel (1)

Affiliations:
1. Clinical Physiology/Nutritional Medicine, Charité - Universitätsmedizin Berlin, Berlin, Germany
2. Institute for Molecular Medicine,Health and Medical UniversityPotsdam, Potsdam, Germany

Abstract:

Salivary secretion is a highly regulated process that integrates receptor-mediated signaling (muscarinic and adrenergic) with downstream Ca²⁺ and cAMP pathways to orchestrate ion and fluid transport. Mutations in the tight junction protein Claudin-10 (Cldn10) are associated with HELIX syndrome, a rare disorder characterized by hypohidrosis, electrolyte imbalance and salivary gland dysfunction. We hypothesize that deficiency of the Claudin-10b isoform impairs Na⁺ secretion in the secretory units (acini) of salivary glands, leading to reduced saliva volume. To investigate this, we established a functional organoid model derived from inducible Cldn10 knockout (iCldn10 KO) mice. Organoids were cultured both in 3D Matrigel and as 2D monolayers on permeable filters, enabling electrophysiological assessment using novel intraluminal potential measurements and Ussing chamber. Stimulation with carbachol and forskolin induced distinct short-circuit currents, while specific inhibitors — such as diphenylamine-2-carboxic acid, flufenamic acid, thapsigargin, ouabain, CFTRinh-172 and bumetanide — provided insights into the underlying ion transport mechanisms. Live-cell imaging of luminal swelling confirmed functional secretory responses, while immunostaining validated the presence of acinar, ductal, myoepithelial and progenitor cell populations. These findings establish salivary gland organoids as a physiologically relevant system to study epithelial ion transport. Integration of intraluminal potential measurements provides a novel real-time readout of luminal bioelectrical properties, complementing Ussing chamber analyses. This system sets the stage for future experiments using Claudin-10b-deficient organoids to uncover how tight junction defects disrupt glandular secretion.

Authors: Miller P (1), Meyer K V (1,2,3,4), Bahnemann J (2,5), Meissner A (1,3,4,5)

Affiliations:
1. University of Augsburg, Institute of Theoretical Medicine, Division of Physiology & Vascular Biology, Augsburg, Germany
2. University of Augsburg, Institute of Physics, Augsburg, Germany
3. Lund University, Department of Experimental Medical Science, Faculty of Medicine, Lund, Sweden
4. Lund University, Wallenberg Centre for Molecular Medicine, Lund, Sweden
5. University of Augsburg, Centre for Advanced Analytics and Predictive Sciences (CAAPS), Augsburg, Germany

Abstract:

The blood-brain barrier (BBB) protects the central nervous system (CNS) from potentially harmful substances in the bloodstream through specialised endothelial cells that form a highly selective interface. In addition to endothelial cells, other cell types and their interactions are essential for proper BBB function. This multicellular system, which includes glia cells, neurons, and the basement membrane, is referred to as the neurovascular unit (NVU). A detailed understanding of NVU function is crucial, as many CNS disorders are associated with a disruption of the BBB. A wide range of cell-based in vitro models have been developed over the last decades, with Transwell-based systems being the most used. However, 2D-models cannot accurately represent the complex cellular organisation in an in vivo environment. Astrocytes, the most abundant glia cell type in the CNS that plays an important role in tight junction formation and intercellular communication, do not show physiological morphology under 2D-culture conditions. To more closely mimic in vivo conditions, advanced 3D-culture systems are therefore required. Hydrogels represent a promising strategy to support astrocyte morphology and function in 3D; however, the vast variety of hydrogels are not equally suitable. This study systematically compares different hydrogel systems for their suitability as matrices for the 3D-culture of primary mouse astrocytes and 2D-culture of murine brain endothelial cells. Three hydrogel types are investigated: an ECM-derived hydrogel comparable to Matrigel, a semi-synthetic hydrogel based on methacrylate-functionalised gelatin, and a fully synthetic polysaccharide-based hydrogel. The hydrogels are evaluated based on their ability to preserve astrocyte morphology, suppress endothelial tube formation, and maintain cell viability. In addition, the impact of the different matrices on typical gene expression patterns of endothelial cells and astrocytes is assessed. This study may provide an overview of the various hydrogel systems available, making it easier to select a suitable option for future NVU models.

Authors: Elena Miglioranza (1), Marco Emili (2), Chiara Berteotti (1), Hafiza Rohma Ahmed (1), Stefano Bastianini (1), Dario Coraci (1), Sandra Guidi (3), Viviana Lo Martire (1), Alessandro Silvani (1), Fiorenza Stagni (2), Emilia Volino (1), Renata Bartesaghi (3), Giovanna Zoccoli (1)

Affiliations:
1. PRISM Lab, Department of Biomedical and Neuromotor Sciences, Alma Mater Studiorum – University of Bologna, Bologna, Italy
2. Department for Life Quality Studies, University of Bologna, Rimini, Italy
3. Department of Biomedical and Neuromotor Sciences, Alma Mater Studiorum - University of Bologna, Bologna, Italy

Abstract:

Sleep disturbances and neurodegenerative diseases are linked by a strong bidirectional relationship, with sleep alterations increasingly recognized as a risk factor for Alzheimer’s disease (AD). Individuals with Down syndrome (DS) frequently show sleep disturbances and are at high risk of developing AD. However, whether chronic sleep disruption causally contributes to accelerating neurodegeneration in DS remains unclear. The aim of this study was to investigate whether long-term sleep alterations promote neurodegenerative changes in the Ts65Dn (Ts) mouse, a widely used model of DS that exhibits early AD-like neuropathology and sleep-disordered breathing. Three-month-old Ts and euploid (Eu) control mice were either exposed to a validated sleep fragmentation protocol (FR) for three months or maintained under control conditions (CO). At the end of FR, mice underwent 24-hour whole-body plethysmography to assess hypnic and ventilatory phenotypes, followed by histological and morphometric analyses of brain regions critically involved in AD, including the dentate gyrus (DG), hippocampal fields CA1 and CA3, entorhinal (LEC) and perirhinal (PRC) cortices. Microglial activation was assessed by analysis of IBA1-positive cells in the same areas. After three months of FR, Ts-FR and Eu-FR mice showed only marginal alterations in hypnic and ventilatory phenotype compared to controls. Sleep fragmentation induced a marked reduction in neuronal density in the DG and CA1 in both Ts-FR and Eu-FR mice. Neuronal loss in the PRC and LEC was observed selectively in Ts-FR mice compared to Ts-CO. CA3 field was relatively spared from degeneration. In parallel, sleep fragmentation was associated with a significant enlargement of IBA1-positive microglial soma in hippocampal regions. Results demonstrated that chronic sleep fragmentation causes widespread neuronal loss and microglial activation in brain structures essential for long-term memory in a mouse model of DS. These findings support a causal role of sleep disruption in accelerating neurodegenerative processes in predisposed individuals.

Authors: Mónica Navarro Sánchez (1), Zineb Bourgane (2), Francisco Eliseo Olucha Bordonau (1), Mohamed Zahran (1), Danna Rubio (1), Isis Gil Miravet (1), Jose Hidalgo (1), Josephine Lira López (1), Esther Castillo Gómez (1), Liana Fattore (3)

Affiliations:
1 Universidad Jaume I
2 Cadi Ayad University
3 CNR, IN, Cagliari

Abstract:

Exposure to nanoplastics represents a growing health concern, particularly regarding their potential impact on neurological function. While previous studies have shown accumulation of nanoplastics in various organs, their effects on neural activity remain unclear due to the protective role of the blood-brain barrier (BBB). However, during development or in specific brain regions, the BBB can be permeable. This study examined whether maternal exposure to nanoplastics during pregnancy, lactation, or both periods alters offspring behavior. Pregnant rats were exposed to nanoplastics or saline during these windows, and their offspring underwent behavioral testing in adulthood, including the open field, elevated plus maze, 3-chamber test, and contextual fear conditioning. Nanoplastic exposure produced significant behavioral alterations with sex-dependent effects. Male offspring exposed during pregnancy or lactation showed increased locomotor activity, an effect absent in combined exposure. Anxiety-like behaviors increased in both sexes in the pregnancy group. Social assessments revealed impaired sociability: offspring exposed during lactation or both periods failed to discriminate between social and non-social stimuli, while those exposed during pregnancy or both periods did not recognize novel social targets. Contextual fear conditioning further showed disrupted memory processes. Male offspring from pregnancy-exposed mothers failed to acquire context-fear associations, whereas those exposed during lactation could learn but not extinguish fear responses. Females exhibited comparable impairments with subtle differences. Overall, maternal exposure to nanoplastics during critical developmental stages induced long-lasting behavioral changes in offspring, underscoring potential neurodevelopmental risks associated with early-life nanoplastic exposure.

Authors: Szabó J (1,2), Blahútová A (1,2), Ovcharenko K (1,2), Feješ A (2), Borbélyová V (2), Celec P (2)

Affiliations:
1. Institute of Medical Physics and Biophysics, Medical Faculty, Comenius University, Bratislava, Slovakia
2. Institute of Molecular Biomedicine, Medical Faculty, Comenius University, Bratislava, Slovakia

Abstract:

Loneliness and social isolation are a global phenomenon with significant neuropsychiatric impact, linked to reduced neuroplasticity, neuroendocrine dysfunction, and neuroinflammation. However, whether these alterations cause or only accompany behavioral pathology remains unclear. This project evaluates the neurobehavioral consequences of long-term social isolation in rats, focusing on the neuroinflammation and neuroplasticity. Following a 2-month social isolation protocol, adult Wistar rats (Isolated, n=30) and the controls (Ctrl, n=30) of both sexes underwent behavioral testing for locomotion, anxiety-like behavior, cognition and sociability. Subsequently, plasma concentrations of GFAP, S100B, and BDNF were analyzed. Isolated male rats showed 40% higher incidence of aggressive behaviors in comparison to isolated females and controls (p<0.05). No other behavioral effects of the social isolation were observed. However, both isolated groups showed elevated concentrations of plasmatic S100B (p<0.05), while isolated males further showed elevated plasmatic concentrations of GFAP (p<0.01), indicating underlying astrocytosis. Curiously, while isolated males showed reduced BDNF in plasma (p<0.05), isolated females had higher plasmatic BDNF concentrations, in comparison to controls (p<0.05). Additionally, sex differences were observed among the groups, regardless of social isolation. Female rats showed twice as much locomotor activity (p<0.001), 45% less anxiety-like behaviors (p<0.01) and 40% more social interactions (p<0.01) than male rats. Our results suggest a 2-month social isolation was not sufficient to produce notable behavioral effects in adult Wistar rats. The subtle shift towards aggressive behaviors in socially isolated males and its potential implications to sex hormones, need to be further elucidated. Nevertheless, the sex-specific differences in markers of neuroinflammation and neuroplasticity might indicate that specific molecular alterations at the neurocellular level might precede the onset of behavioral effects. The ongoing evaluation of the endocrine profiles of the isolated rats will help further clarify the sex-specific characteristic of our findings. Research was supported by Slovak Agency of Research and Development No: APVV-24-0274.

Authors: Scordino M (1), Urone G (1), Frinchi M (1), Seidita L (1), Nuzzo D (2), Di Carlo M (2), Belluardo N (1), Mudò G (1), Di Liberto V (1)

Affiliations:
1. Department of Biomedicine, Neuroscience and Advanced Diagnostics, University of Palermo, Italy
2. Institute for Biomedical Research and Innovation (IRIB), National Research Council (CNR), Palermo, Italy

Abstract:

Alzheimer’s disease (AD) is a multifactorial, age-related neurodegenerative disorder classically associated with the formation of senile plaques and neurofibrillary tangles and characterized by chronic oxidative stress and neuroinflammation. Previous studies have shown that Oxotremorine-M (Oxo), a non-selective muscarinic acetylcholine receptor agonist, exerts neurotrophic effects in primary neurons and reduces basal oxidative stress and neuroinflammation in the rat brain. In differentiated SH-SY5Y neuroblastoma cells, Oxo treatment upregulates HSP70 and SOD1 expression, thereby enhancing resistance to oxidative stress–induced cell death. Moreover, in Aβ₁₋₄₂–challenged SH-SY5Y cells, Oxo preserves cell viability and neurite integrity and prevents DNA fragmentation through the attenuation of ROS production, restoration of mitochondrial function, and recovery of SOD activity. On the basis of these findings, the effects of Oxo were investigated in a rat model of AD induced by bilateral intra-hippocampal Aβ₁₋₄₂ injection. In vivo, Oxo markedly reduces oxidative stress by decreasing ROS levels and lipid peroxidation while restoring SOD activity. In addition, Oxo attenuates microglial activation, reduces the expression of pro-inflammatory cytokines, including IL-1β and IL-6, and increases the levels of the anti-inflammatory cytokine IL-10. Finally, Oxo significantly ameliorates functional alterations and rescues Aβ₁₋₄₂–induced cognitive impairment. Altogether, these results indicate that Oxo, by modulating cholinergic neurotransmission, oxidative stress responses, mitochondrial function, and neuroinflammatory pathways, may represent a promising multi-target therapeutic strategy for Alzheimer’s disease.

Authors: Juan C. Cárdenas-Arciniegas (1), Wiebke Maurer (1), Ahmed Wagdi (2), Christof Lenz (3, 4), Tobias Bruegmann (1), and Dörthe M. Katschinski (1)

Affiliations:
(1) Institute of Cardiovascular Physiology, University Medical Center, Georg-August University, Göttingen, Germany
(2) Clinic of Cardiology and Pneumology, University Medical Center, Georg-August University, Göttingen, Germany
(3) Department of Clinical Chemistry, University Medical Center, Georg-August University, Göttingen, Germany
(4) Bioanalytical Mass Spectrometry Group, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany

Abstract:

Introduction Reactive oxygen species (ROS) mediate cellular signaling and stress responses. However, how the main ROS (O2·−and H₂O₂) are compartmentalized and whether they differentially impact cell homeostasis is unclear. We established tools to selectively manipulate mitochondrial O2·− and H₂O₂ in HEK cells and explored their compartmentalization and effect on mitochondrial transcription and nuclear stress signaling. Finally, we explored how mitochondrial and cytoplasmic H₂O₂ sources impact the cysteine proteome of iPSC-cardiomyocytes. Materials and methods HEK-293 cells expressing mitochondrial-matrix targeted SuperNova or D-amino acid oxidase (DAO) were stimulated with light (578/20 nm, 0.39 - 2.5 mW/mm²) or D-alanine (2.5-50 mM) to generate O2·− or H₂O₂ respectively. EPR spectroscopy of mitoTEMPO was performed to assess O2·− production, while MitoSox-Green and HyPer7 were imaged to spatially resolve O2·− and H₂O₂ transients in real time. Mitochondrial transcripts and KEAP1-Nrf2 targets were quantified by RT-qPCR. Resin-assisted thiol-enrichment redox proteomics was performed in iPSC-cardiomyocytes expressing mitochondrial matrix or cytoplasmic DAO. Results SuperNova stimulation quenched mitoTEMPO spectra, indicating specific O2·− production, while MitoSox imaging confirmed a localized and dose-responsive effect. At a 2.5 mW/mm² dose, HyPer7 imaging evidenced modest downstream H₂O₂ production confined to the mitochondrial matrix, closely resembling low-dose (1.5 mM) D-alanine stimulation of DAO. Neither mitochondrial nor KEAP1-Nrf2 transcription was impacted, suggesting physiological “eustress” conditions. In iPSC-CMs, mitochondrial H₂O₂ production by DAO resulted in minimal cysteine oxidation, while cytoplasmic H₂O₂ significantly impacted thiols, including a subset of actin-binding sarcomeric proteins. Discussion Our preliminary findings showcase opto- and chemogenetic tools used in parallel to selectively produce different ROS from different organelles and resolve specific roles in signaling. We achieved ROS levels compatible with physiological signaling conditions and with comparable downstream H₂O₂ production, potentially allowing O2·− specific signaling effects to be isolated. Finally, our initial forays into redox proteomics highlight the relevance of different H₂O₂ sources in iPSC-cardiomyocytes.

Authors: Amelie A. Zimmermann (1,2) , Zhirong Jiang (1), Robert B. Thomson (1), Felix Knauf (3), Peter S. Aronson (1)

Affiliations:
1. Yale School of Medicine, New Haven, CT, United States.
2. Charité - Universitätsmedizin Berlin, Berlin, Germany.
3. Mayo Foundation for Medical Education and Research, Rochester, MN, United States.

Abstract:

Background: Inorganic sulfate deficiency has been implicated in the pathogenesis of musculoskeletal, neurodevelopmental, and metabolic disorders. Slc26a1 is a sulfate transporter that is highly expressed in multiple mammalian tissues of which three have been proposed to play key roles in sulfate homeostasis: sulfate is reabsorbed in the renal proximal tubule, absorbed in the intestine, and taken up by the liver. The relative roles of Slc26a1 expressed in each of these tissues in sulfate homeostasis are not known. Methods: CRISPR-Cas9 technology was used to generate founder mice with LoxP sites flanking critical 5’ exons of Slc26a1. Mice with global, liver-, intestine-, or kidney-specific disruption of Slc26a1 were created by crossing founder mice with β-actin-, albumin-, villin-, or Pax8-cre mice, respectively. Plasma and urine sulfate concentrations were determined by turbidimetry. Tissue expression levels of Slc26a1, Na-sulfate cotransporter Slc13a1 and anion transporter Slc26a3 were evaluated by qPCR. Results: Mice with global- or kidney-specific Slc26a1 deletion had similar 2/3 reductions in plasma sulfate and 3-fold increases in fractional excretion of sulfate (FESO4) relative to respective wild-type or flox/flox littermate controls. In contrast, no difference in either plasma sulfate or FESO4 relative to respective flox/flox controls was observed in mice with liver- or intestine-specific deletion of Slc26a1. Sulfate excretion rate, reflected by urine sulfate/creatinine ratio, was unaffected in all four groups. No compensatory changes were detected in expression of Slc13a1 in kidney and intestine, or of Slc26a3 in intestine of mice with global Slc26a1 deletion. Conclusion: The similarly severe hyposulfatemia with markedly elevated FESO4 observed in mice with global and kidney-specific Slc26a1 deletion strongly suggests that the hyposulfatemia in mice with global Slc26a1 deletion is primarily due to a defect in renal sulfate reabsorption. This conclusion was supported by the lack of detectable effects of Slc26a1 deletion from the liver or intestine on sulfate homeostasis.

Authors: Ammar Ahmedani (1) , Eva Savulkina (1) , Lin Ma (1) , Simang Champramary (1) , Alessia Di Pauli (1) , Elina Valkonen (1) , Samuli Laasanen (1) , Riikka Palimo (1) , Ruben Aju George (1) , Keshav Thapa (1), Tiina Lehtiniemi (1) , Ann-Kristin Dicke (2), Birgit Stallmeyer (2), Frank Tüttelmann (2), Juho-Antti Mäkelä (1) , and Noora Kotaja (1)

Affiliations:
1. Institute of Biomedicine, Research Centre for Integrative Physiology and
Pharmacology, University of Turku, Finland
2. Institute of Reproductive Genetics, Centre of Medical Genetics, University of Münster, Münster, Germany

The author did not consent to the publication of the abstract.

Authors: Cöln M. (1), Maity U. (1), Kiehn S.M. (1), Wagdi A. (1,2), Sathyanarayanan U. (1), Rizzoli S.O. (3), Brügmann T. (1)

Affiliations:
1. Institute of Cardiovascular Physiology, University Medical Center Göttingen, Göttingen, Germany
2. Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany
3. Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany

Abstract:

In cardiomyocytes, the Ca²⁺ dynamics between the cytosol and different intracellular compartments are not entirely understood yet. This is mainly due to technical limitations of state-of-the-art Ca²⁺ indicators. Our multiplexing approach using HaloTag-ed Ca²⁺ indicators combines specific targeting with fast kinetics of chemical Ca²⁺ indicators. Stable monoclonal HEK cell lines with the HaloTag targeted to the cytosol, nucleus, and mitochondria were generated by lipofection, antibiotic selection and single cell dilution. hiPSC-derived cardiomyocytes were transiently transfected with the same constructs. Co-localization analysis was performed with TMRHalo, DAPI, and immunostaining. In HEK cells, ATP-induced transients were measured with Fluo4-AM as the cytosolic Ca²⁺ indicator, while compartment-specific transients were measured with the HaloTag-bound far-red spectrum indicator SiRMaP-Ca²⁺. Diffusion of Ca²⁺ into mitochondria was blocked using 1 µM Ru265, while the efflux was blocked with 10 µM CGP37157. HaloTag was targeted successfully into the cytosol, nucleus and mitochondria in both HEK cells and hiPSC-cardiomyocytes and simultaneous loading with both dyes did not alter Ca²⁺ transients in any of the compartments. We observed that mitochondrial Ca²⁺ had a faster onset (p<0.0001, n=350 cells in 14 ROIs) than nuclear Ca²⁺ (p=0.2810, n=350 cells in 8 ROIs) compared to cytosolic Ca²⁺ (n=400 cells in 12 ROIs). Blocking the influx of Ca²⁺ into mitochondria caused a delay in the onset (p=0.0935; n=300 cells in 15 ROIs), while blocking the efflux significantly prolonged the decay of mitochondrial Ca²⁺ transients (p= 0.0087; n=250 cells in 12 ROIs) compared to untreated cells (n=350 cells in 14 ROIs). The HaloTag-targeted Ca²⁺ indicator SiRMaP-Ca²⁺ can be used to measure compartment-specific Ca²⁺ transients. Its far-red spectrum enables multiplexing to compare the diffusion kinetics distinctively between two compartments. In the future, this approach will be applied to adult cardiomyocytes, providing better insight into Ca²⁺ handling in subcellular compartments and nanodomains under physiological and pathophysiological conditions.

