The current study targets three major aspects central to the phenomenon of Iridium (Ir) catalyst dissolution in polymer electrolyte membrane water electrolyzers; the stabilization over time, the influence of current density and the mechanisms of transport and deposition of dissolved Ir species. Quasi-online measurements of Ir dissolution at the anode side of single-cells using single-pass water flow reveal that oxygen molecules produced per Ir dissolved increases by up to two orders of magnitude with current density and then level off. Experiments under water recirculation show that Ir ions are back-transported into the catalyst coated membrane. Model-aided analysis suggests that Ir species re-deposited into the catalyst layer are stabilized at elevated concentrations of dissolved Ir ions. Application of single-pass flow is recommended for stability testing. An observed stabilization of over two orders of magnitude with time provides promising insight into the applicability of low Ir loadings under steady-state conditions.

DNA-binding protein-A (DbpA; gene: Ybx3) belongs to the cold shock protein family with known functions in cell cycling, transcription, translation, and tight junction communication. In chronic nephritis, DbpA is upregulated. However, its activities in acute injury models, such as kidney ischemia/reperfusion injury (IRI), are unclear. To study this, mice harboring Ybx3+/+, Ybx3+/- or the Ybx3-/- genotype were characterized over 24 months and following experimental kidney IRI. Mitochondrial function, number and integrity were analyzed by mitochondrial stress tests, MitoTracker staining and electron microscopy. Western Blot, immunohistochemistry and flow cytometry were performed to quantify tubular cell damage and immune cell infiltration. DbpA was found to be dispensable for kidney development and tissue homeostasis under healthy conditions. Furthermore, endogenous DbpA protein localizes within mitochondria in primary tubular epithelial cells. Genetic deletion of Ybx3 elevates the mitochondrial membrane potential, lipid uptake and metabolism, oxygen consumption rates and glycolytic activities of tubular epithelial cells. Ybx3-/- mice demonstrated protection from IRI with less immune cell infiltration, endoplasmic reticulum stress and tubular cell damage. A presumed protective mechanism was identified via upregulated antioxidant activities and reduced ferroptosis, when Ybx3 was deleted. Thus, our studies reveal DbpA acts as a mitochondrial protein with profound adverse effects on cell metabolism and highlights a protective effect against IRI when Ybx3 is genetically deleted. Hence, preemptive DbpA targeting in situations with expected IRI, such as kidney transplantation or cardiac surgery, may preserve post-procedure kidney function.

Background Calcium (Ca 2+ ) signaling regulates various vital cellular functions, including integrin activation and cell migration. Store-operated calcium entry (SOCE) via calcium release-activated calcium (CRAC) channels represents a major pathway for Ca 2+ influx from the extracellular space in multiple cell types. The impact of JAK2-V617F and CALR mutations which are disease initiating in myeloproliferative neoplasms (MPN) on SOCE, calcium flux from the endoplasmic reticulum (ER) to the cytosol, and related key signaling pathways in the presence or absence of erythropoietin (EPO) or thrombopoietin (TPO) is poorly understood. Thus, this study aimed to elucidate the effects of these mutations on the aforementioned calcium dynamics, in cellular models of MPN. Methods Intracellular Ca 2+ levels were measured over a time frame of 0–1080 s in Fura-2 AM labeled myeloid progenitor 32D cells expressing various mutations (JAK2-WT/EpoR, JAK2-V617F/EpoR; CALR-WT/MPL, CALR-ins5/MPL, and del52/MPL). Basal Ca 2+ concentrations were assessed from 0–108 s. Subsequently, cells were stimulated with EPO/TPO in Ca 2+ -free Ringer solution, measuring Ca 2+ levels from 109–594 s (store depletion). Then, 2 mM of Ca 2+ buffer resembling physiological concentrations was added to induce SOCE, and Ca 2+ levels were measured from 595–1080 s. Fura-2 AM emission ratios (F340/380) were used to quantify the integrated Ca 2+ signal. Statistical significance was assessed by unpaired Student's t-test or Mann–Whitney-U-test, one-way or two-way ANOVA followed by Tukey's multiple comparison test. Results Following EPO stimulation, the area under the curve (AUC) representing SOCE significantly increased in 32D-JAK2-V617F cells compared to JAK2-WT cells. In TPO-stimulated CALR cells, we observed elevated Ca 2+ levels during store depletion and SOCE in CALR-WT cells compared to CALR-ins5 and del52 cells. Notably, upon stimulation, key components of the Ca 2+ signaling pathways, including PLCγ-1 and IP3R, were differentially affected in these cell lines. Hyper-activated PLCγ-1 and IP3R were observed in JAK2-V617F but not in CALR mutated cells. Inhibition of calcium regulatory mechanisms suppressed cellular growth and induced apoptosis in JAK2-V617F cells. Conclusions This report highlights the impact of JAK2 and CALR mutations on Ca 2+ flux (store depletion and SOCE) in response to stimulation with EPO and TPO. The study shows that the JAK2-V617F mutation strongly alters the regulatory mechanism of EpoR/JAK2-dependent intracellular calcium balance, affecting baseline calcium levels, EPO-induced calcium entry, and PLCγ-1 signaling pathways. Our results reveal an important role of calcium flux in the homeostasis of JAK2-V617F positive cells.