Authors: Roshani Narayan Singh, Marin Juko, Ami Kampshoff, Wolf-Michael Weber, Heymut Omran, Jörg Große-Onnebrink

Affiliations:
Department of General Paediatrics, University Hospital Münster, Albert-Schweitzer-Campus 1, D-48149 Münster, Germany

Abstract:

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-regulated epithelial anion channel that plays a central role in airway transepithelial ion transport, epithelial barrier properties, and mucus hydration. Impaired CFTR function leads to reduced epithelial conductance, altered junctional regulation, and abnormal mucus characteristics. In this study, we investigated CFTR-dependent epithelial physiology using primary human nasal air–liquid interface (ALI) cultures derived from patients with cystic fibrosis (pwCF) and healthy controls. Transepithelial electrophysiological properties were assessed using a modified Multi Transepithelial Current Clamp (MTECC) Ussing chamber, complemented by immunofluorescence analysis of CFTR localisation, epithelial barrier markers, and mucus-related proteins. CF airway cultures exhibited markedly reduced transepithelial conductance and altered epithelial barrier characteristics compared with healthy controls. Restoration of CFTR activity following chitosan-mediated wild-type CFTR (wtCFTR) mRNA therapy resulted in a significant increase in transepithelial conductance (ΔGt), from −0.01135 ± 0.04027 mS/cm² to 0.2566 ± 0.04621 mS/cm² (N = 11 donors, n = 34 inserts), along with recovery of apical CFTR localisation. In parallel, CF cultures showed elevated MUC5AC expression, increased mucus viscosity, and enhanced claudin-1 expression, consistent with altered epithelial tightness and mucus properties. Restoration of CFTR function led to a reduction in mucus viscosity, normalisation of MUC5AC levels, and partial recovery of epithelial barrier integrity. Together, these findings demonstrate a direct functional relationship between CFTR-mediated ion transport, transepithelial conductance, junctional regulation, and mucus properties in human airway epithelia, and highlight the utility of combined electrophysiological and cellular approaches for investigating epithelial homeostasis.

Authors: Abraham Tettey-Matey(1), et al., ... Raul E. Guzman(2*) and Michael Pusch(1*)

Affiliations:
1. Istituto di Biofisica, CNR, Via De Marini 6, 16149 Genova, Italy
2. Institute of Biological Information Processing (IBI-1), Molecular and Cell Physiology, Jülich Research Center, Jülich, Germany
* Correspondence: michael.pusch@ibf.cnr.it (Michael Pusch); r.guzman@fz-juelich.de (Raul E. Guzman)

Abstract:

Endo-lysosomal ClC-3 and ClC-4 transporters, encoded by CLCN3 and CLCN4, respectively, are structurally and functionally related. They form protein dimers to mediate the transmembrane anti-transport of chloride ions (Cl⁻) and protons (H⁺) in endosomes and lysosomes, thereby regulating intracellular biology in health and disease. We recently discovered that the transport activities of ClC-3 and ClC-4 are inhibited by closely related single-span transmembrane proteins, Tmem9A and 9B (Festa et al., 2024), potentially helping reduce their overactivity to prevent CLC-related disorders. Therefore, ClC-3 and -4 variants not inhibited by Tmem9A and 9B are likely associated with CLCN3/4-related disorders. In fact, variants in CLCN3 and CLCN4 are linked to neurodevelopmental disorders with broad phenotypic variability. Over sixty CLCN4 variants have been functionally characterized, showing gain- or loss-of-function (GoF or LoF) effects. While ClC-3 can function as a homodimer, ClC-4 depends on heterodimerization with ClC-3 for efficient endosomal trafficking. CLCN4, located on the X chromosome, exhibits diverse pathogenic outcomes: complete LoF variants often cause non-syndromic presentations in hemizygous males and are asymptomatic in heterozygous females, whereas certain missense variants with partial or complete LoF produce severe syndromic phenotypes in both sexes. In Tettey-Matey et al. 2025, we demonstrate dominant effects of four disease-associated CLCN4 variants within ClC-3/ClC-4 heterodimers using two-electrode voltage-clamp recordings in Xenopus laevis oocytes and whole-cell patch-clamp recordings in mammalian cells co-expressing both proteins via a bicistronic IRES construct. Our findings provide the first evidence of dominant-negative effects of CLCN4 within ClC-3/ClC-4 complexes and establish a platform for functional analysis of novel or additional disease-associated variants. Moreover, we functionally characterized existing and identified novel neurodevelopmental disease-associated loss- and gain-of-function variants in CLCN3 (Tettey-Matey et al., unpublished) and concluded that toxic gain-of-function is emerging as the major pathogenic mechanism in CLCN3- and CLCN4-mediated neurodevelopmental diseases.

Authors: Chaimae Benkerdagh (1), Jonathan Ralph (2), Nathaniel Smith (2), Han Sun (2), Thomas Baukrowitz (1) & Marcus Schewe (1)

Affiliations:
1. Institute of Physiology, Kiel University, Kiel, Germany
2. Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany

Abstract:

Two-pore domain potassium (K2P) channels are widely expressed across tissues and respond to a broad range of physiological stimuli. Consequently, they participate in numerous physiological and pathophysiological processes, including pain, making them attractive therapeutic targets. The K2P family comprises several members with distinct functional, structural, and pharmacological properties. Here, we focus on the TREK subfamily, for which selective pharmacological targeting remains challenging, particularly for TRAAK. In this context, a recent study identified aprepitant as a novel and selective activator of TRAAK based on high-throughput screening and electrophysiological validation. Using a combination of molecular modeling and functional electrophysiological approaches, we investigated the pharmacological action of aprepitant and characterized its selectivity, putative binding site, and gating mechanism. Although aprepitant was previously reported to be selective for TRAAK, our data show that it also activates TREK-2 to a comparable extent, while remaining inactive toward TREK-1. Computational modelling suggest that aprepitant binds within the modulator pocket of TRAAK, at a site analogous to the ML335 binding site in TREK-1. Consistently, electrophysiological analyses identified key residues within the modulator pocket that are critical for aprepitant binding. Together, variations in binding-pocket architecture provide a framework for the rational design of more selective and, consequently, safer therapeutics for pain management.

Authors: Cansu Gülbahar(1), Andrea Jansen-Grabowski(1), Inge van Soest(1), Stefanie Bungert-Plümke(1), Arne Franzen(1), Thomas Gensch(1), Christoph Fahlke(1)

Affiliations:
1.Institute of Biological Information Processing (IBI-1), Molekular- und Zellphysiologie, Forschungszentrum Jülich GmbH, Germany

Abstract:

Introduction: Lysosomes are not only crucial for degrading intracellular and extracellular macromolecules, but they also play significant roles in cell signaling. Lysosomal function relies on a balance of ion gradients, maintained by ion channels and transporters. Recently, SLC26A11 with predominant lysosomal localization was shown to function as H⁺/SO₄^2–:Cl^– exchanger and as Cl^- channel that is gated by this transporter. SLC26A11 was therefore proposed to serve as lysosomal SO₄^2- exchanger, but also as an anion channel. We here test how SLC26A11 affects lysosomal pH and Cl^- homeostasis. Methods: We combined fluorescence lifetime imaging microscopy (FLIM) with chloride-sensitive SPQ dyes to measure lysosomal chloride concentrations in mammalian cells cultured in medium, and perform intensity measurements with fluorescently labelled dextran to quantify lysosomal pH. We modified expression levels of lysosomal channels or transporters using overexpression or shRNA or CRISPR/Cas -mediated knockdown or knockout. Results & Discussion: Under physiological conditions, lysosomes in HEK293T cells contain a high chloride concentration (~150 mM), whereas the cytoplasmic chloride concentration is lower, at ~ 56 mM. Overexpression of SLC26A11 increases pHlyso and reduces [Cl^-]lyso. In contrast, CRISPR/Cas-mediated knock-out of SLC26A11 increases [Cl^-]lyso (to ~ 190 mM). These two results suggest that SLC26A11 anion channels regulate lysosomal [Cl^-] by permitting Cl^- efflux in their open state. ClC-7 is believed to accumulate Cl^- in the lysosome acting as electrogenic 2Cl^-:H⁺ exchanger. To test for possible interactions of these two Cl^- transport proteins, we additionally knocked-down ClC-7 in SLC26A11 KO cells, however, without additional effects on [Cl^-]lyso. Surprisingly, overexpression of ClC-7 in SLC26A11 KO cells reduced the [Cl^-]lyso (to ~ 120 mM). ClC-7 is electrogenic and thus depends on the voltage across the lysosomal membrane. Expression levels of other transporters or channels might thus modify pHlyso by regulating the lysosomal membrane potential.

Authors: Tobias Wang

Affiliations:
Zoophysiology, Aarhus University

Abstract:

Snakes provide a fascinating animal model to study homeostatic regulation during their transition from prolonged fasting to the sudden digestion of very large meals. Ingestion of food leads to rapid restoration of the functional capacity and the mass of the gastrointestinal organs. The very large rise in postprandial metabolism (specific dynamic action), where the rate of oxygen consumption can increase four- to six-fold, is due to a global rise in protein synthesis in all tissues, and the stimulation of tissue turnover involves humoral regulation through a gut-pancreas axis. There are also large increases in cardiac function with higher stroke volume and heart rate, and the gastric acid secretion causes an alkaline tide in blood and tissue.

1. Wang, T. and E. Rindom (2021). The physiological response to digestion in snakes: a feast for the integrative physiologist. Comparative Biochemistry and Physiology 253, 110891.
2. Jensen, B. and T. Wang (2024). The elusive hypertrophy of the python heart. PHYSIOLOGY 39, 88-97.
3. Last, K.B., E. Rindom, I.N. Guagnoni and T. Wang (2024). Proportional increment of oxygen consumption, heart rate and core body temperature in the digesting Python bivittatus. Journal of Experimental Biology 227, jeb248021
4. Rindom, E., K.B. Last, A. Famme, P.G. Henriksen, J. Farup, N. Jessen, F.V. de Paoli and T. Wang (2025). Rapid stimulation of protein synthesis in digesting snakes: Unveiling a novel gut‐pancreas‐muscle axis. Acta Physiologica 241, e700006.

Authors: Nils Pape (1), Annika Klotz (1), Albrecht Schwab (1), Thomas Vogl (2), Oliver Soehnlein (3), Carolina Junho (1), Wolfgang A Linke (1)

Affiliations:
1. Institute of Physiology II, University of Münster, Münster, Germany.
2. Institute of Immunology, University of Münster, Münster, Germany
3. Center for Molecular Biology of Inflammation (ZMBE), University of Münster, Münster, Germany.

Abstract:

Background: Although titin (TTN) has traditionally been recognized for its role in muscle elasticity, emerging evidence from T-lymphocytes indicates additional functions in cytoskeletal organization, cell migration, and resistance to mechanical stress, implicating TTN in broader aspects of immune cell mechanics¹. Here, we investigated the contribution of TTN to neutrophil mechanobiology and functional competence. Methods: We established a neutrophil progenitor line derived from HoxB8 cells of HaloTag-TEV knock-in mice, in which a TEV cassette is inserted into the LTTN2 (leucocyte titin isoform 2) locus, enabling targeted and inducible proteolytic cleavage of TTN by TEV protease (TEVp)². These cells, termed TC cells, received intracellular TEVp via liposomal delivery (LipTEVp). Control groups included wild-type (WT) neutrophils and cells treated with calcein-loaded liposomes (LipCtrl). TTN cleavage efficiency was quantified by flow cytometry 24 h after treatment. Mechanical properties were assessed using AFM-based nanoindentation, complemented by functional assays of chemotaxis and phagocytosis. Results: AFM nanoindentation revealed a significant reduction in cortical stiffness and increased indentation depth in LipTEVp-treated TC cells, whereas WT neutrophils showed no mechanical alterations under any condition. Functionally, TTN-cleaved TC cells exhibited marked reductions in migratory speed, overall displacement, and path length during chemotaxis toward KC chemokine, while directional persistence and chemotactic index remained unchanged. Phagocytic activity was modestly reduced by liposome treatment alone and was further impaired following TTN cleavage in TC cells (~40% reduction relative to untreated controls), an effect not observed in WT cells. Conclusion: Collectively, these findings identify TTN for the first time as a key regulator of neutrophil mechanobiology, controlling cortical stiffness, migratory capacity, and phagocytic efficiency. Our results expand the functional repertoire of TTN beyond striated muscle, establishing it as a critical cytoskeletal scaffold for immune cell performance. Ref: 1 Toffali et al., Cell Rep 42:112516 (2023). 2 Rivas-Pardo et al., Nat Commun 11:2060 (2020).

Authors: Bianco F. (1,2), Savio E. (1), Galvagno F. (4,5), Gilardino A. (1), Puliafito A. (4,5), Barbero N. (3), Prevarskaya N.(6) , Chinigò G. (1), Fiorio Pla A. (1)

Affiliations:
1. Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy.
2. Department of Chemistry, Biology and Biotechnology University of Perugia, Perugia, Italy.
3. Department of Chemistry, University of Turin, Turin, Italy.
4. Department of Oncology, University of Torino, Turin, Italy.
5. Candiolo Cancer Institute, FPO-IRCCS, TO, Candiolo, Italy.
6. INSERM, U1003-PHYCEL-Physiologie Cellulaire, University of Lille, F-59000 Lille, France.

Abstract:

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the deadliest tumors worldwide, due to the characteristics of its tumor microenvironment (TME), which high stiffness promotes hypoxia and pH lowering. Particularly, acidity favour the resistance to gemcitabine-based chemotherapy. To overcome this, alternative therapies are under investigation: among them, Photodynamic Therapy (PDT) low invasiveness, high specificity and tunability makes it a promising candidate. Specifically, it is based on the use of photo-switchable molecules called photosensitizers (PS), that induce ROS-dependent tumor specific cell death. In this context, we characterized Br-SQ-C4 as promising RED-absorbing PS using PDAC cell lines maintained at physiological media (pHe 7.4, CT), and acidic (pHe 6.6, pH selected). Therefore, we performed Ca2+ and ROS imaging and assessed the phototoxicity when irradiated using LEDs emitting at 640nm. Specifically, we demonstrated Br-SQ-C4 ability to induce Ca2+ signals, consisting in the ion release from the ER, which influx into mitochondria further boost ROS, creating a self-maintaining loop, crucial for the PS phototoxicity in both 2D and 3D PDAC models. This ER release promotes the influx of Ca2+ from the extracellular medium, due to the involvement of SOCs (Store-Operated Channels). Among them, Orai1 is the best characterized in PDAC progression and we studied its crucial involvement in the maintenance of ROS and Ca2+ signals triggered by Br-SQ-C4 photoactivation. Precisely, inhibiting Orai1 caused a reduction in both cytosolic ROS and Ca2+ signals in the cell lines, but the ER and Mitochondrial Ca2+ levels are affected differently in pH selected and CT PDAC cells. Overall, the inhibition of Orai1, using Synta66 specific inhibitor, resulted in significantly higher phototoxicity in both cell lines. In conclusion, therapeutic disruption of ORAI1-dependent Ca²⁺ in combination with PDT may overcome microenvironment-driven resistance in PDAC. This work was supported by PRIN 2022 n.20227YTZE3 “AdaPtiviTy”.

Authors: Mangazeev N (1), Kiel A (2), Kralemann-Köhler A (2), Plain Reyes A (1), Kern T (1), Bergmeier M (1), Schulte am Esch (2,3), Lee W (1)

Affiliations:
1. Physiology & Pathophysiology of Cells and Membranes, Medical School OWL, Bielefeld University
2. Department of General and Visceral Surgery, Medical School OWL, Bielefeld University
3. Department of General and Visceral Surgery, Protestant Hospital of Bethel Foundation, University Hospital OWL of the Bielefeld University

Abstract:

Cadmium (Cd) is a toxic heavy metal, which is released by industrial activities, entering the human food chain as well as tobacco plants. After intestinal uptake, Cd is transported via the portal vein to the liver. The current dogma states that Cd is released as Cd-metallothionein (MT) upon hepatocyte lysis. However, no direct evidence exists. In fact, Cd and MT preferably accumulated in hepatic macrophages, known as Kupffer cells (KCs), after Cd(-MT) exposure in vivo. This project investigates uptake pathways of different Cd species, including ionic Cd (CdCl2) or Cd bound to transferrin (Tf), albumin (Alb) or MT, in the immortalized Kupffer cell line KUP5. Compared to hepatocytes, KUP5 were more sensitive to Cd effects (CdMT < CdTf < CdAlb < CdCl2). While ionic Cd caused ~70% cell death (MTT, trypan blue), Tf- and Alb-bound Cd only alter cellular metabolism (MTT) and KC morphology with no significant changes in cell number or trypan blue-positive cells. Live-cell imaging confirmed fluorescent Tf and Alb uptake within 1 h of exposure in KUP5. The endocytic receptor megalin was not detected in primary or immortalized KCs. Candidate receptors for protein-bound Cd include transferrin receptor 1 (TFR1) and the lipocalin-2 receptor SLC22A17, while divalent metal transporter 1 (DMT1) and the zinc transporters ZIP8 and ZIP14 likely mediate ionic Cd uptake. In conclusion, KCs internalize multiple species of Cd, employing cell-surface receptors and metal transporters. Possible fates of Cd handling in the hepatic cell niche will be discussed.

Authors: Savio E (1), Bianco F (1,2), De Toni S (1), Amadei G (1), Belleudi F (3), Chinigò G (1), Fiorio Pla A (1)

Affiliations:
1. Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy.
2. Department of Chemistry, Biology and Biotechnology University of Perugia, Perugia, Italy.
3. Department of Clinical and Molecular Medicine, University of Rome “La Sapienza”, Rome, Italy

Abstract:

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies, with poor prognosis mainly due to its peculiar tumor microenvironment (TME) and resistance to chemotherapy. Consistent with previous evidence, we demonstrated that extracellular acidosis exerts a critical selective pressure within PDAC TME by actively driving tumor cell invasiveness and resistance to gemcitabine, the standard care. This study aims to deepen the role of TME acidosis in enhancing PDAC aggressiveness via Ca²⁺ signaling modulation and to uncover molecular mechanisms allowing PDAC cells to cope with an acidic environment as well as gemcitabine-driven oxidative stress. Specifically, we investigated the Ca²⁺ signaling contribution to acidosis-driven chemoresistance, focusing on the store-operated Ca²⁺ entry (SOCE) channel ORAI1, previously shown to exert a pro-survival role in PDAC cells by impairing gemcitabine-induced apoptosis. Using human PANC-1 cells cultured at physiological (7.4) extracellular pH (pHe) or exposed for one month to acidic pHe (6.6), we observed that ORAI1 is the main contributor of SOCE in both models. Although the absolute SOCE magnitude is comparable between conditions, pHₑ-selected cells exhibit a significantly higher SOCE-to-ER Ca²⁺ release ratio, reflecting a more efficient functional coupling between ER Ca²⁺ depletion and ORAI1 activation. Notably, this optimized Ca²⁺ signaling arises despite the downregulation of ORAI1 mRNA and protein expression in pHₑ-selected cells, suggesting that acidosis selects for a Ca²⁺ signaling state optimized for efficiency rather than channel abundance. Moreover, we found that pharmacological inhibition of ORAI1 with Synta66 significantly reduces FBS-induced intracellular Ca²⁺ oscillations and selectively impairs pHₑ-selected cell viability, without affecting control cells. These findings indicate a rewiring of Ca²⁺ influx pathways and increased dependency on ORAI1-mediated signaling in pHₑ-selected, suggesting a protective role of ORAI1 in supporting survival under acidic stress. Therefore, targeting Ca²⁺ signaling in combination with chemotherapy may represent a promising strategy to overcome therapeutic resistance in PDAC.

Authors: I. Groth (1, 2), B. Nitzsche (2), M. Höpfner (2), M. Schrader (1)

Affiliations:
(1) MSB Medical School Berlin, Hochschule für Gesundheit und Medizin, Berlin, Germany
(2) Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Physiology, Berlin, Germany

Abstract:

Histone deacetylases (HDACs) are epigenetic regulators of chromatin condensation and decondensation and, when dysregulated, HDACs can exert effects on the proliferation and spread of cancer cells. HDACs are overexpressed in prostate cancer (PC) and repress the expression of tumor suppressor genes leading to uncontrolled proliferation and migration. Inhibition of HDACs by specific inhibitors (HDACi) has been shown as an effective treatment strategy in hematologic cancers but less so in solid tumors. Here we investigated the enhanced effectiveness of a newly designed chimeric HDACi Broxcyclobam (BCB). BCB is a conjugate of HDAC-inhibiting hydroxamic acid and a cytoskeleton integrity disrupting pharmacophore. BCB, was tested for its anticancer and antiangiogenic properties in PC cell lines (DU145, PC3 and LNCaP) reflecting different types of castration-resistant and metastatic PCs. The underlying modes of action of BCB were evaluated by cell- and molecular biological methods (crystal violet staining, cytotoxic- and apoptosis assays, WB; ROS-, Scratch-, LDH- and HDAC-Assays). Cytotoxic and antiangiogenic effects were assessed in vivo/ovo by chorioallantoic membrane (CAM) assays of fertilized chicken eggs. First results revealed BCB time- and dose-dependently inhibited PC cell proliferation, with IC50 values of <100 nM (DU145) and ~200 nM (PC3 and LNCAP after 48 h) and provided first indications of apoptosis induction without inducing unspecific cytotoxicity. Moreover, BCB significantly inhibited cell migration of the prostate cancer cells. Preliminary CAM assay data showed antiangiogenic and antitumorigenic effects and morphological alterations of the microvessel structure after treatment. Notably, development and survival of the chicken embryos were not affected by BCB, indicating antiangiogenic efficacy at none/low systemic toxicity. Further in vitro and in vivo investigations will focus on the in-depth characterization of additional underlying modes of action (cell cycle arrests), HDAC specificity and the portion of the cytoskeleton-disrupting pharmacophore on promising anticancer effects of BCB in PC cells.

Authors: Alexandr Melnikov (1), Philip D. Solymosi (1,2), Paul-Lennard Perret (1,3), Szandor Simmons (1,3), Wolfgang M. Kuebler (1,3,4,5,6)

Affiliations:
1 Institute of Physiology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
2 Deutsches Institut für Ernährungsforschung Potsdam-Rehbrücke, Nuthetal, Germany
3 German Center for Cardiovascular Research (DZHK), Partner Site Berlin, Berlin, Germany
4 German Centre for Lung Research, Giessen, Germany
5 Keenan Research Centre, St. Michael´s Hospital, Toronto, ON, Canada
6 Departments of Physiology and Surgery, University of Toronto, Toronto, ON, Canada

Abstract:

Background: The primary cilium (PC) is a hair-like cellular protrusion responsible for mechano- and chemosensation. We recently identified PC loss and shortening in pulmonary vascular cells as both hallmark and driver of the characteristic pathological phenotype in pulmonary arterial hypertension (PAH) in vitro and in vivo. Conversely, PC elongation by lithium alleviated the pro-proliferative and pro-migratory phenotype of pulmonary vascular cells from PAH patients in vitro. Thus, we hypothesized that lithium treatment attenuates pulmonary hypertension in chronic hypoxic mice.
Methods: Male 10-12 weeks old C57BL/6J mice were kept under hypoxia (10% O2) or normoxia for five weeks to develop a PAH phenotype or serve as controls, respectively. For lithium treatment, the mice were fed chow containing 0.2% Li2CO3 for five weeks in the “prophylactic”, or only for the last 2 weeks in the “therapeutic” treatment groups. After 5 weeks, PAH was evaluated by echocardiographic assessment of tricuspid annular plane systolic excursion (TAPSE) and by invasive measurement of right ventricular systolic pressure (RVSP) via right heart catheterization. Pulmonary vessel wall thickness as measure of vascular remodeling was determined in H&E-stained lung sections.
Results: Mice exposed to 5 weeks of hypoxia developed characteristic signs of PAH, including an increase in RVSP, a decreased TAPSE, and histological evidence of pulmonary vascular wall thickening relative to normoxic mice. In animals receiving lithium throughout the 5 weeks of hypoxia, these changes were largely normalized. Lithium treatment initiated in the fourth week was less effective but still resulted in reduced vessel wall thickness, a 20.2% decrease in RVSP and a 39.4% increase in TAPSE relative to hypoxic controls.
Conclusion: Consistent with our previous in vitro findings, these data demonstrate that lithium-mediated restoration of PC length alleviates PAH development and lung vascular remodeling in chronic hypoxic mice, identifying PC elongation as a novel therapeutic strategy for PAH.

Authors: Polona Kovačič (1), Urška Marolt (2), Andraž Stožer (1), Jurij Dolenšek (1,3)

Affiliations:
1 University of Maribor, Faculty of Medicine, Maribor, Slovenia
2 Clinical department for abdominal and general surgery, University Medical Centre Maribor, Maribor, Slovenia
3 University of Maribor, Faculty of Natural Sciences and Mathematics, Maribor, Slovenia

Abstract:

Background and aims: Precisely regulated Ca²⁺ signaling in pancreatic acinar cells is essential for coordinated digestive enzyme secretion, and its disruption is a key early event in pancreatitis. Ethanol (EtOH) is a major risk factor, yet the link between altered Ca²⁺ dynamics and structural tissue injury remains incompletely defined. We aimed to determine how acute vs. prolonged EtOH exposure affects acinar Ca²⁺ signaling and how these changes relate to tissue damage in an ex vivo slice model. Methods: Pancreatic tissue slices (140 µm) were prepared from male NMRI mice (≤12 months) after agarose perfusion via the common bile duct and vibratome sectioning in ice-cold carbogenated extracellular solution. Slices were loaded with 10 µM Calbryte™ 520 AM and imaged during stimulation with 50 nM acetylcholine (ACh). Three protocols were compared: (i) control—ACh for 20 min; (ii) acute EtOH—ACh 10 min followed by ACh + 30 mM EtOH for 10 min; (iii) chronic EtOH—pre-incubation with 30 mM EtOH for 3–7 h, then ACh for 20 min. After imaging, the same slices underwent LIVE/DEAD staining, re-imaging, and FIJI-based necrotic area quantification. Results: Chronic EtOH significantly reduced the active time of Ca²⁺ oscillations compared with both control (p = 0.0027) and acute EtOH (p < 0.0001), indicating sustained Ca²⁺ dysregulation after prolonged exposure. Acute EtOH did not produce a significant overall disruption of oscillatory activity versus control. Chronic EtOH also increased tissue necrosis versus control (p = 0.0444), while differences between control vs acute and acute vs chronic were not significant in this dataset. Conclusions: Exposure duration critically determines EtOH-induced injury: prolonged (chronic) EtOH drives Ca²⁺ dysregulation accompanied by increased necrosis, whereas acute exposure has minimal impact on tissue integrity. These data support Ca²⁺ dysregulation as a mechanistic contributor to alcohol-related pancreatic damage.

Authors: ALEXIA MYRIDAKI

Affiliations:
curently Erasmus Dotoresa in the Embryology Lab the University of Teramo in Italy

Abstract:

Sheep embryonic fibroblasts (SEFs) are widely used in developmental and cellular biology due to their genetic stability. Their chromosomal integrity is essential for downstream applications such as cellular reprogramming, since SEFs can be converted into induced pluripotent stem cells (iPSCs) capable of generating multiple cell types. The aim of this project was to systematically investigate how culture conditions affect cell proliferation , metaphase arrest efficiency ,and chromosomal stability . Cell cultures were conducted under aseptic conditions to maintain cell viability. SEFs were arrested in metaphase by incubation with colcemid for 3 hours at 37°C. A hypotonic solution was then applied to swell the cells and facilitate nuclear isolation, followed by cold fixative treatment; preparations were stored at −20°C. Chromosomes were visualized on glass slides following fixation and Giemsa staining. Our observations demonstrated that superconfluent cultures were unsuitable for karyotyping, as limited space impaired proper cell proliferation and reduced the metaphase arrest efficiency. In contrast, subconfluent cultures produced clearer and more abundant metaphase spreads. Maintaining a stable temperature was also crucial for normal SEF physiology. According to our data an optimal confluency percentage of 70%-80% resulted in efficient yield of metaphase cells.However reports support the presence of chromosomal abnormalities in early‑passage fibroblasts, with approximately 33% showing monosomy 8 and 48.3% disomy 8, while later passages stabilize toward a predominantly normal 46,XY karyotype. A small proportion of early‑passage cells also exhibit metaphases with monosomy 18 (Nikitina et al., 2021). In conclusion, optimizing culture conditions is essential for reliable cytogenetic analysis of SEFs. Ensuring chromosomal stability is particularly important for applications such as reprogramming into iPSCs. These findings contribute to a deeper understanding of SEF physiology and their functional behavior in vitro.

Authors: Tamara Lena Tacey (1), Ben Murphy (1), Dr. Eric Lucking (1), Anthony Marullo (1), Prof. Ken O'Halloran (1)

Affiliations:
1. Department of Physiology, School of Medicine, College of Medicine &amp; Health, University
College Cork, Cork, Ireland.

Abstract:

Structural and functional characterisation comparing D2-mdx mice at 4 and 8 months. Duchenne muscular dystrophy (DMD) is a x-linked genetic disease-causing severe degeneration and weakness of skeletal muscles. It affects 1-in-3500 male births. Most common cause of death is cardio-respiratory failure. Respiratory research is scarce in this area. D2-mdx model was chosen as it is considered a more severe and translatable than the classical BL10 model. The following study aimed to describe the histopathology and the functional characteristics of the diaphragm in the D2-mdx mouse model. DBA/J2 and D2-mdx mice were compared at 4- and 8-months of age. Ex-vivo diaphragm muscle function was assessed. To assess percentage of area of collagen deposition, calcium deposition, percentage of infiltrate and the number of centrally nucleated fibres Sirius red, Alizarin red and Haematoxylin and Eosin was used respectively. Ongoing analysis of immunofluorescent labelling of laminin, type-1, type-IIa, and type-IIx myosin heavy chain isoforms will be used to assess fibre type populations and muscle fibre cross-sectional area. Statistical comparisons were performed via 2-way ANOVA with Sidak pairwise comparisons, using GraphPad Prism (v.10.5.0). The percentage of calcium deposition and % area infiltrate was significantly increased in 4-month D2 mice with P values of (p = 0.0190) and (P = 0.0497) respectively. A significant increase of central nucleation and collagen deposition was seen in D2-mdx mice at both 4- (p < 0.0001) and 8-months (p < 0.0001) respectively. There was no change between 4-to 8-month of age. Compared to DBA/2J mice diaphragm power production was significantly lower in 4- (P < 0.0001) and 8-months-old (P < 0.0001) animals, but no change was seen from 4- to 8-months of age in D2-mdx genotype. Decrease in power production was seen parallel with and increase in collagen deposition of D2-mdx mice, there was no change between 4- and 8-month.

Authors: Shobik R (1), van der Veen R (1), Bieck M (1), Sezer I (1), Haucke V (1, 2), Lehmann M (1)

Affiliations:
1. Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin
2. Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin

Abstract:

Tight junctions (TJs) are important in establishing barriers that are either impermeable or selectively permeable to water, ions or small molecules. While selective permeability of TJs depends on Claudin proteins (Cldns), the function of another tetraspan membrane protein – Occludin (Ocln) – remains unclear. Ocln has two extracellular loops (ECLs) and a mutation in ECL2 has been linked to brain calcification in humans. Complete knockout of Ocln in mice results in a similar phenotype. Cldns seal the intercellular space through the formation of complex polymerized structures (i.e. strands), and Ocln has been shown to contribute to the complexity of these strands, thereby modulating the strength of the paracellular epithelial barrier. In this research, we investigate how Occludin, and especially its ECLs, supports the Cldn-based TJ barrier. Using stimulated emission depletion (STED) microscopy, we demonstrate that Ocln attaches to Cldn strands in vitro and in vivo. We confirm that presence of aromatic amino acids in first and second ECL mediate Claudin-Occludin interaction through liquid-liquid phase separation. Moreover, replacement of ECLs with fragment from an evolutionary unrelated but structurally similar protein preserves the colocalization of chimeric Ocln with Cldn2. The functional importance of the ECLs was tested in epithelial MDCKII Ocln KO cells through reconstitution of WT, mutant and chimeric protein and ion permeability measurements. In this study, we describe how Ocln incorporates into Cldn polymers, and identify a role of liquid-liquid phase separation of ECLs in barrier formation.

Authors: Landstorfer H (1), Koch PA (1), Meçi X (1), Kirschner KM (1), Scholz H (1)

Affiliations:
1. Institute of Translational Physiology, Charité – Universitätsmedizin Berlin

Abstract:

Obesity represents a major global health burden and is strongly associated with metabolic disorders including type 2 diabetes and cardiovascular disease. In particular, expansion of visceral white adipose tissue (WAT) correlates with increased morbidity and mortality, whereas browning of WAT is considered metabolically beneficial. The transcription factor Wilms’ tumor 1 (WT1) is expressed in progenitor cells of visceral WAT and has been implicated in maintaining a white adipocyte cell fate, as heterozygous Wt1 knockout mice display browning of visceral WAT accompanied by improved glucose tolerance and reduced hepatic steatosis [1]. To further elucidate the role of WT1, we generated mice with a visceral fat–specific deletion of Wt1 (Wt1fl/fl; Pdgfrα-Cre). Unexpectedly, conditional knockout mice developed obesity characterized by enlarged visceral and subcutaneous fat depots, hepatic steatosis, and hypertrophy of white adipocytes. Bulk RNA sequencing of primary preadipocytes following Wt1 silencing identified Wnt4 and Cyp26a1 as candidate WT1 target genes. Consistently, retroviral overexpression of Wnt4 in 3T3-L1 cells markedly impaired adipogenic differentiation in vitro, indicating a functional role of Wnt4 in suppressing adipogenesis. In parallel, bioinformatic analysis of publicly available single-nucleus RNA sequencing data (GSE176171) revealed heterogeneity among preadipocyte populations in visceral WAT. One of four preadipocyte clusters was enriched for gene signatures associated with adipogenesis and angiogenesis, whereas WT1 expression was confined to mesothelial cells and a distinct preadipocyte subpopulation characterized by reduced adipogenic potential. Collectively, these findings suggest that WT1 marks and regulates a specialized preadipocyte subtype that limits adipogenic differentiation in visceral adipose tissue. Integrating in vivo genetic models with single-cell transcriptomic and in vitro approaches provides a powerful framework to dissect transcriptional networks governing adipose tissue homeostasis. 1. Kirschner, K.M., et al., Wt1 haploinsufficiency induces browning of epididymal fat and alleviates metabolic dysfunction in mice on high-fat diet. Diabetologia, 2022. 65(3): p. 528-540.

Authors: Vivienne Schneider, Joachim Fandrey, Nora Koll & Sandra Winning

Affiliations:
Institute of Physiology, University of Duisburg-Essen

Abstract:

Urinary bladder carcinoma (UBC) is associated with i) chronic inflammation and ii) a drop of oxygen in the tumor microenvironment. Thus, the hypoxia-inducible factor 1 (HIF-1) is a potential prognostic marker for poor outcome in UBC holding an important role in the urothelium. Ninety percent of UBC arise from urothelial cells, which can be categorized into basal, intermediate, and umbrella cells. In this project, urinary bladder organoids will be used to analyze the underlying signaling pathways of UBC formation and the additional role of urinary tract infections. Urinary bladder organoids were generated under normoxic conditions and characterized under Roxadustat stimulation using microscopy and RNA analysis for their cellular composition and HIF target gene expression. This revealed the expression of typical urothelial cell markers. Further, we plan to infect the organoids with uropathogenic E. coli and treat them with carcinogens to analyze critical aspects of UBC formation. The resulting data will help to deepen our understanding of the relationship between HIF, urinary tract infections and UBC.

Authors: Cong Xie (1), Henriette Dähnhardt (1), Carina Wagner (2), Sarah Dittrich (1), Qing Wu (1), Madita Vahrenbrink (1), Sylvia J. Wowro (1), Achim Lass (2), Michael Schupp (1)

Affiliations:
(1) Charité Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Pharmacology, Cardiovascular Metabolic Renal (CMR)- Research Center, Berlin, Germany.
(2) Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria.

The author did not consent to the publication of the abstract.

Authors: Gimeno-Ferrer F (1), Matthes F(1,2,3) & Meissner A (1,2,3)

Affiliations:
1 Division of Physiology & Vascular Biology, Institute of Theoretical Medicine, Faculty of Medicine, University of Augsburg, 86159 Augsburg, Germany

2 Department of Experimental Medical Science, Faculty of Medicine, Lund University, 221 84 Lund, Sweden

3 Wallenberg Center for Molecular Medicine, Faculty of Medicine, Lund University, 221 84 Lund, Sweden

Abstract:

Current research increasingly emphasizes the development and application of in vitro models for the investigation of complex physiological processes. Studying the blood-brain barrier (BBB) in particular requires physiologically relevant systems that enable controlled assessment of how different triggers affect BBB integrity, resistance, and activation, as well as their relevance to diseases such as stroke. Here, we present an in vitro pipeline for the generation of a transwell-based BBB model, combined with multiple complementary techniques to assess key parameters of brain vasculature. The BBB model was established using transwell inserts with sequential culture of primary mouse astrocytes and cerebral endothelial cells (bEnd.3) in the basal and apical compartments, respectively, enabling physiologically relevant co-culture interactions. Monocultures of primary astrocytes and bEnd.3 cells, maintained under identical conditions and time-courses, served as controls. Barrier integrity was assessed by trans-endothelial electrical resistance (TEER) measurements and permeability assays using fluorescent tracers (FITC-Dextran), while molecular alterations were analyzed by flow cytometry and qPCR. BBB co-cultures exhibited higher TEER values and reduced permeability compared with endothelial monocultures, reflected by reduced apical-to-basal FITC-Dextran flux. At the molecular level, co-culture conditions shifted both cell types away from a monoculture-associated stress phenotype towards a more developmentally regulated, BBB-like state. In endothelial cells, BBB co-culture led to a significant downregulation of Tjp1, Cdh5, Pecam1, and Selplg genes, consistent with astrocyte-induced endothelial maturation. Conversely, astrocytes in co-culture displayed decreased expression of Gfap, Sox9 and S100b, alongside increases Vim expression, indicating reduced astrocytic reactivity and morphological adaptation towards a structural support phenotype. This BBB model demonstrates that astrocyte–endothelial interaction drive maturation towards a BBB-like state and provides a versatile platform to investigate how diverse triggers affect BBB integrity at both functional and molecular levels.

Authors: Dittrich S (1), Wu Q (1), Dähnhardt H (1), Xie C (1), Wowro SJ (1), Schupp M (1)

Affiliations:
(1) Charité Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Pharmacology, Max Rubner Center (MRC) for Cardiovascular Metabolic Renal Research, Berlin, Germany

Abstract:

Background: Retinol Saturase (RetSat), an endoplasmic reticulum-localized oxidoreductase, is highly expressed in in all major types of adipose tissue. We reported previously that adipose RetSat expression is induced via β-adrenergic (β-AR) signalling and that its deletion in brown adipose tissue impairs adaptations to an acute cold challenge. Brown and white adipocytes lacking RetSat showed lower lipolytic activity upon β-AR stimulation. Aims: We asked whether the depletion of RetSat in adipocytes affects protein kinase A (PKA) and the phosphorylation of downstream targets involved in lipolysis. Since PKA activation also promotes transcriptional activity of cAMP responsive element binding protein (CREB), we tested whether RetSat depletion alters the expression of CREB target genes. Methods: Mature 3T3-L1 adipocytes were electroporated with siRNA to deplete RetSat expression. 72 hours later, adipocytes were exposed to the pan-β-agonist isoproterenol or the adenylate cycle activator forskolin for 4 hours. Expression of RetSat and proteins involved in lipolysis was analysed by qPCR and immunoblotting. Results: Phosphorylation of PKA substrates upon β-AR stimulation was strongly reduced in adipocytes depleted of RetSat. This was mirrored by lower phosphorylation of hormone sensitive lipase, a major lipase in adipocytes and known target of PKA. Similar effects were observed in adipocytes stimulated with forskolin or a synthetic analogue of cAMP, suggesting that defective PKA activation due to RetSat depletion is downstream of β-AR receptors. Furthermore, CREB target genes such as Pgc1a and Cebpb were downregulated in adipocytes depleted of RetSat. Conclusions: We found that RetSat is required for PKA/CREB signalling, which likely explains defective lipolysis in RetSat-depleted adipocytes. Targeting RetSat may carry therapeutic potential to correct dysregulated lipolysis.

Authors: Christina Buchberger (1), Petra Kameritsch (2), Hanna Mannell (1) and Kristin Pogoda (1)

Affiliations:
1. Physiology, Institute of Theoretical Medicine, University of Augsburg, Augsburg, Germany.
2. Walter Brendel Center of Experimental Medicine, University Hospital, LMU Munich, Munich, Germany

Abstract:

Question: Endothelial progenitor cells (EPC) support neovascularization and endothelial repair and show a strong angiogenic potential. EPC specifically express connexin (Cx) 43. By forming gap junctions (GJ), Cx allow the direct exchange of ions and small molecules between adjacent cells. Here, we studied the interaction of EPC with EC and the role of GJ in the formation of capillary-like structures in vitro. Methods: EPC and EC (HUVEC, PAEC, HMEC) were co-cultured and the angiogenic network formation was studied in long-term co-cultures or on Geltrex. Cx43 localisation was assessed by immunofluorescence stainings (IF). The gap junctional coupling (GJC) was investigated by dye-injection studies and FACS analysis. GJC was inhibited pharmacologically using different blockers as heptanol combined with meclofenamic acid (GJB) or carbenoxolone (CBX). Cell viability was tested after treatment with GJ inhibitors to rule out potential non-specific toxic effects. Results: Angiogenic networks were observed in co-cultures after 3-6 days on uncoated dishes (n=4-7). In contrast, monocultures only formed angiogenic networks on an angiogenic basement matrix (Geltrex). FACS analysis and dye-injection studies demonstrated a time-dependent GJC of EC in co-culture with EPC (n=4, p<0.01). IF stainings confirmed a localisation of Cx43 at contact sites between EC and EPC. The angiogenic network formation was significantly reduced by inhibition of GJC in EC/EPC co-cultures (n=4-7, p<0.05 vs. control) without affecting the cell viability. Conclusions: Our results suggest that the direct interaction via GJ involving Cx43 in co-cultures is necessary for the spontaneous formation of angiogenic networks. Therefore, Cx43 is a potential target for regulating angiogenesis and exploring new cell-based treatment approaches in clinical therapy.

Authors: F. Zantow (1), H. Scholz (1), K. M. Kirschner (1)

Affiliations:
1. Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Translational Physiology, Berlin, Germany

The author did not consent to the publication of the abstract.

Authors: Caihong Li (1), Lasti Erfinanda (1,2), Wolfgang M. Kuebler (1-3)

Affiliations:
1. Institute of Physiology, Charité – Universitätsmedizin Berlin, 10117 Berlin, Germany
2. German Center for Lung Research (DZL), associated partner site, 10117 Berlin, Germany
3. German Centre for Cardiovascular Research (DZHK), partner site, 10117 Berlin, Germany

Abstract:

Rationale: Pneumonia is a leading cause of death worldwide. Recently, we showed that loss of endothelial CFTR drives lung barrier failure in Streptococcus pneumoniae infection, while the potentiator ivacaftor prevents CFTR downregulation and edema (Erfinanda et al., Sci Transl Med 2022). This study investigates the mechanism by which ivacaftor stabilizes CFTR, focusing on the ubiquitination/deubiquitination pathway. Methods: Human PMECs and murine precision-cut lung slices (PCLS) were pretreated with ivacaftor (10 μmol/L) and stimulated with pneumolysin (PLY, 0.25 μg/mL). MG132 or bafilomycin A1 (BafA1) were used to block proteasomal or lysosomal degradation. E3 ubiquitin ligases and USP10 were analyzed by qPCR, Western blot, and deubiquitinase (DUB) activity assays. USP10 function was assessed using spautin-1, siUSP10, and cycloheximide (CHX) chase. Results: PLY induced K48- and K63-linked CFTR polyubiquitination in human PMECs and increased total protein ubiquitination. Prevention of PLY-induced CFTR loss by MG132 or BafA1 suggests degradation via both proteasomal and lysosomal pathways. Ivacaftor stabilized CFTR in PLY-stimulated human PMECs and PCLS, reduced CFTR polyubiquitination, and decreased total protein ubiquitination. Mechanistically, PLY upregulated E3 ligases HRD1 and RFFL while downregulating the DUB USP10. Ivacaftor increased general DUB and USP10-specific activity, restoring the expression of CFTR and other USP10 targets. USP10 inhibition abolished the protective effects of ivacaftor, as ivacaftor no longer stabilized CFTR in CHX chase or reduced PLY-induced CFTR polyubiquitination in the presence of spautin-1. Finally, ivacaftor rescued the PLY-induced reduction of USP13, which cooperates with USP10 in CFTR regulation. Conclusion: PLY causes endothelial CFTR degradation via ubiquitination. Ivacaftor counteracts this by activating the DUBs USP10 and USP13, thereby stabilizing CFTR and normalizing the deubiquitination machinery. Project funding: WMK is supported by the German Research Foundation (SFB 1449, project ID 431232613, sub-project B01; SFB 1470, project ID 437531118, sub-project A04; operational grants KU1218/11-2, KU1218/12-1, KU1218/14-1) and the DZHK (projects 81Z0100214, 81X2100294).

Authors: Nastja Murko (1), Jasmina Kerčmar (1), Lidija Križančić-Bombek (1), Eva Paradiž-Leitgeb (1), Sandra Postić (2), Johannes Pfabe (2), Ya-Chi Huang (2), Andraž Stožer (1), Dean Korošak (1), Xaver Kozisek (4), Monika Perisic (5), Markus Muttenthaler (5,6), Christian W. Gruber (4), Marjan Slak Rupnik (1,2)

Affiliations:
(1) University of Maribor, Faculty of Medicine, Institute for Physiology, Maribor, Slovenia
(2) Institute of Physiology, Center for Physiology and Pharmacology, Medical University of Vienna, Austria
(3) The University of Montreal Hospital Research Centre, Montreal, Canada
(4) Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Austria
(5) Institute of Biological Chemistry, Faculty of Chemistry, University of Vienna, Austria
(6) Institute for Molecular Biosciences, The University of Queensland, Brisbane, Australia

Abstract:

The role of arginine vasopressin (AVP) in the regulation of pancreatic α and β cell function has been controversial. We investigated the effects of AVP and its receptor-specific agonists and antagonists on α and β cells using pancreatic tissue slices from C57BL/6J mice. Slices were imaged by confocal microscopy and stimulated with 8 mM glucose in the presence of AVP or AVP receptor agonists/antagonists together with cAMP-modulating agents. Cytosolic Ca²⁺ events were recorded and analyzed as described previously (1). AVP exerted glucose-dependent effects on both α and β cells. At low glucose concentrations, AVP selectively activated α cells without significantly affecting β cells. At stimulatory glucose concentrations, AVP enhanced activity in both cell types, leading to increased intracellular Ca²⁺ signaling. Depletion of cAMP levels in β cells by epinephrine suppressed AVP-induced responses, indicating a permissive role of AVP. AVP exhibited a bell-shaped concentration dependence, with lower concentrations enhancing and higher concentrations diminishing cellular responses, consistent with known activation and inactivation properties of IP₃ receptors. Notably, increased α-cell activity observed at higher AVP concentrations could coincided with reduced δ-cell activity, suggesting that enhanced α-cell responses may arise, at least in part, from diminished paracrine inhibition by δ cells rather than from direct α-cell stimulation alone. Pharmacological profiling and RNAscope further indicated that AVP acts predominantly via V1b receptors. Together, these findings indicate that AVP modulates pancreatic α and β cell activity in a glucose- and cAMP-dependent manner and that its effects on α cells may involve indirect regulation through δ-cell–mediated paracrine interactions. This highlights AVP as an important modulator of intra-islet communication and glucose-dependent hormone secretion. (1) Postic S, Sarikas S, Pfabe J, Pohorec V, Krizancic Bombek L, Sluga N, et al. High-resolution analysis of the cytosolic Ca(2+) events in beta cell collectives in situ. Am J Physiol Endocrinol Metab. 2023;324(1):E42-E55.

Authors: García-Dávila S (1), de la Cruz-Morcillo MA (2), Cifuentes C (1), Carmona M (2), LLorens S (1)

Affiliations:
1. School of Medicine of Albacete, Biomedicine Institute UCLM (IB-UCLM), Instituto de Investigación Sanitaria de Castilla-La Mancha (IDISCAM), Universidad de Castilla-La Mancha, Albacete, Spain

2. Institute for Regional Development (IDR), Universidad de Castilla-La Mancha, Albacete, Spain.

Abstract:

Introduction: Guayule (Parthenium argentatum) resin contains little-explored bioactive compounds, including guayulins. Their influence on adipocyte biology, key in obesity-related dysfunction, remains unknown. Our aim is to evaluate the effects of guayulins B and D (GB, GD; 20 µM) on the differentiation and adipokine secretion profile of 3T3-L1 cells. Material and Methods: 3T3-L1 preadipocytes were differentiated with the standard induction cocktail, DMI, (dexamethasone, methylisobutylxanthine, insulin) in the presence or absence of GB or GD. Upon maturation (Day 8), cytotoxicity was assessed by MTT assay and intracellular lipid accumulation by Oil Red O (ORO) staining. The secretion profile of 17 adipokines was analyzed using a proteome profiler array. Results were normalized to the DMI control (100%). Results: Neither guayulin affected cell viability. ORO staining normalized to cell viability (ORO/MTT) revealed that GB reduced intracellular lipid content, while GD increased it. Notably, both compounds significantly induced the secretion of Pref-1 (DLK1), an inhibitor of adipogenesis, which was undetectable in control cells. Furthermore, GB and GD potently and significantly suppressed the pro-inflammatory chemokine MCP-1 (to ~62% and ~58% of control, respectively). In contrast, GD uniquely and markedly increased the secretion of the pro-inflammatory adipokine Resistin (to ~169%), while GB caused only a mild increase. Conclusions: Guayulins B and D are specific modulators of adipocyte biology. Both induce Pref-1 and suppress MCP-1. However, they exert opposing effects on lipid accumulation and inflammatory signaling, with GD promoting a lipogenic and potentially pro-inflammatory state (high Resistin). GB emerges as the compound with a more favorable profile, reducing lipid content while minimally affecting Resistin. These findings revalue guayule resin as a source of precision tools for studying adipose tissue biology.

Authors: Sophia Schwarz (1), Tina Lehrich (1), Mia Lorenzen (1), Maria Reichenbach (2), Anaclet Ngezahayo (1, 3)

Affiliations:
1. Institute of Cell Biology and Biophysics, Department of Cell Physiology and Biophysics, Leibniz University Hannover, Hannover, Germany
2. Symrise AG, Scent & Care Global Innovation – Cosmetic Ingredients, Holzminden, Germany
3. Center for Systems Neuroscience (ZNS), University of Veterinary Medicine Hannover Foundation, Hannover, Germany

Abstract:

Connexins (Cx) are transmembrane proteins comprising 21 isoforms known in humans. They oligomerise into hexameric hemichannels (connexons) which are permeable to ions and small metabolites (< 1.5 kDa) including nucleotides such as ATP, thereby contributing to autocrine and paracrine signalling. Two connexons of adjacent cells may dock together to form gap junction channels, which facilitate gap junctional intercellular communication (GJIC). This mechanism stresses the important role played by Cxs for the maintenance of tissue homeostasis and physiological as well as pathophysiological processes including the innate immune response. The skin epithelium represents a barrier against microbial or environmental threats. Keratinocytes bind pathogen-associated molecular patterns (PAMPs), including bacterial lipopolysaccharide (LPS) or peptidoglycan (PGN), via their toll-like receptors (TLRs) and activate inflammatory cascades, culminating in the release of damage-associated molecular patterns (DAMPs) such as adenosine. Since it was found that inflammatory conditions favour Cx hemichannel activity, research started aiming to understand the mechanistic relationship between Cx hemichannels and inflammation. The immortalised HaCaT keratinocyte cell line was used to analyse the expression of Cx isoforms and to assess Cx hemichannel activity and GJIC. Treatment with the specific TLR agonists LPS and Pam₃CSK₄ resulted in a significant increase in Cx hemichannel activity, accompanied by a marked reduction in GJIC. While the expression of relevant TLRs and Cx isoforms was confirmed in HaCaT cells, the overall Cx expression pattern remained largely unchanged following the treatment with either agonist. Together, these results indicate the functional relevance of Cxs in keratinocyte-mediated inflammatory responses and provide a basis for future studies investigating the contribution of chronically hyperactive Cx hemichannels to inflammatory skin diseases and pathologies such as Keratitis-Ichthyosis-Deafness (KID)-syndrome.

Authors: Tina Lehrich (1), Sophia Schwarz (1), Ilka Anja Kröber (1), Katharina Hoffmann (2), Maria Reichenbach (2), Christos C. Zouboulis (3), Anaclet Ngezahayo (1,4)

Affiliations:
1. Institute of Cell Biology and Biophysics, Department of Cell Physiology and Biophysics, Leibniz University Hannover, Hannover, Germany
2 .Symrise AG, Scent & Care Global Innovation – Cosmetic Ingredients, Holzminden, Germany
3. Departments of Dermatology, Venerology, Allergology and Immunology, Dessau Medical Center, Brandenburg Medical School Theodor Fontane and Faculty of Health Sciences Brandenburg, Dessau, Germany
4. Center for Systems Neuroscience (ZNS), University of Veterinary Medicine Hannover Foundation, Hanover, Germany

Abstract:

Connexins (Cx) are transmembrane proteins with 21 known isoforms in humans. They oligomerize to a hexameric hemichannel, the connexon. These hemichannels are permeable to ions and small metabolite (<1.5 kDa) including nucleotides such as ATP, therefore contributing to autocrine and paracrine signaling. When two connexons of adjacent cells dock together, they form a gap junction channel, which facilitates cell-cell-communication. A balanced regulation between activity of Cx hemichannel activity a gap junction coupling is essential for tissue homeostasis and normal physiological functions. Especially in the skin, dysregulation of Cx channels has been linked to severe pathologies. Hyperpermeable Cx26 hemichannels as observed in Keratitis-Ichthyosis-Deafness (KID)-syndrome correlate with disruption of epidermal structure and barrier function, while Cx30 hemichannel hyperpermeability, which is associated with the Clousten-Syndrome, leads to enlarged sebaceous glands in mice. The sebaceous gland is an important structure within the epidermis, producing sebum to protect the skin from desiccation and environmental insults. Sebum secretion depends on sebocytes differentiation, lipid accumulation and subsequent release by holocrine secretion. Despite the clinical relevance, little is known about the role of Cx in sebaceous gland biology and sebocyte differentiation. To address this, Cx expression and function were analysed in immortalized SZ95 sebocytes and primary human sebocytes (pSEBs). Undifferentiated SZ95 cells express several Cx isoforms and exhibited strong hemichannel activity but lacked gap junctional intercellular communication (GJIC). In contrast, pSEBs displayed a distinct Cx expression pattern, showed functional GJIC but almost no hemichannel activity. Differentiation was induced by linoleic acid (LA) and changes in Cx expression and function were investigated. The results indicate that Cx channel composition and function differ between sebocyte models and differentiation states and provide a basis for future research investigating the role of Cxs in sebaceous gland development and physiology.

Authors: Lennart Evers (1), Johannes Riebeling (1,2), David Zipf (1), Markus Vogt (1), Lena-Christin Conradi (2), Michael Ghadimi (2), Robert Patejdl (3), Tobias Bruegmann (1)

Affiliations:
1. Institute for Cardiovascular Physiology, University Medical Center Göttingen, Göttingen, Germany

2. Clinic for General, Visceral and Pediatric surgery, University Medical Center Göttingen, Germany,

3. Department of Medicine, Health and Medical University Erfurt, Erfurt, Germany

Abstract:

Gastroparesis is characterized by severely impaired gastric emptying without mechanical obstruction and is most commonly caused by dysfunction of the enteric nervous system (ENS) and interstitial cells of Cajal (ICC). Typical symptoms include early satiety, postprandial fullness, nausea, vomiting, bloating, and epigastric pain. Currently, no treatment reliably restores gastric emptying. We demonstrate direct optogenetic stimulation of murine gastric smooth muscle cells (SMCs) expressing Neuropsin (hOPN5) or Channelrhodopsin 2 (ChR2). In transgenic ChR2-eYFP and OPN5-eYFP mouse models, illumination of SMC strips with blue light (470 nm) or UV light (385 nm) induced force and intragastric pressure. Comparative analysis revealed that light effects propagated from positive to negative cells only in ChR2 strips, whereas OPN5 responses were restricted to OPN5-positive cells. Force generation in hOPN5 SMCs depended critically on all-trans retinal (ATR) supplementation. To identify electrical stimulation paramters selectively stimulation the ENS and not the SMC, SMC strips from healthy wild-type mice were stimulated by electrical field stimulation pre and post tetrodotoxin-induced neural blockade at varying electrical energies and frequencies. At 10 V and 100 Hz reptition rate, we did not see any response after TTX incubation but found a reproducible force induced before which was 3.56±0.47 bigger than spontaneous contractions (N=5; n=12, p=<0.0001). Human SMC strips, retrieved intraoperatively from patients undergoing gastrectomy for stomach cancer, showed substantially higher variability indicative for an injured ENS in the cases in which ENS stimulation was not as efficient. In conclusion, two optogenetic approaches effectively induce gastric contractions in transgenic mice and we found electrical stimulation parameters selectively stimulating the ENS allowing to screen for ENS dysfunction.

Authors: Janek Haus (1), Julia Schewe (1), Aurelian Falk (1), Emily Kim (2), Eric Blanc (1), Dieter Beule (1), Matthias Wuttke (2), Anna Köttgen (2), Ute Scholl (1)

Affiliations:
1. Berlin Institute of Health at Charité, Charité Universitätsklinikum Berlin
2. Institute of Genetic Epidemiology, Universitätsklinikum Freiburg

Abstract:

Objectives Primary aldosteronism (PA) is one of the main causes of secondary hypertension. Almost all cases occur sporadically and are caused by aldosterone producing adenomas or bilateral aldosteronism. In rare cases, PA is inherited as a Mendelian disorder (familial hyperaldosteronism, FH-I-IV). The aim of this study is to identify and characterize new primary aldosteronism disease genes and investigate their effects on aldosterone production. Methods We performed exome sequencing of families without mutations in known disease genes and computational analysis of the UK Biobank cohort to identify phenotypic associations of candidate disease genes. For functional studies, we used an aldosterone producing adrenocortical cell lines (H295R, Hac15), which we transfected with the wild type and mutant cDNA as well as with a knock-down shRNA. We analyzed the effect on CYP11B2 (encoding aldosterone synthase) expression using quantitative real-time PCR and RNA-Seq to gain more insight into the underlying regulatory pathways. We investigate the effect of the mutations on the resulting catalytic activity in functional assays (substrate digestion). Results In two families without mutations in known disease genes, we identified heterozygous mutations in a new gene previously not associated with FH or hypertension; one of these occurred de novo. These mutations are located in close proximity to each other and at a highly conserved region that is involved in the catalytic activity of the resulting protein. Among UK Biobank probands, rare variants in the disease gene were associated with secondary hypertension and hyperaldosteronism, and a lead candidate variant was identified. The gene product shows nuclear expression throughout the adrenal gland, including the zona glomerulosa, where aldosterone is produced. The gene of interest apparently down-regulates aldosterone synthase expression, and mutations found in patients with familial hyperaldosteronism lead to knock-down-like CYB11B2 expression in qPCR and potentially altered enzymatic activity in the gene product.

Authors: Maruša Plesnik Rošer (1), Nika Polšak (1), Jasmina Jakopiček (1), Jurij Dolenšek (1), Andraž Stožer (1), Maša Skelin Klemen (1)

Affiliations:
1. Institute of Physiology, Faculty of Medicine, University of Maribor, Slovenia

Abstract:

Pancreatic tissue slices constitute a ex vivo approach for investigating pancreatic function and disease while maintaining the native cellular organization and microenvironment. Owing to their close anatomical and physiological resemblance to humans, porcine pancreatic tissue represents a highly relevant experimental model. This preparation enables functional assessment of multiple pancreatic cell types, such as endocrine β cells and exocrine acinar cells, under conditions that mimic in vivo physiology. Nevertheless, the use of tissue slices derived from porcine pancreas has not yet been widely characterized. The objective of this study was to optimize the preparation of viable porcine pancreatic tissue slices and to evaluate their applicability for functional calcium imaging and immunohistochemical characterization. Fresh porcine pancreatic tissue was collected and sectioned into slices using a vibratome. Tissue viability was assessed by live/dead staining. Intracellular calcium dynamics in β cells and acinar cells were monitored using calcium-sensitive fluorescent indicators and fluorescence microscopy. Immunohistochemical labeling was performed to visualize and distinguish specific pancreatic cell populations. Porcine pancreatic tissue slices with preserved morphology and cellular viability were successfully obtained. Calcium imaging demonstrated functional responses in endocrine and exocrine compartments. β cells displayed glucose-induced calcium activity, whereas acinar cells responded to acetylcholine stimulation. Immunohistochemical analyses enabled identification of distinct pancreatic cell types, corroborating the structural and functional integrity of the slices. In conclusion, porcine pancreatic tissue slices represent a physiologically relevant ex vivo model for studying pancreatic function. The combination of real-time calcium imaging and immunohistochemistry offers a powerful platform for future investigations into pancreatic physiology, diabetes, and other pancreatic disorders. Marciniak, A., et al., Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology. Nature protocols, 2014. 9(12): p. 2809-2822. Andraž, S., et al., Confocal laser scanning microscopy of calcium dynamics in acute mouse pancreatic tissue slices. 2021.

Authors: Sabinari I (1), Horakova O (1), Karkucinska-Wieckowska A (2), Kleinova V (1), Pakula B (3), Jakubek P (3), Wieckowski M R (3), Rossmeisl M (1)

Affiliations:
1. Laboratory of Adipose Tissue Biology, Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 14200 Prague, Czech Republic

2. Department of Pathology, The Children's Memorial Health Institute, Warsaw, Poland

3. Laboratory of Mitochondrial Biology and Metabolism, Nencki Institute of Experimental Biology of Polish Academy of Sciences, Warsaw, Poland

Abstract:

Introduction and Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the liver manifestation of obesity and metabolic syndrome. Steatosis can progress to steatohepatitis, which increases the risk of end-stage liver disease. N-3 polyunsaturated fatty acids (omega-3 PUFA) may be an effective nutritional strategy for reducing liver fat, and possibly also in the prevention of advanced stages of MASLD. However, further research is needed to understand the effects of different chemical forms of omega-3 PUFA supplementation on MASLD progression. Materials and methods: Male C57BL/6J mice (n=6-8) were fed for 20 weeks on a Western-type diet (WD; 21% milk fat, 40.5% sucrose, 1.25% cholesterol) or WD supplemented with omega-3 PUFA (EPA+DHA content ~18.2 mg/g diet) in the form of triacylglycerols, ethyl esters or phospholipids (krill oil) always combined with a sweet drink. Mice fed a standard diet (SD) were used as controls. Body composition, liver fat content, gene expression and histopathological changes were analysed. Additional animals from each group (n=4-5) were dissected to isolate primary hepatocytes for ex vivo mitochondrial respiration measurements. Results: Compared to SD-fed mice, WD promoted weight gain and visceral adiposity while lean body mass was reduced. WD-induced obesity was accompanied by hepatic steatosis and liver injury associated with inflammatory changes and tissue remodeling, as determined by histopathological analysis of the liver. In addition, WD administration was associated with impaired mitochondrial COX activity in primary hepatocytes, which was partially restored by omega-3 PUFA. Importantly, compared to other lipid forms of omega-3 PUFA supplementation, krill oil phospholipids were more effective in reducing liver weight and steatosis, and in suppressing inflammatory responses and tissue remodeling. Conclusion: Omega-3 PUFA, especially when supplemented as phospholipids from krill oil, are able to partially prevent MASLD progression in WD-fed obese mice. Funding: Czech Science Foundation (23-04100L) and National Science Centre (UMO-2021/43/I/NZ3/00510).

Authors: Qing Wu (1), Sarah Dittrich (1), Yueming Meng (1), Cong Xie (1), Henriette Dähnhardt (1), Madita Vahrenbrink (1), Sylvia J. Wowro (1), Michael Schupp (1)

Affiliations:
(1) Charité Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Pharmacology, Cardiovascular Metabolic Renal (CMR)- Research Center, Berlin, Germany

The author did not consent to the publication of the abstract.

Authors: Pashyna A (1,2), Knauf F (2,3), Najenson AC (2)

Affiliations:
1. Freie Universität Berlin, Department of Biology, Chemistry, Pharmacy, Institute of Biology, Berlin, Germany
2. Charité – Universitätsmedizin Berlin, Department of Nephrology and Internal Intensive Care Medicine, Berlin, Germany
3. Mayo Clinic, Division of Nephrology and Hypertension, Rochester, MN, USA

Abstract:

Background: Calcium oxalate (CaOx) crystals are the main mineral component of kidney stones, and their deposition in renal tissue is a hallmark of oxalate nephropathy. Macrophages play an important role in crystal clearance by internalizing and dissolving CaOx crystals, yet the molecular mechanisms governing this process remain incompletely understood. While epithelial oxalate transport is well characterized, the contribution of immune cells and oxalate transporters has received less attention. This study aimed to establish and validate a robust in vitro workflow for quantifying CaOx crystal uptake, retention, and clearance in macrophages, providing the basis for future functional studies of the anion exchanger SLC26A6. Methods: Murine bone marrow-derived macrophages (BMDMs) were exposed to CaOx crystals and analysed using fluorescence and polarization microscopy. Key methodological parameters were systematically optimized, including washing efficiency, fixation method, timing of staining, coating conditions, CaOx concentration, and incubation time. Results: A stringent washing step was identified as essential to prevent continued uptake of surface-bound crystals and ensure accurate quantification. Fixation with 4% paraformaldehyde combined with immediate staining provided optimal crystal preservation, whereas methanol fixation led to crystal loss during storage. Fibronectin coating did not significantly affect crystal uptake or retention. Dose-response experiments identified 100 µg/mL CaOx as the optimal concentration, and a consistent reduction in intracellular crystal burden was observed after 48 hours, indicating active macrophage-mediated clearance. Conclusions: Using the optimized protocol, macrophages were shown to efficiently internalize and process CaOx crystals, confirming their suitability as an in vitro model for crystal clearance. This validated workflow will enable future comparative experiments using wild-type and SLC26A6-deficient macrophages to directly assess the contribution of SLC26A6 to intracellular crystal dissolution and oxalate release. The established approach provides a reliable foundation for mechanistic studies of macrophage-mediated oxalate handling and may be extended to human macrophage systems.

Authors: Najenson AC (1), Wagner T (2), Thomas C (1), Pashyna A (1), Bachmann S (3), Thomson RB (4), Knauf F (1,5), Aronson PS (4)

Affiliations:
1. Department of Nephrology and Medical Intensive Care, Charité - Universitätsmedizin Berlin, Germany.
2. NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany
3. Institute of Functional Anatomy, Charité - Universitätsmedizin Berlin, Germany.
4. Department of Internal Medicine, Section of Nephrology, Yale University School of Medicine, New Haven, Connecticut, USA.
5. Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN, USA

Abstract:

Background: Macrophages are crucial for recovery from oxalate-induced nephropathy. In vivo and in vitro studies showed that they can engulf and dissolve calcium oxalate crystals, yet the mechanisms mediating crystal clearance by macrophages remain unknown. We previously showed that macrophages express the SLC26A6 transporter, which functions as a Cl-oxalate exchanger. Our studies indicated that under steady-state conditions, SLC26A6 mediates net oxalate efflux and prevents intracellular oxalate accumulation in macrophages. Here, we analyzed the role of SLC26A6 in mediating the release of soluble oxalate from macrophages following the internalization of calcium oxalate crystals. Methods: SLC26A6 expression in primary murine and human macrophages was confirmed via qPCR and immunoblotting. Functional studies using radioactively labeled oxalate were conducted in wild-type (WT) and SLC26A6-deficient (Slc26a6-/-) macrophages. To assess crystal clearance, cells were exposed to calcium oxalate crystals for 48 hours. Internalization was analyzed using transmission electron microscopy (TEM), and soluble oxalate release was quantified using enzymatic assays. Results: Primary macrophages robustly expressed functional SLC26A6. Under steady-state conditions, Slc26a6-/- macrophages exhibited higher oxalate accumulation than WT controls, confirming SLC26A6 mediates net efflux. TEM analysis showed both genotypes internalized crystals, with intracellular retention decreasing over time. Importantly, after crystal internalization, Slc26a6-/- macrophages showed a significantly reduced ability to release soluble oxalate compared to WT cells. Conclusion: We conclude that the oxalate transporter SLC26A6 mediates oxalate efflux from macrophages. Moreover, it mediates the release of soluble oxalate from macrophages after the internalization of oxalate crystals. Macrophage SLC26A6 may thereby play a critical role in the clearance of oxalate crystals from tissues.

Authors: Das S (1), Schermuly I.I (1), Romanet S (1), Amasheh S (1), Zentek J (2) & Aschenbach J.R (1)

Affiliations:
1. Institute of Veterinary Physiology, Freie Universität Berlin
2. Institute for Animal Nutrition, Freie Universität Berlin

Abstract:

Dietary fiber is the indigestible fraction of plant-derived feed and a major component of pig diets. It can be classified as fermentable or non-fermentable based on its susceptibility to microbial fermentation in the gastrointestinal tract. Fiber alters the characteristics of digesta and through microbial fermentation, generates bioactive metabolites that influence intestinal physiology and nutrient transport. This study aimed to investigate the effect of dietary fiber source (fermentable & non-fermentable), fiber particle size and weaning age on the mRNA expression of nutrient transport proteins in the piglet jejunum. Sixteen German Landrace sows and their 64 piglets were assigned to four dietary treatments containing either non-fermentable (hay) or fermentable (sugar beet pulp; SBP) fiber in fine or coarse particle sizes. Piglets were weaned at either 28 or 42 days of age. Two weeks post weaning, middle jejunal tissues were collected to quantify the mRNA expression of glucose transporters (SLC5A1, SLC2A2, SLC2A5) amino acid transporters (SLC6A20, SLC3A1, SLC6A19, SLC1A5, SLC6A14, SLC7A9) and dipeptide transporter SLC15A1 using qRT-PCR. Data were compared by three-way ANOVA (weaning age, fiber type and particle size), followed by Student-Newman-Keul’s post-hoc tests. Late weaning significantly increased the expression of glucose transporters SGLT1 (SLC5A1) (P = 0.001), GLUT2 (SLC2A2) (P = 0.002) and GLUT5 (SLC2A5) (P < 0.001) compared to early weaning. Similarly, amino acid transporters B0AT1 (SLC6A19) (P = 0.008) and rBAT (SLC3A1) (P = 0.015) as well as dipeptide transporter PEPT1 (SLC15A1) (P < 0.001) were significantly upregulated in late weaned piglets. A significant fiber effect (P = 0.027) was observed for amino acid transporter ASCT2 (SLC1A5) expression, being higher in piglets fed SBP compared with Hay. This study can conclude that late weaning significantly enhances the gene expression of several intestinal nutrient transporters, while fermentable fiber from SBP may further support intestinal nutrient transport capacity in piglets.

Authors: Bernhardt E (1), Stölting G (1)

Affiliations:
(1) Center of Genomic Medicine, Berlin Institute of Health at Charité - Universitätsmedizin Berlin

Abstract:

Calcium signaling represents a central regulator of aldosterone production in adrenal zona glomerulosa cells. Under physiological conditions, aldosterone synthesis is stimulated by factors such as angiotensin II or elevated extracellular potassium, which increase intracellular calcium levels via receptor-mediated signaling pathways. However, it remains unclear whether calcium influx alone is sufficient to induce aldosterone related gene expression independently of upstream signaling pathways. The aim of this study was to investigate the impact of optogenetically induced calcium influx through CapChR2 on aldosterone-related gene expression and to establish experimental conditions suitable for reliable long-term stimulation. HAC15 cells were genetically modified using a PiggyBac Tet-On system to achieve stable expression of the light-activated calcium channel CapChR2. Calcium dynamics were monitored using the genetically encoded calcium indicator PinkyCaMP and compared with the chemical indicator Fura-2-AM, while different light sources and stimulation protocols were evaluated to determine optimal experimental settings. Both microscope-based illumination and external LED stimulation induced reproducible calcium responses. Based on these observations, a standardized LED-based stimulation protocol was established for long-term experiments. Gene expression analysis following two and four hours of optogenetic stimulation revealed strong induction of the immediate early genes NR4A1, NR4A2, EGR1, and FOS after two hours, accompanied by an increase in CYP11B2 expression. After four hours, CYP11B2 expression increased strongly, while immediate early gene expression declined, indicating a temporal transition from early transcriptional responses toward sustained activation of the aldosterone synthase gene CYP11B2. Overall, these findings demonstrate that optogenetically induced calcium influx alone is sufficient to activate aldosterone-relevant signaling pathways and drive time-dependent regulation of gene expression. The established model provides a controlled experimental platform for investigating calcium-dependent mechanisms underlying aldosterone production.

Authors: Cara J Gebauer (1), Frederike Butz (2), Ute I Scholl(1), Gabriel Stölting (1)

Affiliations:
(1) Center of Genomic Medicine, Berlin Institute of Health at Charité - Universitätsmedizin Berlin
(2) Chirurgische Klinik, CCM, Charité - Universitätsmedizin Berlin

Abstract:

The adrenal zona glomerulosa (ZG) regulates aldosterone production via a calcium-dependent steroidogenic pathway. In rodent models, stimulation with angiotensin II and/or elevated extracellular potassium evokes electrical activity that recruits voltage-gated calcium entry and results in intracellular calcium oscillations. Primary aldosteronism, a frequent cause of hypertension, is commonly driven by gain-of-function mutations in ion channel genes that increase calcium signaling and promote (semi-)autonomous aldosterone production. Important species-specific differences limit direct translation from murine models to human physiology. Notably, KCNJ5, the most frequently mutated ion channel gene in human aldosterone-producing adenomas, is absent in the murine ZG. To better define human physiology, we studied calcium dynamics in fresh human adrenal tissue obtained from adrenalectomies performed for diverse clinical indications. Human ZG cells exhibited angiotensin II–evoked intracellular calcium fluctuations, with oscillation patterns and frequencies that differed from those described for mice. Current work focuses on establishing a protocol for recording and characterizing these calcium signals. We want to establish a functional framework for human ZG physiology and investigate the contribution of voltage-gated calcium channels in human ZG cells. Unlike in mice, the adult human ZG is not a continuous layer but occurs in sparse nodular regions across the outer adrenal cortex. We assessed calcium activity in CYP11B2 (aldosterone synthase) expressing ZG nodules. Relating calcium activity to CYP11B2 expression and the somatic genotype of these nodules is relevant for distinguishing early stages of primary aldosteronism from healthy adrenal states and for interpreting functional heterogeneity across clinical backgrounds. Together, this work aims to refine physiological models of adrenal calcium signaling by anchoring them in human tissue, providing a basis for future mechanistic and disease-oriented studies.

Authors: Maity U. (1), Cöln M. (1), Kiehn S.M. (1), Wagdi A. (1,2), Sathyanarayanan U. (1), Rizzoli S.O. (3), Brügmann T. (1)

Affiliations:
1. Institute of Cardiovascular Physiology, University Medical Center Göttingen, Göttingen, Germany
2. Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany
3. Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany

Abstract:

In cardiomyocytes, the Ca²⁺ dynamics between the cytosol and different intracellular compartments are not entirely understood yet. This is mainly due to technical limitations of state-of-the-art Ca²⁺ indicators. Our multiplexing approach using HaloTag-ed Ca²⁺ indicators combines specific targeting with fast kinetics of chemical Ca²⁺ indicators. Stable monoclonal HEK cell lines with the HaloTag targeted to the cytosol, nucleus, and mitochondria were generated by lipofection, antibiotic selection and single cell dilution. hiPSC-derived cardiomyocytes were transiently transfected with the same constructs. Co-localization analysis was performed with TMRHalo, DAPI, and immunostaining. In HEK cells, ATP-induced transients were measured with Fluo4-AM as the cytosolic Ca²⁺ indicator, while compartment-specific transients were measured with the HaloTag-bound far-red spectrum indicator SiRMaP-Ca²⁺. Diffusion of Ca²⁺ into mitochondria was blocked using 1 µM Ru265, while the efflux was blocked with 10 µM CGP37157. HaloTag was targeted successfully into the cytosol, nucleus and mitochondria in both HEK cells and hiPSC-cardiomyocytes and simultaneous loading with both dyes did not alter Ca²⁺ transients in any of the compartments. We observed that mitochondrial Ca²⁺ had a faster onset (p<0.0001, n=350 cells in 14 ROIs) than nuclear Ca²⁺ (p=0.2810, n=350 cells in 8 ROIs) compared to cytosolic Ca²⁺ (n=400 cells in 12 ROIs). Blocking the influx of Ca²⁺ into mitochondria caused a delay in the onset (p=0.0935; n=300 cells in 15 ROIs), while blocking the efflux significantly prolonged the decay of mitochondrial Ca²⁺ transients (p= 0.0087; n=250 cells in 12 ROIs) compared to untreated cells (n=350 cells in 14 ROIs). The HaloTag-targeted Ca²⁺ indicator SiRMaP-Ca²⁺ can be used to measure compartment-specific Ca²⁺ transients. Its far-red spectrum enables multiplexing to compare the diffusion kinetics distinctively between two compartments. In the future, this approach will be applied to adult cardiomyocytes, providing better insight into Ca²⁺ handling in subcellular compartments and nanodomains under physiological and pathophysiological conditions.

Authors: Loveline M (1), Schmiderson A (2)

Affiliations:
National School of Nurses of Port-au-Prince (1),
Saint Francis of Assisi University of Haiti (2)

Abstract:

Physiology, the science of life in action, unfolds as a field of inquiry where experimental heritage meets the horizons of innovation. As a foundational discipline of biomedical sciences, it no longer confines itself to the descriptive study of organic functions, but now asserts itself as an integrative matrix, capable of linking molecular dynamics to systemic regulations and adaptive behaviors. Recent methodological developments bear witness to this transformation. Functional multimodal imaging, bioinformatics applied to physiological networks, and the use of organoids as translational models constitute milestones toward a predictive physiology. These tools not only refine the understanding of neuronal plasticity, endocrine regulation, and cardiovascular adaptation, but also anticipate the trajectories of human health in the face of environmental and societal constraints. Beyond technical sophistication, physiology proves indispensable for addressing contemporary challenges: endocrine disruptors, oxidative stress, and metabolic imbalances tied to modern lifestyles. By situating its research within the perspective of personalized and sustainable medicine, it combines experimental rigor with social responsibility. The discipline thus becomes a mediator between fundamental knowledge and clinical applications, between laboratory and society. This dynamic is accompanied by a fertile interdisciplinary dialogue. Biologists, clinicians, engineers, and philosophers intersect their approaches, enriching theoretical paradigms and refining methodologies. Far from being an isolated science, physiology emerges as a space of convergence where the boundaries of the living and the conditions of its intelligibility are redefined. Thus, contemporary physiology presents itself as both heir and innovator: heir to a rigorous experimental tradition, innovator through its capacity to integrate emerging technologies and respond to ethical and societal challenges. It embodies a science in motion, carrying a renewed vision of biomedical research and its role in shaping a more enlightened and sustainable future. Physiology merges tradition and innovation, shaping biomedical futures through dialogue, ethics, creativity, resilience, and deeper understanding of life’s complexity.

Authors: Hanna Goenawan (1), Putri Teesa Radhiyanti (1), Ingrid Naomi Adelia Panjaitan (2), Ronny Lesmana (1), Yuni Susanti Pratiwi (1)

Affiliations:
1. Physiology Division, Department Biomedical Sciences -- Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia
2. Anti Aging and Aesthetic Medicine Department -- Faculty of Medicine, Universitas Padjadjaran, Bandung Indonesia

The author did not consent to the publication of the abstract.

Authors: Cristina Baio (1), Diego Pastene (1, 2), Simon Wiegert (1), Andrey Formozov (1)

Affiliations:
(1): Department of Neurophysiology, Medical Faculty Mannheim, Mannheim Centre for Translational Neuroscience, Heidelberg University, Mannheim, Germany

(2): Core Facility for Live Cell Imaging (CF-LIMa), Medical Faculty Mannheim of Heidelberg University, Mannheim, Germany.

Abstract:

How living organisms store and retrieve information about the external world is one of the central questions in neuroscience. In existing theories and associated models of memory formation and recall, the physiological parameters of a biological network of interest – such as neuronal connectivity - play a fundamental role, determining overall memory capacity. Thus, the experimental determination of these parameters is of great importance. As the number of connections between neurons scales approximately as the square of the number of neurons, even for relatively small networks, their characterization requires substantial effort. In this regard, the use of high-throughput techniques is essential. A classical approach to the electrophysiological characterization of connectivity is patch-clamp whole-cell recordings with multiple pipettes, capable of stimulating presynaptic neurons and recording excitatory and inhibitory synaptic potentials in postsynaptic partners. However, this process requires physical attachment of the pipette to the cell, making it time-consuming, labor-intensive, and prone to failure. Optogenetics is a powerful technique that uses genetically encoded light-sensitive proteins, called opsins, to selectively manipulate neuronal activity using targeted illumination. It offers an alternative approach to stimulate presynaptic neurons for connectivity characterization, and combining optogenetics with whole-cell recordings increases the overall throughput of the process. In my work, I investigated synaptic connectivity in hippocampal organotypic slice cultures, combining temporally and spatially precise two-photon holographic optogenetic stimulation with classical whole-cell patch-clamp recordings. This approach provides a powerful platform for dissecting hippocampal circuits in vitro and for further investigating the fundamental questions of their operation.

Authors: Anna Aurora Taddei (1), Laura Bellingacci (1,2), Valentina Imperatore (1), Alessandro Tozzi (2), Lucilla Parnetti (3,4), Andrea Mancini (3), Miriam Sciaccaluga (5), Cinzia Costa (1,3)

Affiliations:
1. Laboratory of Experimental Neurology, Department of Medicine and Surgery, University of Perugia, Perugia, Italy
2. Section of Physiology and Biochemistry, Department of Medicine and Surgery, University of Perugia, Perugia, Italy
3. S.M. della Misericordia Hospital, Section of Neurology, Department of Medicine and Surgery, Perugia, Italy
4. Section of Neurology, Department of Medicine and Surgery, University of Perugia, Perugia, Italy
5. Department of Life Science, Health, and Health Professions, Link Campus University, Rome, Italy

Abstract:

Background: Lafora Disease (LD) is a rare genetic form of progressive myoclonus epilepsy caused by mutations in the Emp2a or Emp2b genes, which encode laforin and malin, respectively. These two proteins are involved in glycogen metabolism, and their dysregulation leads to the accumulation of insoluble forms of glycogen, known as Lafora bodies (LBs), in the brain and peripheral tissues. The exact mechanism is still not fully elucidated, but these aggregates drive epilepsy, neuroinflammation, and neurodegeneration. Several mouse models of Lafora disease have been developed, and extensive biochemical and molecular characterizations have been performed. However, electrophysiological investigations remain limited, representing a critical gap in the understanding of disease mechanisms. Aim. The present work aimed to provide a comprehensive electrophysiological characterization of four Lafora disease mouse models: knock-out (KO) models lacking laforin (Emp2a-/-) or malin (Emp2b-/-); and knock-in (KI) models carrying the most frequent patient-derived mutations, Emp2aR240X and Emp2bD148N. Methods: Ex vivo electrophysiological recordings were performed from hippocampal slices. Epileptic-like activity was induced using a pro-epileptogenic artificial solution, and synaptic plasticity was assessed by eliciting long-term potentiation (LTP) through a high-frequency stimulation protocol. Results: Epileptic-like activity was similarly elevated in Emp2a-/- and Emp2aR240X animals, while Emp2b-/- and Emp2bD148N animals showed an intermediate level of hyperexcitability, higher than wild-type (WT) but lower than Emp2a-/- and Emp2aR240X. Interestingly, both KI models showed aberrant hyperplasticity (h-LTP) while KO models showed either unaltered (Emp2b-/-) or a reduced LTP (Emp2a-/-). Conclusions: Distinct Emp2a and Emp2b genotypes differentially affect hippocampal network excitability and synaptic plasticity. Among them, the KI mice carrying patient-derived mutations exhibit the most severe and disease-relevant phenotype. These findings indicate that KI models as an optimal tool for investigating Lafora disease pathophysiology.

Authors: Fesseler L (1), Heinz V (1,2), Pilz N (3), Bothe TL (4)

Affiliations:
1. Institute of Physiology, Charité – Universitätsmedizin Berlin
2. Institute of Integrative Neuroanatomie, Charité - Universitätsmedizin Berlin
3. Department of Cardiology and Angiology, Hannover Medical School
4. Sydney School of Health Sciences, Faculty of Medicine and Health, University of Sydney

Abstract:

Ultra-endurance cycling offers a natural laboratory for studying physiological responses under sustained extreme load. Continuous in-race monitoring is rarely reported. The aim of this study was to investigate the feasibility of a multimodal framework of physiological parameters including metabolic, cardiovascular, and muscle-mechanical patterns during an ultra-endurance event. Approach: This study stress-tests a multimodal framework of physiological parameters of a 58-year-old male athlete during the Race Across America (RAAM) 2024, covering 4,933 km in 11 days from Oceanside, California, to Atlantic City, New Jersey. Parameters included energy expenditure, continuous blood glucose levels, heart rate, power output, passive muscle stiffness and resting tone, as well as sleep times. Main Results: The multimodal monitoring toolkit proved feasible and provided continuous, physiological measurements throughout the RAAM, enabling the observation of the following physiological changes: The athlete lost 2.3 kg of total weight and had an estimated energy deficit of 21,169 kcal. Blood glucose levels decreased over the course of the RAAM (0.92 mg/dl/d, p < 0.001), with an increased time spent below 100 mg/dl (p < 0.001). Heart rate during cycling progressively decreased, stabilising at a plateau of 94 bpm. Power output-to-heart rate ratio initially dropped until day 7 before peaking on day 11. Mean passive muscle stiffness and resting tone increased during the race compared to baseline levels, with distinct response patterns observed between two leg muscles and one lower back muscle. The total sleep deficit was 65 hours during the RAAM. Significance: Continuous, multimodal in-race physiological monitoring during the RAAM proved feasible and operationally useful, enabling real-time adjustments to pacing, fuelling and recovery. This framework offers a field-deployable template for ultra-endurance events. Future research should focus on larger, multi-participant studies and long-term follow-up to characterise the physiological responses to extreme endurance.

Authors: Abdou Jammeh

Affiliations:
Ministry of Health, Banjul, The Gambia

Abstract:

Cardiovascular diseases (CVDs) remain a leading cause of morbidity and mortality globally, with a rapidly increasing burden in low- and middle-income countries. Hypertension, a major modifiable risk factor for cardiovascular disease, often develops silently and is influenced by complex interactions between genetic, physiological, and environmental factors. This abstract explores the physiological mechanisms of cardiovascular regulation and the early risk factors contributing to hypertension, with particular relevance to low-resource settings such as The Gambia. Normal cardiovascular homeostasis is maintained through coordinated regulation of cardiac output, vascular resistance, autonomic nervous system activity, and renal function. Disruption of these mechanisms—through endothelial dysfunction, increased sympathetic activity, impaired baroreceptor sensitivity, and altered renin–angiotensin–aldosterone system activity—can lead to sustained elevation of blood pressure. In low-resource environments, these physiological disturbances are often exacerbated by lifestyle transitions, including reduced physical activity, increased salt intake, urbanization, psychosocial stress, and limited access to preventive healthcare. Early identification of cardiovascular risk factors such as prehypertension, obesity, physical inactivity, and metabolic abnormalities is critical for preventing disease progression. However, challenges including limited screening programs, low public awareness, and inadequate integration of physiological knowledge into primary healthcare systems hinder early intervention efforts. This presentation emphasizes the importance of applying core principles of cardiovascular physiology to inform early detection, prevention strategies, and public health interventions. Strengthening physiologically informed health education, promoting lifestyle modification, and improving primary healthcare screening can significantly reduce the long-term burden of hypertension and cardiovascular disease. Understanding cardiovascular regulation within the local context is essential for developing sustainable, cost-effective prevention strategies in low-resource settings.

Authors: Angelica Magni (1), Jana Frahm (2), Sven Dänicke (2), Korinna Huber (1), Helga Sauerwein (3), Jana Seifert (1), Franziska Dengler (1)

Affiliations:
1. Institute of Animal Science, University of Hohenheim, Stuttgart, Germany;
2. Institute of Animal Nutrition, Federal Research Institute for Animal Health, Braunschweig, Germany;
3. Institute of Animal Science, Physiology Unit, University of Bonn, Bonn, Germany

Abstract:

To provide dairy cows with the large amount of energy required for lactation, feeding high-energy diets has become a universal strategy. However, this approach interferes with the physiological function of the gastrointestinal system with a consequential accumulation of glucose and short-chain fatty acids that might ultimately result in digestive disorders. Previous studies demonstrate an alteration in the microbial composition due to exposure to high-energy diets with the production of different (microbial) metabolites such as biogenic amines (BA). Trace amine-associated receptors (TAARs) are a class of receptors capable of binding BA and whose activation might contribute to the animal’s adaptation to dietary shifts. Therefore, we aimed to elucidate an involvement of TAARs in the crosstalk between the microbiota and the host epithelium in the bovine gastrointestinal tract. 20 primiparous lactating Holstein Frisian cows were transitioned from a ratio that either met (low energy, LE) or exceeded (high energy, HE) their energy needs to the respective other feeding group. Six weeks after changing the diet, the animals were slaughtered and epithelial samples from the ventral sac of the rumen and jejunum were collected. Gene expression analysis of TAAR1, 2, 3, 4 and 9 as well as immunofluorescent staining and Western blot analysis of TAAR1 were performed. TAARs are present in both tissues with TAAR1 being located in the apical membrane of the jejunum epithelium and intracellularly in the basal layers of the rumen epithelium. Feeding the HE diet tended to decrease the mRNA expression levels for TAAR4 and protein abundance of TAAR1 compared to the LE diet in the jejunum epithelium, but otherwise we observed no differences. However, sensing of BA and regulation of transepithelial transport mechanisms might not only involve an upregulation of TAARs, but rather intracellular signalling pathways downstream of it that remain to be elucidated in future studies.

Authors: Lampkemeyer M (1), Spahiu F (1), Hagemann M (1), Ottlik M (1), Helbig LC (1), Stöhr EJ (1,2)

Affiliations:
1. COR-HELIX, Institute of Sports Science, Leibniz University Hannover
2. Division of Cardiology, Columbia University Irving Medical Center, New York, New York, USA

Abstract:

Background: Ventilatory thresholds (VT1/VT2) are widely used to describe cardiometabolic transitions, yet they offer limited insight into how cardiac reserve is utilised as intensity rises. Because VT1/VT2 guide exercise prescription and CPET interpretation, mechanistic context is clinically and practically relevant. Here, we aligned individual cardiac ceiling points in LV volumes and deformation with VT1 and VT2 on a common %HR_peak axis. Methods: Eighteen healthy recreationally active adults completed individualized step-wise incremental supine cycling (40%W_peak, +10%W_peak every 3 min to volitional exhaustion). Exercise echocardiography quantified LV end-diastolic volume (EDV), stroke volume (SV), ejection fraction (EF), longitudinal strain (LS), apical circumferential strain (ACS) and LV twist. Breath-by-breath gas exchange determined VT1 (V-slope), VT2 (ventilatory equivalents and end-tidal CO₂ behaviour; visual assessment by two investigators), and VO₂peak. Individual ceiling points were identified via polynomial regression versus %HR_peak and summarized as group-level mean values. Results: Group-level mean ceiling points suggested a volume-to-deformation sequence across intensities. Volumetric ceilings occurred first (EDV 65 and SV 70%HR_peak), followed by deformation ceilings (LS 71 and ACS 78%HR_peak), with EF and LV twist peaking later (both 87%HR_peak). VT1 occurred at 68%HR_peak, close to the SV ceiling; at the individual level, VT1 correlated moderately with the EF ceiling (ρ=0.55, p=0.0375). VT2 occurred at 91%HR_peak and strongly correlated with the ACS ceiling (ρ=0.86, p=0.0002). Inter-individual heterogeneity was substantial, with some deformation ceilings absent. Conclusion: Aligning cardiac ceiling points with ventilatory thresholds reveals a structured shift from volumetric reserve to deformation-based support as exercise intensity rises, potentially reflecting progressive constraints on diastolic filling and preload recruitment at higher heart rates. VT1 clustered around early volumetric ceilings, whereas VT2 tracked apical mechanical ceilings. This framework anchors VT1/VT2 in cardiac reserve domains (volume-to-deformation), thereby providing clinically and practically relevant mechanistic context for VT-based CPET interpretation and exercise prescription.

Authors: Hamidi Wiam (1), El Hangouche Abdelkader Jalil (1,2)

Affiliations:
1. Departement of Physiology, Faculty of Medicine and Pharmacy of Tangier, Abdelmalek Essaâdi University, Morocco
2. University of New England, Tangier, Morocco

The author did not consent to the publication of the abstract.

Authors: Taiek Nora (1), El Hangouche Abdelkader Jalil (1,2)

Affiliations:
1. Department of Physiology, Faculty of Medicine and Pharmacy of Tangier, Abdelmalek Essaâdi University, Morocco

2. University of New England, Tangier, Morocco

The author did not consent to the publication of the abstract.

Authors: Emílio-Silva MT (1), Solon IG (1), Trajano IP (1), Passaglia P (1), Donadeli MFS (1), Costa LHA (1), Salvador SLS (2), Branco LGS (1)

Affiliations:
1. Department of Basic and Oral Biology, Dental School of Ribeirão Preto, University of São
Paulo, Avenida Do Café s/n, Ribeirão Preto, SP, Brazil
2. Department of Clinical Analyses, Toxicology and Food Science, School of Pharmaceutical Sciences Ribeirão Preto, University of São Paulo, Avenida Do Café s/n, Ribeirão Preto, SP, Brazil

Abstract:

Periodontitis is a chronic inflammatory disease with both local and systemic effects. Tissue damage affecting the oral cavity contributes to systemic implications, particularly in other parts of the gastrointestinal tract, which can lead to an imbalance in immune homeostasis. Citral, a monoterpene present in essential oils, exhibits important pharmacological properties, such as anti-inflammatory and immunomodulatory properties. Thus, our aim was examined the chronic preventive effect of oral treatment with citral during Porphyromonas gingivalis-induced systemic inflammation in C57BL-6J mice against oral, intestinal and systemic alterations. Male mice (n = 10 per group) were administered P. gingivalis (10⁹ UFC/mL, gavage route) or 2% carboxymethylcellulose (vehicle; 200 µL per animal) to induce periodontitis for 12 days, concomitantly with treatment with citral (25, 100, and 300 mg/kg; gavage route) or vehicle (1% Tween 80 at 10 mL/kg) for 42 days. After euthanasia, alveolar bone loss (cementoenamel junction-bone crest distance), colon morphometric, intestinal permeability (FITC-Dextran assay) and plasma cytokines levels (IL-1β and IL-10) were assessed (protocol #2025.1.123.58.3). Mice exposed to P. gingivalis showed greater bone loss and intestinal permeability than controls (p<0.001), characterizing the periodontitis model. Treatment with citral (25 and 100 mg/kg) prevented gingival and intestinal damage, bone loss (p<0.05 and p<0.0001, respectively), and increased intestinal permeability (p<0.05) compared to the periodontal control group. Consequently, it prevented the systemic increase in IL-1β, regardless of IL-10 levels, indicating the anti-inflammatory effect of the monoterpene. Citral (100 and 300 mg/kg) also prevented systemic IL-1β elevation without altering IL-10 levels, indicating its anti-inflammatory effect. Overall, citral prevented systemic inflammation and reduced bone loss, likely mediated by a reduction in intestinal damage associated with periodontitis. These findings may help develop new treatment methods for chronic inflammatory diseases for clinical use.

Authors: Oswald M.S. (1), Märtin K (1), Scholz C.C.

Affiliations:
1. Institute of Physiology, University Medicine Greifswald, Germany

The author did not consent to the publication of the abstract.

Authors: Krishnacoumar B (1), Schild Y (1), Malyshkina A (1), Henning Y (1), Auger JP (3), Garibagaoglu H (3), Stenzel M (2), Winning S (1), Fandrey J (1)

Affiliations:
1. Institute of Physiology, University of Duisburg-Essen, University Hospital Essen, Hufelandstr. 55, 45122 Essen, Germany

2. Office for Research and Strategic Development, University of Duisburg-Essen, University Hospital Essen, Hufelandstr. 55, 45147 Essen, Germany

3. Department of Rheumatology and Clinical Immunology Charité-Universitätsmedizin Berlin, Charitéplatz1, 10117 Berlin, Germany

The author did not consent to the publication of the abstract.

Authors: Yves Schild (1), Joachim Fandrey(2), Anna Wrobeln(3)

Affiliations:
Yves Schild (1) - Universität Duisburg-Essen
Joachim Fandrey(2) - Universität Duisburg-Essen
Anna Wrobeln(3) - Universität Duisburg-Essen

Abstract:

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies, largely due to its profoundly immunosuppressive tumor microenvironment (TME). A defining feature of the PDAC TME is severe hypoxia, driven by dense desmoplastic stroma and the abundance of cancer-associated fibroblasts (CAFs), which collectively impair antitumor immune responses. This hypoxic milieu hinders the survival, infiltration, and cytotoxic function of ex vivo–expanded Vγ9Vδ2 T cells, limiting their therapeutic efficacy. Given their unique MHC-independent cytotoxicity and broad tumor reactivity, Vγ9Vδ2 T cells hold strong potential as a broadly applicable cancer immunotherapy. Therefore, elucidating how hypoxia shapes Vγ9Vδ2 T-cell function is critical for developing strategies to enhance their antitumoral activity in PDAC. Hypoxia-inducible factors (HIFs), particularly HIF-2α, orchestrate many of these adaptions. HIF-2α regulates a distinct set of target genes and can be pharmacologically modulated, making it of clinical interest. To investigate the role of HIF-2α in immune evasion, we established 3D PDAC-CAFs-spheroids with hypoxic cores, co-cultured with Vγ9Vδ2 T cells from healthy volunteers. To assess the function of HIF-2α in CAFs, we generated HIF-2α knockout CAFs and pharmacologically modulated HIF-2α activity with inhibiting Belzutifan or activating Roxadustat. We then assessed changes in the medium/TME and the Vγ9Vδ2 T cell state and functionality. Here, we present the successful establishment of a human PDAC spheroid model that enables detailed analysis of HIF-2α-driven immune modulation. This system provides new insights into how HIF-2α in CAFs contributes to immune evasion and highlights potential therapeutic avenues for restoring Vγ9Vδ2 T cell activity in PDAC.

Authors: X. Meçi (1), H. Landstorfer (19, H. Scholz (1), S. Kelterborn (2)

Affiliations:
1. Institute of Translational Physiology, Charité – Universitätsmedizin Berlin
2. Life Sciences and Technology, Berliner Hochschule für Technik

Abstract:

Neuroblastoma is a malignant tumor arising in the peripheral sympathetic nervous system of children. In addition to genetic drivers, local tissue hypoxia has been reported to promote aggressive tumor growth. Here, we set out to explore the transcriptional response of neuroblastoma cells to hypoxia. For this purpose, MYCN-amplified Kelly neuroblastoma cells were edited using CRISPR/Cas9 to delete hypoxia-inducible factor (HIF)-1α, HIF-2α, or their shared HIF-β subunit. Neuroblastoma cells with and without deletion of HIFs were exposed to either 21% (normoxia) or 0.5% (hypoxia) ambient oxygen for 16 h and their transcriptome were analyzed by bulk RNA-sequencing. HIF binding sites in the genome of neuroblastoma cells were mapped by CUT&RUN-sequencing. A total of 289 genes including CA9 and FOXE3 prove as HIF-1α targets, whereas 1630 genes comprising EPO and FLT1 are regulated by HIF-2α. The number of genes that are controlled by both, HIF-1α and HIF-2α, amounts to 66. While HIF-1α targets are assigned to GO terms such as “immune regulation” and “cytotoxicity”, HIF-2α-regulated genes are functionally related to developmental and nervous system processes. Data analysis of 478 neuroblastomas revealed significantly (p<0.001) reduced survival rates and advanced clinical stages in patients with high tumor levels of HIF-1α. The detrimental effect of MYCN-amplification on the clinical outcome of neuroblastoma prognosis is enhanced by the simultaneous high-level expression of HIF-β in tumors. In conclusion, CRISPR/Cas9 genome editing combined with RNA-sequencing allows for the classification of specific HIF target genes in neuroblastoma cells with potential implications for disease progression.

Authors: Isabelly G. Solon (1), Patrícia Passaglia (1), Ísis P. Trajano (3), Wanderson S. Santos (1), Sergio L. Salvador (4), Daniela B. Pailioto (2),Glauce C Nascimento (1) and Luiz G. S. Branco (1)

Affiliations:
1 Department of Basic and Oral Biology, Dental School of Ribeirão Preto, University of São Paulo, Avenida Do Café s/n, 14040-904 Ribeirão Preto, SP, Brazil
2 Department of Oral and Maxillofacial Surgery and Periodontology, School of Dentistry of Ribeirão Preto, University of São Paulo - USP, Ribeirão Preto, SP, Brazil
3 Program of Physiology, Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
4 Department of Clinical Analyses, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo - USP, Ribeirao Preto, SP, Brazil

Abstract:

Periodontal disease is a chronic inflammatory condition resulting from both local alterations in the oral cavity and systemic changes in the host. Its development depends on a dysregulated host immune response associated with the presence of periodontopathogenic bacteria in the subgingival environment, such as Porphyromonas gingivalis (Pg), which plays a central role in alveolar bone destruction and the loss of periodontal supporting tissues. Given the multifactorial and systemic nature of periodontal disease, several therapeutic strategies have been investigated to control its progression, including complementary approaches based on natural compounds with anti-inflammatory properties. In this context, citral, a bioactive compound derived from lemongrass essential oil, has emerged as a promising therapeutic agent. Therefore, this study aimed to evaluate the effects of citral (100 mg/kg) on Pg-induced periodontitis in male BALB/c mice (approval number: 2024.1.57.58.0). Periodontitis was induced by six intermittent Pg gavages over 12 days, concomitant with 12 citral gavages. Forty-two days after the first gavage, all animals were euthanized. Citral significantly reduced systemic and local inflammation, as demonstrated by decreased IL-6 levels in plasma and reduced IL-6, IL-10, and TNF-α levels in gingival tissue. Furthermore, citral attenuated alveolar bone loss in treated animals compared to the experimental group (p < 0.05), as evidenced by histological and micro-computed tomography analyses. Collectively, these findings suggest that citral may represent a promising alternative therapeutic strategy for periodontal disease induced by systemic microbial challenge.

Authors: Schneider P (1), Fandrey J (1), Tertel T (2), Leu T (1)

Affiliations:
1. Institute of Physiology, University of Duisburg-Essen - University Hospital Essen
2. Group for Translational EV Research, University of Duisburg-Essen - University Hospital Essen

Abstract:

Primary cilia, often referred to as “antennae of the cell”, are centrosome-derived sensory organelles that function as central components in the detection and integration of extracellular signals and are ubiquitously present on nearly all mammalian cell types. They also play a crucial role throughout the central nervous system, where they maintain neuroplasticity. Recent work by Leu et al. demonstrated a functional dependence of primary cilia on HIF2alpha in the context of cellular adaptation to hypoxic conditions. The aim of this study was to investigate the role of hypoxia-inducible factors (HIFs) in the context of primary cilia in more detail. In the first part of this work, a ciliary processed variant of HIF2alpha with an approximated molecular weight of 66 kDa was identified following the isolation of primary cilia from SH-SY5Y cells. The reduced molecular weight was attributed to prior proteasomal processing. On a functional level, inhibition of the proteasome prevented hypoxia-induced elongation of primary cilia, indicating a central role of proteasomal regulation in ciliary HIF2alapha function. Based on these findings, the second part of this study focused on the analysis of the previously unknown functional relationship between primary cilia and HIF1alpha. A Hif1a knockout was generated using a specific Cre/LoxP system and validated by Western blotting and qPCR analyses. The results revealed a pronounced HIF1alpha-dependent effect on the expression of selected components of cilia-associated signaling pathways. In particular, the expression of NeuroD1 and Sav1 significantly increased following Hif1a knockout induction, which was associated with enhanced neuronal differentiation, reduced proliferative activity, and an increased rate of ciliarization. In summary, this study demonstrates that HIF1alpha and HIF2alpha exert distinct cilia-specific functions and highlights the importance of primary cilia in hypoxic signal transduction. Whether these mechanisms are universally applicable or specific to the central nervous system remains to be determined by future studies.

Authors: A. D. Dorsch, A.-L. Herrmann, A. L. Baum, P. Lutze, C.C. Scholz

Affiliations:
Institute of Physiology, University Medicine Greifswald

The author did not consent to the publication of the abstract.

Authors: Leu T (1), Denda J (1), Kromat S (1), Wrobeln A (1), Fandrey J (1)

Affiliations:
Institute of Physiology, University Duisburg-Essen, 45147 Essen, Germany

Abstract:

Primary cilia are microtubule-based sensory organelles that translate extracellular signals into intracellular responses. They regulate fundamental processes like proliferation and migration via pathways such as Shh and TGF-β. Ciliary integrity is maintained by intraflagellar transport (IFT), and changes in length often indicate impaired function. During hypoxia, such as in ischemic stroke, rapid signal regulation is important for cell survival. This response is driven by hypoxia-inducible factors (HIFs). Specifically, HIF-2 promotes neurogenesis and protects neuronal stem cells, making it essential for regenerating cerebral injuries. Recent research demonstrates that HIF-2α localizes to the ciliary axoneme in neuronal cells, inducing elongation through direct interaction with the protein IFT88. Furthermore, MEK/ERK signaling and downstream gene expression only occur when primary cilia are intact and HIF-2α has accumulated within them. These findings highlight that ciliary HIF-2 signaling is a key regulator of cellular recovery and regenerative processes. In contrast to the neural cells examined, immune cells demonstrate a divergent morphology of primary cilia. Immunofluorescence stainings revealed consistently localized, overlapping signals for markers of the ciliary base, axoneme, and membrane in the majority of immune cells observed. It is of particular interest to note that these structures manifested predominantly in pairs and were potentially intracellular, thus indicating an association with the centrosome. Preliminary functional results further indicate that these structures are associated with TGF-beta signaling. In response to stimulation, a variety of SMADs have been observed to accumulate at the ciliary structure. The collective findings of our research underscore the function of primary cilia as cell-type-specific regulators of cellular responses to hypoxic stress. It is recommended that further investigation be given to additional ciliary structures, such as the specialized ciliary proteasome.

Authors: Borgardt J (1), Räwer M (1), Pape L (1), Jägers J (1)

Affiliations:
1. Department for Paediatrics II, University Hospital Essen, Hufelandstr. 55, 45147 Essen

Abstract:

Introduction For kidney transplantation, organs are stored on ice for transportation without perfusion. As a result, the organs are exposed to hypoxic stress for a longer period of time, inducing ischemia-reperfusion-injury as main causes for primary graft dysfunction. The presence of Hypoxia-inducible factor1α (HIF1a) after transplantation, correlates with increased graft survival. Pretreatment of donor rats with HIF1a stabilising compounds before organ retrieval showed improved graft survival. This study aims to investigate, if treatment during cold storage time renders a similar effect. Methods NRK cells were flushed with cold medium containing different concentrations of Roxadustat, ranging from 10 - 150 mM, and stored in a fridge. After 24h the cells were flushed with medium (37 °C) and incubated for 5 min, 30 min, 2h and 24h. Kidneys from 20 Wistar rats were perfused with chilled UW-solution. The left or the right kidney were assigned to either the control group or the treatment group. The control kidney was dissected after the first flush, while the treatment kidney, remained in the body for a second perfusion of chilled UW-solution containing different Roxadustat concentrations. Western blot and immune histochemistry was applied to determine HIF1a stabilisation and nuclear localisation, and expression of kidney injury markers such as NGAL and KIM1. The expression of HIF1a target genes was determined using qPCR. For assessment of oxidative stress TBARs assay was applied. Results/Discussion Preliminary results show no significant protective effects of Roxadustat during cold ischemia, which may be du to limited impact of HIF1a in cold temperatures. Future analyses will show, if prolonged HIF1a expression will help the cells to recover from reperfusion injury and therefore decrease delayed graft function and primary graft non-function.

Authors: Nelson Niski, Joachim Fandrey, Anna Wrobeln

Affiliations:
Institute of Physiology, University of Duisburg-Essen, University Hospital Essen

Abstract:

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia and impaired immune responses, caused by mutations in genes involved in the transforming growth factor-beta (TGF-β) signaling pathway. Hypoxia-inducible factors (HIFs) are central regulators of cellular adaptation to hypoxia and play a key role in immune regulation and cellular metabolism. Crosstalk between TGF- β and HIF signaling is essential for immune homeostasis. Recent studies have revealed that leukocytes from HHT patients show significantly reduced HIF-1α expression and transcription of HIF target genes, accompanied by decreased HIF-1α protein levels and cellular metabolism. Pharmacological stabilization of HIF using the prolyl hydroxylase inhibitor Roxadustat has been shown to restore HIF-1α levels and metabolic function. In this study, we investigated the mechanisms underlying HIF-1α suppression in HHT with a focus on CD4+ T cells, key players of adaptive immunity. Ex vivo treatment with Roxadustat increased HIF-1α protein levels and gene expression, resulting in upregulation of HIF target genes. Importantly, metabolic profiling demonstrated restoration of both mitochondrial respiration and glycolysis in CD4+ T cells derived from HHT patients. To extend these findings to innate immunity, monocytes were analyzed as central effector cells. Pharmacological HIF stabilization similarly rescued monocyte bioenergetic function, characterized by improved mitochondrial respiration and glycolytic capacity. In summary, these findings underscore the central role of HIF-1α signaling in regulating immune cell metabolism and function and identify pharmacological HIF stabilization as a promising therapeutic strategy to correct immune dysfunction in HHT.

Authors: Wrobeln A (1,2), Tenbohlen L (1), Tebbe B (3), Schönberger T (1), Schild Y (1), Heinrich A (1), Kleine-Möllhof L (1), Leu T (1), Fandrey J (1)

Affiliations:
1. Institute of Physiology, University of Duisburg-Essen, University Hospital Essen, Hufelandstr. 55, 45147 Essen, Germany
2. Institute of Physiological Chemistry, University of Duisburg-Essen, University Hospital Essen, Hufelandstr. 55, 45147 Essen, Germany
3. Department of Medical Intensive Care 1, University of Duisburg-Essen, University Hospital Essen, Hufelandstr. 55, 45147 Essen, Germany

Abstract:

Roxadustat, a hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor, is used to treat renal anemia by stabilizing HIF and inducing target genes such as EPO. Beyond erythropoiesis, HIF plays a pivotal role in leukocyte metabolism and immune regulation. Here, we investigated the effects of Roxadustat on human leukocytes both in vitro and in critically ill patients with chronic kidney disease (CKD) and renal anemia. Peripheral blood mononuclear cells (PBMCs) treated with Roxadustat exhibited HIF-1α accumulation, upregulation of HIF target genes, and a metabolic shift towards glycolysis, indicating an activated leukocyte state. Combined treatment with lipopolysaccharide further enhanced inflammatory gene expression, suggesting a potential for modulating immune responses. Importantly, Roxadustat did not impair cell viability. In parallel, blood samples from CKD patients receiving Roxadustat were analyzed for leukocyte composition, activation markers, HIF stabilization, gene expression, and inflammatory cytokines. Early data suggest Roxadustat modulates leukocyte function in vivo, potentially shaping inflammatory responses in vulnerable patient populations. Our findings integrate mechanistic insights with clinical observations, highlighting that Roxadustat-induced HIF stabilization has both metabolic and immunomodulatory consequences. Understanding these effects is critical to optimizing anemia treatment while minimizing infection and inflammatory risks in CKD patients.

Authors: Wowro SJ (1), Dähnhardt H (1), Dittrich S (1), Wu Q (1), Xie C (1), Vahrenbrink M (1), Labes R (2), Kulow VA (2), Fähling M (2), Schupp M (1)

Affiliations:
(1) Charité Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Pharmacology, Max Rubner Center (MRC) for Cardiovascular Metabolic Renal Research, Berlin, Germany
(2) Charité Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute Translational Physiology, Berlin, Germany

Abstract:

Background: Adaptation to dynamic environments is a hallmark of living cells. Simple sugars are sensed by the transcription factor carbohydrate response element–binding protein (ChREBP) to enhance carbohydrate utilization. In hepatocytes, ChREBP and sterol regulatory element-binding protein (SREBP) are major transcriptional drivers of lipogenic gene expression and hepatic lipid homeostasis. Whether ChREBP contributes to cellular adaptation to low oxygen is unknown. Aim: To determine how hypoxia regulates lipogenic transcription factors in hepatocytes. Methods: Primary mouse hepatocytes were exposed to hypoxia (1% O₂) for 8h. Protein expression was assessed by immunoblotting, gene expression by qPCR, and ChREBP genomic binding by chromatin immunoprecipitation. ChREBP and Egln3 were depleted using siRNA. Mice were gavaged daily with the HIF stabilizer daprodustat for 4 weeks and changes in liver protein and mRNA investigated. Results: Hypoxia stabilizes ChREBP protein, increases its genomic binding, and induces its target genes in primary hepatocytes. This induction was reduced when proline hydroxylase domain 3 (Phd3/Egln3)—an oxygen-sensing protein previously shown to interact with ChREBP—was depleted. In contrast, hypoxia strongly reduced protein abundance of SREBP, revealing major differences in hypoxic regulation of these lipogenic transcription factors. This appears physiologically relevant, as certain ChREBP target genes in mouse liver are enriched in low-oxygen pericentral hepatocytes, whereas SREBP and its targets are enriched in oxygen-rich periportal hepatocytes. Chronic administration of daprodustat induced a subset of ChREBP target genes in mouse liver, indicating coordinated activation of hepatic HIF and ChREBP by this class of antianemic drugs. Conclusion: ChREBP functions as a hypoxia-responsive transcription factor whose protein stability, genomic binding, and transcriptional activity increase under low O₂. In contrast, SREBP is suppressed by hypoxia, demonstrating opposing regulatory responses to oxygen limitation. These findings reveal a new link between hepatic oxygen sensing and lipid metabolism and suggest that pharmacological HIF activation can modulate ChREBP activity in vivo.

Authors: Göpel A (1), Bendler C (1), Bauer L (1), van Belle GJ (1), Nanadikar M (1), Krüger R (2), Naas S (2), Schödel J (2), Katschinski DM (1), Zieseniss A (1)

Affiliations:
1 Institute of Cardiovascular Physiology, University Medical Center, Georg-August University, Göttingen, Germany
2 Department of Nephrology and Hypertension, Uniklinikum Erlangen und Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany

The author did not consent to the publication of the abstract.

Authors: Annika Schubert (1), Maria Eduarda Lobo Barbosa da Silva (1), Tabea Ambrock (1), Orbel Terosian (1), Anna Malyshkina (1), Claudia Padberg (1), Safa Larafa (2), Johann Matschke (2,3) , Joachim Fandrey (1), Yoshiyuki Henning (1)

Affiliations:
1. Institute of Physiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
2. Institute of Cell Biology (Cancer Research), University Hospital Essen, University of Duisburg-Essen, Essen, Germany
3. German Cancer Consortium (DKTK) partner site Essen a partnership between DKFZ and University Hospital, Essen, Germany

Abstract:

Age-related macular degeneration (AMD) is the most common blinding disease in the elderly population, which primarily affects central vision because the photoreceptors in the macula degenerate due to a dysfunction and atrophy of the retinal pigment epithelium (RPE). However, AMD pathophysiology is still not fully understood. Major hallmarks of AMD are oxidative stress and hypoxia in RPE cells. Both are associated with accumulation of hypoxia-inducible factors (HIFs), key regulators of cellular adaptation to hypoxia. In a previous study, we have combined oxidative stress and HIF stabilization (hypoxidative stress) in a human RPE cell line and demonstrated that HIF stabilization exacerbates oxidative damage leading to ferroptosis, an iron-dependent cell death mode. In the present study, we have therefore investigated the respective roles of HIF-1 and HIF-2 in hypoxidative stress-induced ferroptosis, by applying siRNA-mediated RNA interference to target HIF1A and HIF2A mRNA. We also tested pharmacological approaches using commercially available HIF inhibitors. We discovered that HIF-1 silencing protected RPE cells by modulating iron metabolism and downregulating intracellular iron levels. Furthermore, antioxidative mechanisms were induced and cell metabolism was improved by HIF-1α silencing. In addition, we identified a pharmacological inhibitor (Vorinostat) that rescued RPE cells from ferroptosis, by downregulation of transferrin receptor 1 (TFR1) and heme oxygenase-1 (HO-1) as well as by improvement of cell metabolism. Our results highlight the importance of considering targeted HIF inhibition as a potential treatment approach against AMD. Therefore, we are currently transferring these findings to preclinical models of AMD.

Authors: Henning Y (1), Schubert A (1), Sunny S (3), Hausherr J (2), Padberg C (1), Grosche A (3), Fandrey J (1), Krishnacoumar B (1), Leu T (1)

Affiliations:
1. Institute of Physiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
2. Department of Thoracic and Cardiovascular Surgery, West German Heart and Vascular Center, University of Duisburg-Essen, Essen, Germany
3. Department of Physiological Genomics, Biomedical Center, Ludwig-Maximilians-Universität München, Germany

Abstract:

Dysregulated metabolic homeostasis in the retina is increasingly recognized as an early pathophysiological driver of retinal degenerative diseases such as age-related macular degeneration (AMD), which affects the photoreceptors in the macula of the retina, leading to the loss of central, high-acuity vision. Hypoxia and the consequent stabilization of hypoxia-inducible factors (HIFs) are major hallmarks of AMD known to impair energy metabolism in the long-term. We have characterized retinal metabolism of a transgenic mouse model (Hif2a^fl/fl x Nes-Cre) that lacks Hif2a in the retina, which encodes the α-subunit of the HIF-2 isoform. For this purpose, we have conducted Metabolic Flux Analyses of the retina using retinal punches in a Seahorse XFe24 Bioanalyzer. Furthermore, high resolution fluorescence microscopy, as well as immunohistochemical and gene expression analyses were performed. In addition, we have treated porcine retinal organotypic cultures with Belzutifan, an FDA-approved HIF-2 inhibitor, to evaluate the translational potential of HIF-2 inhibition. We found that HIF-2α is predominantly expressed in Müller glia and ganglion cells of the retina. Aged Hif2a^-/- mice had an improved metabolic phenotype in terms of mitochondrial respiration and glycolytic flux compared to age-matched wildtype animals. Furthermore, we observed elevated glutamine synthetase levels in Müller glia, increased levels of pyruvate dehydrogenase (PDH) in mitochondria and better pyruvate utilization in Hif2a^-/- mice. Consistent with these findings, the inhibition of HIF-2 signaling by Belzutifan treatment enhanced the metabolic phenotype of the porcine retina. Our findings suggest that targeting HIF-2 in the retina is a promising strategy to counteract hypoxia-associated metabolic impairment.

Authors: Agnes Greven, Joachim Fandrey, Katrin Prost-Fingerle, Sandra Winning

Affiliations:
Institute of Physiology, University Duisburg-Essen

Abstract:

Hypoxia inducible factors (HIF) are transcription factors and consist of a constitutively expressed, but oxygen labile α-subunit and a constitutive β-subunit. Under hypoxia, HIF-1α accumulates and translocates to the nucleus, where it dimerizes via PAS-domain with HIF-1β. Through the basic-helix-loop-helix (bHLH) domain HIF binds to DNA, leading to transcription of target genes. There are 3 isoforms known for HIF-α: HIF-1α (expressed in most tissues), HIF-2α (predominates in some tumours) and HIF-3α (presumably regulatory function). Tumours can form a hypoxic environment, where HIFs are involved in their development and progression. High HIF target gene expression negatively affects patient’s prognosis. The specific HIF-2 Inhibitor PT2977, which is intended for the treatment of clear cell renal cell carcinoma (ccRCC), glioblastoma and advanced solid tumours, promises to affect the dimerization of the HIF-subunits by causing a conformational change in the HIF-2a PAS-domain. To investigate whether PT2977, as proposed, interferes with the dimerization of HIF-2α and HIF-1β under hypoxic conditions, Fluorescence resonance energy transfer (FRET) will be measured using fluorophore-tagged HIF-subunits, Fluorescence lifetime imaging microscopy (FLIM) and Co-Immunoprecipitation (Co-IP). In addition, effects on target gene expression will be analysed by qPCR and effects on protein stability by western blotting. Not only the HIF-2 target genes are of great interest, but also the impact of the inhibitor on the expression and stability of HIF-1α. By inhibiting HIF-2 dimerization, increased levels of unbound HIF-1β may become available, which could interact with HIF-1α.

Authors: El-Khabbaz J (1, 5), Piollet M (1), Rizzo G (2), Sakalli ET (2), Krammer T (3), Leipold AM (3), Saliba AE (3,4 ), Zernecke A (2), Cochain C (1)

Affiliations:
1. Université Paris Cité, Inserm, PARCC, F-75015 Paris, France
2. Institute of Experimental Biomedicine, University Hospital Würzburg, Würzburg, Germany
3. Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Center for Infection Research (HZI), Würzburg, Germany
4. Institute for Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany
5. Freie Universität, Berlin, Germany

Abstract:

Background: Neutrophils are among the earliest immune responders following myocardial infarction (MI) and have traditionally been viewed as short-lived, predominantly pro-inflammatory cells. However, emerging evidence suggests that neutrophils can acquire tissue-adaptive and reparative functions. The mechanisms governing their temporal and spatial reprogramming during cardiac injury and repair remain poorly defined. Methods: To resolve neutrophil heterogeneity across tissues and time, we performed single-cell RNA sequencing (scRNA-seq) combined with CITE-seq on neutrophils isolated from bone marrow, blood, spleen, and heart of mice at 1 and 4 days after experimental MI. Integrated cross-tissue datasets were analyzed using complementary pseudobulk and single-cell approaches to characterize population dynamics, transcriptional trajectories, and functional state transitions. Results: We identified a coordinated maturation continuum from bone marrow progenitors to peripheral and heart-infiltrating neutrophils. An early interferon-responsive population (Isg15⁺, Ifit3⁺) transiently expanded during the acute inflammatory phase and diverged from subsequent cardiac trajectories. In contrast, heart-infiltrating neutrophils underwent marked transcriptional reprogramming, shifting from hypoxia- and stress-response programs toward enhanced oxidative phosphorylation and lipid metabolism. By day 4 post-MI, the cardiac neutrophil compartment was dominated by a Tnf⁺/SiglecF⁺ population characterized by elevated expression of pro-inflammatory markers and anti-apoptotic Bcl2a1 family genes, indicative of prolonged survival and sustained activity. Pseudotime analysis revealed progressive differentiation toward this long-lived, metabolically adaptive state as a terminal fate of the cardiac neutrophil response. Conclusion: This study defines a continuous transcriptional trajectory underlying neutrophil adaptation after MI and identifies long-lived Tnf⁺/SiglecF⁺ neutrophils as terminal effectors of the post-infarction response. These findings refine current concepts of neutrophil plasticity in sterile inflammation and suggest temporal modulation of neutrophil states as a potential therapeutic strategy to improve cardiac repair outcomes.

Authors: Nadja Jauert¹², Katrin Schilling-Ziese¹, Antje Meyer², Melanie Heigl², Carmen Scheibenbogen¹, Wolfram Doehner²³⁴, Michael Boschmann⁵

Affiliations:
¹ Institute of Medical Immunology, Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum (CVK), Berlin, Germany
² Berlin Institute of Health Center for Regenerative Therapies, Charité – Universitätsmedizin Berlin, Berlin, Germany
³ German Heart Center of the Charité, Department of Cardiology, Angiology and Intensive Care Medicine, Berlin, Germany
⁴ German Center for Cardiovascular Research (DZHK), Partner Site Berlin, Berlin, Germany
⁵ Experimental and Clinical Research Center (ECRC), Charité – Universitätsmedizin Berlin, Berlin, Germany

Abstract:

Background: Severe weakness and exertional intolerance are hallmark symptoms of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Impaired endothelial function, reduced tissue perfusion, and altered skeletal muscle metabolism have been proposed to contribute to muscular fatigue. Insufficient substrate delivery and utilization may therefore underlie impaired muscle performance in ME/CFS. Methods: Eleven female ME/CFS patients (age 45.7±13.0 y, BMI 24.8±6.3 kg/m²) and eleven healthy female controls matched for age (40.6±12.0 y) and BMI (22.6±2.4 kg/m²) were studied. After overnight fasting, a microdialysis catheter (M71, µDialysis, Stockholm, Sweden) was inserted into the vastus lateralis muscle and perfused with Ringer’s solution (2 µL/min). Fifteen-minute dialysate fractions were collected to analyze glucose (substrate supply), pyruvate and lactate (aerobic and anaerobic glycolysis), and glycerol (lipid mobilization) at baseline and for two hours after an oGL (75 g glucose). Blood glucose and insulin were measured in parallel. Results: HOMA-IR index did not differ significantly between patients (1.5±0.6) and controls (1.4±0.9), indicating comparable systemic insulin sensitivity. Baseline blood and dialysate parameters were similar between groups. After oGL, blood glucose and insulin profiles were also comparable. In muscle, dialysate glucose increased ~2-fold in controls and ~1.5-fold in patients (p < 0.05). Dialysate lactate increased similarly in both groups (~1.5–2.0-fold), while pyruvate rose from ~30 µmol/L to ~80 µmol/L within 90 min. The baseline lactate/pyruvate ratio was ~45 in controls and ~25 in patients but converged to ~20 after 120 min. Dialysate glycerol decreased by ~50% in both groups. Conclusion: Glucose supply to skeletal muscle in ME/CFS appears reduced, possibly reflecting impaired capillary density and tissue perfusion. This may be compensated by enhanced oxidative glucose utilization, as suggested by the lower lactate/pyruvate ratio in patients. These findings indicate that muscular fatigue in ME/CFS may involve complex hemodynamic disturbances rather than mitochondrial dysfunction alone.

Authors: Xiaochen Niu (1), Wangming Zeng (1), Stefan Gründer (1)

Affiliations:
Institute of Physiology, RWTH Aachen University

The author did not consent to the publication of the abstract.

Authors: Müller S (1), Chehab N (2,3), Gimeno-Ferrer F (1) & Meissner A (1,2,3,4)

Affiliations:
1. University of Augsburg, Institute of Theoretical Medicine, Division of Physiology & Vascular Biology, Augsburg, Germany
2. Lund University, Department of Experimental Medical Science, Faculty of Medicine, Lund, Sweden
3. Lund University, Wallenberg Centre for Molecular Medicine, Lund, Sweden
4. University of Augsburg, Centre for Advanced Analytics and Predictive Sciences (CAAPS), Augsburg, Germany

Abstract:

Asthma is a chronic respiratory disorder characterized by airway inflammation and bronchial hyperresponsiveness, however, its effect on brain structure and function remains poorly understood. House dust mite (HDM) exposure is widely used to model allergic airway inflammation and is known to elicit sex-dependent immune responses, with females exhibiting a more pronounced pulmonary inflammatory phenotype. In this study, we investigated whether HDM exposure affects the neurovascular unit, including blood–brain barrier integrity, and induces structural neuronal alterations in mice, with a focus on potential sex-specific effects. Male and female 12-week-old C57BL/6N mice were exposed to HDM for three weeks, followed by brain collection for histological, molecular, and biochemical analyses. Neurovascular unit components were assessed using immunohistochemistry, Golgi–Cox neuronal staining, quantitative PCR, and Western blotting. HDM exposure induced pronounced astrocyte reactivity restricted to the dentate gyrus, as revealed by GFAP-based morphological and coverage analyses. Gene and protein expression analyses of vascular and glial markers demonstrated a significant increase in cadherin-5 expression in HDM-exposed females, indicating sex-specific vascular activation that parallels the stronger inflammatory response observed in the lung. In contrast, neuronal tracing revealed impaired neuronal arborization and reduced dendritic length in both cortex and hippocampus, without major sex-specific differences. HDM exposure was associated with measurable alterations in the neurovascular unit and neuronal structure, including astrocyte activation, sex-specific vascular responses, and impaired neuronal structural complexity, even in the absence of pronounced pulmonary inflammation in male mice.

Authors: Laura Bellingacci (1), Andrea Mancini (2), Miriam Sciaccaluga (3), Cinzia Costa (2), Alessandro Tozzi (1), Teresa Zelante (4), Luigina Romani (4), Maria Teresa Viscomi (5), Lucilla Parnetti (2) and Massimiliano Di Filippo (2)

Affiliations:
(1) Section of Physiology and Biochemistry, Department of Medicine and Surgery, University of Perugia, Perugia, Italy
(2) Section of Neurology, Department of Medicine and Surgery, University of Perugia, Perugia, Italy
(3) Department of Life Science, Health, and Health Professions, Link Campus University, Rome, Italy
(4) Section of Pathology, Department of Medicine and Surgery, University of Perugia, Perugia, Italy
(5) Section of Histology and Embryology, Department of Life Science and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy

Abstract:

Background: Growing evidence highlights a close physiological interaction between the nervous and immune systems mediated by immune soluble factors. Cytokines modulate synaptic transmission and plasticity, regulating neuronal and network function in the hippocampus and striatum under physiological conditions. Notably, these same pathways can be co-opted during neuroinflammation, contributing to cognitive and affective dysfunction in neuropsychiatric disorders. Aim: The present study aimed to investigate the role of specific immune-derived molecules in modulating both physiological plasticity and inflammation-related neuronal/synaptic dysfunction. Methods: Experiments were conducted in control mice and in the experimental autoimmune encephalomyelitis (EAE) model of neuroinflammation. Behavioral tests assessed cognitive performance and affective behavior. Electrophysiological recordings were performed in hippocampal and striatal regions to evaluate synaptic plasticity and network excitability. In selected experiments, interleukin-17 (IL-17) and interferon γ (IFN-γ) signaling were manipulated genetically or pharmacologically to dissect their mechanistic contributions. Results: Synaptic plasticity was found to be dependent on immune molecules (IL-17), which are physiologically expressed and dynamically regulated following inflammatory stimuli. During neuroinflammation, IL-17 contributed to cognitive impairment, while IFN-γ selectively mediated affective and depressive-like behaviors in the earliest disease phases. Electrophysiological analyses revealed that these behavioral alterations were paralleled by time- and region-specific changes in synaptic strength and neuronal excitability, which were largely prevented by blocking the respective cytokine pathways. Conclusions: Immune molecules are fundamental for shaping neural circuit function under physiological conditions; however, when dysregulated, they drive waves of functional disconnection in brain circuits critical for cognition and mood regulation. Dissecting these mechanisms provides insight into immune–neural communication and identifies potential mechanistic targets for alleviating neuropsychiatric symptoms associated with neuroinflammatory disorders. Acknowledgements: LB is supported by a research fellowship FISM - Fondazione Italiana Sclerosi Multipla - cod. 2023/BR/005 and financed or co-financed with the '5 per mille' public funding.

Authors: Löffler M (1), Wiegert S (1), Dieter A (1)

Affiliations:
1. Department of Neurophysiology, MCTN, Medical Faculty Mannheim - Heidelberg University, Mannheim, Germany

The author did not consent to the publication of the abstract.

Authors: D’Silva C (1), Poeta E (2), Monti B (1,2), Massenzio F (1)

Affiliations:
1. Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
2. IRCCS Institute of Neurological Sciences of Bologna, Bologna, Italy

Abstract:

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder, characterized by progressive and irreversible cognitive decline, accompanied by neuroinflammation. A central pathological hallmark of AD is the generation of amyloid-beta (Aβ) peptides, particularly Aβ42, in amyloid plaques. However, how Aβ42 affects adult neurogenesis and whether it impacts extracellular vesicle (EV)–mediated inflammatory signaling remain poorly understood. Subventricular zone (SVZ)-derived neurospheres are an established in vitro model to study neurogenesis and the progeny originating from neural stem cells (NSCs). NSC-derived EVs are emerging as modulators of the neural microenvironment, reflecting properties of their cells of origin and influencing neighboring or distant cells through their molecular cargo, including miRNAs. Using live-cell imaging, we observed that Aβ42 enhanced neurosphere proliferation, followed by an earlier onset of differentiation toward a neuronal lineage. This accelerated differentiation was accompanied by alterations in EV size distribution and miRNA content. Aβ42 treatment increased expression of miR-34a, a miRNA implicated in both the regulation of NSC proliferation and differentiation and in pro-inflammatory signaling. Collectively, these findings indicate that Aβ42 differentially regulates neurogenesis by transiently stimulating proliferation while accelerating neuronal differentiation and independently alters EV signaling profiles in NSC-derived neurospheres. The modulation of inflammation-related miRNAs suggests a potential link between Aβ42 exposure, neurogenic dysregulation, and neuroinflammatory signaling in AD. Understanding these parallel mechanisms may provide new insights into disease progression and identify potential targets to counteract neurodegeneration.

Authors: Isabella S. Balles (1), Annalena Franz (1, 2), Tania Fernández d.V. Alquicira (1), David Owald (1)

Affiliations:
(1) Institute of Neurophysiology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität, Humboldt-Universität, and Berlin Institute of Health, Berlin, Germany
(2) Institute of Biotechnology, Faculty III Process Sciences, Technische Universität Berlin

Abstract:

Cocaine ranks among the top five most harmful drugs, posing significant risks to users' physical health, cognitive function, and socioeconomic well-being. Both short- and long-term cocaine abuse have been linked to cognitive impairments, particularly in learning and memory. While cocaine's rewarding effects resemble those of natural rewards, the extent to which it engages neural and molecular learning mechanisms remains poorly understood, especially in complex mammalian brains. To address this, we employ Drosophila melanogaster as a powerful model organism, leveraging its well-characterized neural circuitry and established classical conditioning paradigms. Building on previous insights into how dopaminergic neurons convey rewarding and punishing reinforcement signals to the fly's Mushroom Body - the primary olfactory learning center - we aim to elucidate how cocaine influences these processes.

Authors: Ricciardi N (1), Di Liberto V (1), Di Majo D (1), Cangelosi A (1), Scordino M (1), Urone G (1), Giglia G (1,2), Sardo P (1), Ferraro G (1) and Gambino G (1)

Affiliations:
1. Department of Biomedicine, Neuroscience and Advanced Diagnostics (Bi.N.D), Section of Human Physiology, University of Palermo, Italy
2. Euro Mediterranean Institute of Science and Technology-I.E.ME.S.T., 90139 Palermo, Italy

Abstract:

Traditionally, metabolic Syndrome (MetS) is defined by peripheral metabolic abnormalities such as insulin resistance, dyslipidemia and obesity. However, its impact on brain physiology and cognition, as well as the mechanisms underlying these alterations, remain elusive. Reframing cognitive impairment as a core feature of MetS is supported by emerging evidence indicating that cognitive alterations may not only represent sequelae of metabolic dysfunction, but also early, mechanistically relevant features of MetS progression. To this aim, we employed a longitudinal high-fat diet (HFD) rat model over 20 weeks. This approach allows us to characterize the temporal evolution of MetS and identify neuro-metabolic markers delineating impaired cognitive dimensions. Rats were subjected to behavioral testing assessing reactivity, anxiety-like behavior, and declarative memory, alongside longitudinal profiling of systemic metabolic and redox markers. Prolonged HFD exposure induced a progressive deterioration of anxiety-related behavior and memory performance that temporally paralleled metabolic impairment. Our findings reveal a progressively shifting MetS phenotype, characterized by an early predominance of metabolic alterations followed by convergence with cognitive and affective dysfunctions. Multivariate analyses showed coordinated neuro-metabolic coupling, indicating that cognitive dysfunction co-varies with altered metabolic burden, oxidative stress, leptin signaling, and ketone body levels. Furthermore, causal modeling supports that the observed effects were mediated by systemic leptin signaling, highlighting a mechanistic link between metabolic load, neuroendocrine dysregulation, and cognitive vulnerability. Collectively, these results demonstrate that cognitive impairment is structurally embedded within the progression of MetS. Rather than arising as a secondary epiphenomenon, cognitive dysfunction contributes to the organization and expansion of the neuro-metabolic phenotype.

Authors: L. Hub; H. May-Simera; M. Cetiner; L. Pape, J. Jägers

Affiliations:
Pediatrics 2, University Hospital Essen, Essen, Germany
Institute of Molecular Physiology, Johannes Gutenberg University, Mainz, Germany

Abstract:

Purpose: Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by multisystem manifestations, including hyperphagia, polydactyly, neurocognitive deficits, and renal abnormalities. In pediatric patients, prominent features include impaired satiety and renal dysfunction, encompassing cystic kidney disease, hyposthenuria, and reduced glomerular filtration rate (GFR). These renal phenotypes may arise from disrupted polarization of renal epithelial cells. Notably, hyposthenuria in BBS occurs independently of NKCC2 dysfunction or aquaporin-2 mistrafficking. A clinical trial evaluating appetite suppressants in BBS patients demonstrated a significant GFR increase within the initial 12 months of therapy. Methods (Methode): BBS6 knockout mice and wild-type littermates served as the experimental model. Kidney tissues were harvested: one hemikidney was fixed for immunohistochemistry, while proteins and mRNA were isolated from the contralateral hemikidney. Western blotting and quantitative PCR (qPCR) assessed protein and mRNA expression levels, respectively; immunohistochemical analyses evaluated epithelial cell polarization in different segments using apical and basolateral membrane transporters. Results: So far, our results show that BBS6 knockout does not induce defective cell polarization. Sodium transporters regulated by insulin are significantly upregulated, whereas all other examined transporters remain unchanged. Conclusion: Together with findings from the clinical study, our data indicate that hyposthenuria in BBS may be associated with hyperphagia-associated hyperinsulinemia.

Authors: Jingyang Liu (1), Wolfgang M. Kuebler (1-5), Laura Michalick (1,2)

Affiliations:
1. Charité - Universitätsmedizin Berlin, Berlin, Germany
2. German Centre for Cardiovascular Research (DZHK), Berlin, Germany
3. German Center for Lung Research (DZL), Gießen, Germany
4. Keenan Research Centre for Biomedical Science, St. Michael´s Hospital, Toronto, Canada
5. University of Toronto, Toronto, Canada

Abstract:

Mechanical ventilation can damage the lung by causing endothelial barrier failure and edema formation. Yet the underlying mechanisms remain unclear. Notably, even protective positive-pressure ventilation imposes repetitive, non-physiological stress that contributes to ventilator-induced lung injury (VILI). In previous work, we identified a critical role for the mechanosensitive Ca²⁺ channel Transient Receptor Potential Vanilloid-type 4 (TRPV4) in lung microvascular barrier failure following mechanical overventilation, with preliminary data further indicating an involvement of thermosensitive TRPV1. As temperature is an increasingly recognized yet underexplored modulator of these injury pathways, we tested the contribution of thermosensitive TRPV1 to mechanosensitive TRPV4-induced lung injury. To assess mechanical and temperature-dependent TRPV1/4 activation, isolated perfused lungs (IPLs) from wild-type, TRPV1-/-, and TRPV4-/- mice were perfused at 20–40°C and ventilated with low (7 ml/kg) or high (20 ml/kg) tidal volumes. Lung injury was assessed by real-time monitoring of lung weight gain and post-mortem as wet-to-dry lung weight ratio. In parallel, human pulmonary microvascular endothelial cells were exposed to static or cyclic stretch (0 or 18% elongation) at 20, 30, or 40°C, in presence or absence of TRPV1 (SB-366791) or TRPV4 (HC-067047) antagonists, and TRPV1–TRPV4 interactions were quantified by proximity ligation assay (PLA). Higher temperatures progressively aggravated VILI in IPLs. Both TRPV1-/- and TRPV4 /-mice developed less injury than wild-type controls. In vitro, cyclic stretch significantly increased physical TRPV1–TRPV4 interactions, and this effect was further enhanced at 40°C. TRPV1 or TRPV4 antagonists reduced PLA signals, indicating that functional blockade of either channel attenuates temperature- and stretch-induced TRPV1–TRPV4 interactions. Here, we identify a thermo- and mechanosensitive role for TRPV1 and TRPV4 in VILI, whereby temperature-dependent enhancement of TRPV1–TRPV4 interactions drives endothelial dysfunction and edema during mechanical ventilation. In contrast, lower inspired gas temperatures may be protective. Therefore, targeting thermo- and mechanosensitive TRP channels represents a new strategy to mitigate VILI.

Authors: Maikel Assaf(1), Maël Blandin(1,2), Christelle Goanvec (1), Sylvain Gourier(1,3), Michael Theron(1), Karine Pichavant-Rafini(1), Karelle Léon(1)

Affiliations:
1- Univ. Brest, Laboratory ORPHY EA4324, IBSAM, F-29200 Brest, France
2- Department of Emergency Medicine (ED, SAMU 29, SMUR), University Hospital Center of Brest, France
3- Department of Anesthesiology, Intensive Care, and Peri‑operative Medicine, Hospital Center, Brest, France

Abstract:

Introduction: Sepsis is a dysregulated systemic inflammatory response to infection that precipitates multiorgan failure and remains the leading cause of death in intensive care units [1]. Cardiovascular dysfunction drives most sepsis‑related mortality through impaired myocardial contractility and altered vasomotricity, causing major systemic hemodynamic disturbances [2]. Although sex‑dependent cardiovascular phenotypes are documented in other pathological contexts, the influence of biological sex on the cardiovascular response to sepsis remains insufficiently characterized [3]. This study investigates sex as a biological variable by examining how sepsis alters myocardial contractile performance and thoracic aortic vasomotricity function in male and female rats. Materials and Methods: Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in 30 male and 30 female Wistar rats. Analyses were realized at 0, 1 and 3 h post-CLP. Cardiac contractile function was assessed ex vivo using myography on left ventricular trabeculae. Thoracic aortic rings were isolated to evaluate vasomotor responses to cumulative concentrations of acetylcholine(10⁻¹⁰–10⁻⁴ M), phenylephrine(10⁻⁹–10⁻⁴ M), and sodium nitroprusside(10⁻¹⁰–10⁻⁴ M). Results: At 1Hz, only female septic rats exhibited a significant increase in left ventricular trabecular contractile force at 3 h compared with female controls at 0 h (p=0.046) and 1 h (p=0.0367). In vascular assays, male aorta at 3 h displayed markedly reduced acetylcholine‑induced vasorelaxation relative to females, with significant differences at 10⁻⁷(p=0.0076), 10⁻⁶(p=0.0053), 10⁻⁵(p=0.0012), and 10⁻⁴ M(p=0.0013). Conclusion: These findings demonstrate clear sex‑dependent differences in the cardiovascular response to acute sepsis. Females show an increase in myocardial contractile force, whereas males exhibit impaired endothelium‑dependent vasorelaxation, suggesting heightened endothelial vulnerability. Distinct cardiac and vascular trajectories may therefore contribute to sex‑specific clinical outcomes and therapeutic responsiveness in sepsis. References: [1]Yu X et al. Intensive Care Med Exp, 2025. [2]Yap JQ et al. Int J Mol Sci, 2023. [3]Shields CA et al. Am J Physiol, 2022.

Authors: Laasanen, Samuli; Lehti, Mari; Eerola, Kim; Bourgery, Matthieu; Mäkelä, Margareeta; Kärnä, Salli; Sonja Järvinen; Hajati, Fatemeh; Lehtiniemi, Tiina; Olotu, Opeyemi; Mäkelä, Juho-Antti; Ma, Lin; Savontaus, Eriika; Kotaja, Noora

Affiliations:
Institute of Biomedicine, University of Turku, Finland

Abstract:

Growing evidence shows that father’s metabolic disorders and other acquired conditions can be transmitted to the offspring via sperm-mediated epigenetic mechanisms, such as small non-coding RNAs. Taking into consideration the increasing prevalence of obesity and associated metabolic disorders, this poses a huge risk for the health of the following generations as the preventability of the harmful epigenetic inheritance remains unknown. Here we performed multigenerational diet studies in mice to investigate if the epigenetic transmission of father’s high-fat diet (HFD)-induced metabolic disorder to the offspring can be prevented by treating the father’s obesity with a healthy diet. We show that HFD-induced changes in sperm small RNAs were reversed back to control levels with the diet intervention. In F1 offspring, the father’s HFD-induced obesity and diet intervention caused variable metabolic alterations, as well as transcriptomic changes in liver and epididymal white adipose tissue. Some negative metabolic consequences of the paternal HFD were prevented with father's diet intervention. Furthermore, father’s HFD induced changes in F1 sperm small RNAs and round spermatid mRNAs, which were all largely prevented with father’s diet intervention. Some influences of the F0 diet intervention were still observed in F2 metabolism and sperm small RNAs. Together, our results suggest that inter- and transgenerational effects of paternal HFD are complex but can be altered by obesity treatment, and that germline RNAs are responsive to these exposures in three generations, while not explaining all of the observed intergenerational effects.