CD4+ T cells play a central role in orchestrating the immune response in asthma, with dysregulated ion channel profiles and altered metabolic signatures contributing to disease progression and severity. An important classification of asthma is based on the presence of T-helper cell type 2 (Th2) inflammation, dividing patients into Th2-high and Th2-low endotypes. These distinct endotypes have implications for disease severity, treatment response, and prognosis. By elucidating how ion channels and energy metabolism control Th cells in asthma, this review contributes to the pathophysiological understanding and the prospective development of personalized therapeutic treatment strategies for patients suffering from distinct asthma endotypes.

The virulence of intracellular pathogens relies largely on the ability to survive and replicate within phagocytes but also on release and transfer into new host cells. Such cell-to-cell transfer could represent a target for counteracting microbial pathogenesis. However, our understanding of the underlying cellular and molecular processes remains woefully insufficient. Using intravital 2-photon microscopy of caspase-3 activation in the Leishmania major-infected (L. major-infected) live skin, we showed increased apoptosis in cells infected by the parasite. Also, transfer of the parasite to new host cells occurred directly without a detectable extracellular state and was associated with concomitant uptake of cellular material from the original host cell. These in vivo findings were fully recapitulated in infections of isolated human phagocytes. Furthermore, we observed that high pathogen proliferation increased cell death in infected cells, and long-term residency within an infected host cell was only possible for slowly proliferating parasites. Our results therefore suggest that L. major drives its own dissemination to new phagocytes by inducing host cell death in a proliferation-dependent manner.

TGR5 (Gpbar-1) is a membrane-bound bile acid receptor considered a metabolic regulator involved in BA synthesis and glucose metabolism. The activation of TGR5 in macrophages is associated with a reduced inflammatory response. Krüppel-like factors (KLFs) play critical roles in the transcriptional regulation of pathways involved in the metabolism of glucose and lipids and the immune response. The present study aims to determine the contribution of TGR5 to metabolism in macrophages during bacterial infection.

Hydrogen crossover in polymer electrolyte membrane electrolysis cells is important concerning faradaic efficiency, flammability hazards, and degradation phenomena. In recent years, steady-state H2-in-O2 measurements have demonstrated that the hydrogen crossover increases with current density, due to mass transport limitations in the cathode catalyst layer. However, hydrogen crossover during dynamic operation has not been investigated yet. Therefore, this study investigates the hydrogen crossover with a dynamic macroscopic 1-D through-plane model of a polymer electrolyte membrane electrolysis cell. The model focuses on the detailed description of the dynamics of the reactions and mass transport of hydrogen in the membrane electrode assembly. Simulated down steps in current density, lead to transient overshoots in the H2-in-O2 content at the anode side. The membrane acts as short-term mass storage for the dissolved hydrogen, and mass transport lags the instant response of the current density. Under specific conditions with high cathode mass transport limitations, the lower explosion limit of H2-in-O2 can be transiently exceeded. This work provides for the first-time insights into transient hydrogen crossover phenomena and is a further step into dynamic model-based analysis of polymer electrolyte water electrolysis cells.

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: In myeloproliferative neoplasms (MPNs), mutations in Janus kinase 2 (JAK2), calreticulin (CALR), and the thrombopoietin receptor (MPL) activate common Janus kinase/signal transducer and activator of transcription signaling (JAK/STAT) pathways. These mutations confer hypersensitivity in cytokine-induced proliferation and drive clonal expansion of hematopoietic progenitor cells. However, the exact mechanisms of action of these mutants have not been fully comprehended. Calcium (Ca2+) is an important intracellular secondary messenger, which regulates diverse essential cellular functions, like activation of integrins, cell migration, exocytosis and many more. One major Ca2+ influx pathway in many cell types is store-operated calcium entry (SOCE) through calcium release-activated calcium (CRAC) channels. Although, Phospholipase Cγ1 (PLCγ1) which controls Ca2+ signaling pathways, has been implicated in the formation of early erythropoiesis, the role of mutated JAK2 and CALR on SOCE has been remained elusive. Aims: The purpose of this study was to determine the effect of JAK2-V617F and CALR mutations on SOCE and key signaling pathways in the absence or presence of erythropoietin (EPO) or thrombopoietin (TPO), respectively. Methods: 32D cells (JAK2WT/EpoR, JAK2-V617F/EpoR; CALRWT/MPL, CALR-ins5/MPL and del52/MPL) were labeled with Fura-2 AM for 30 minutes and then seeded in 0.01% poly-L-lysine pre-coated 96-well plates. Intracellular Ca2+ measurements were performed using Synergy H1 plate reader. Total Ca2+ measurement time frame was from 0sec to 1080sec, where basal Ca2+ concentration was measured between 0sec-108sec. Subsequently, cells were stimulated with EPO/TPO in Ca2+-free Ringer solution and Ca2+ measurement was performed from 109sec-594sec (store depletion), followed by addition of 2mM Ca2+ Ringer solution to induce SOCE where the measurement was acquired from 595sec-1080sec. Fura-2 emission ratios (F340/380) were quantified by analyzing the integrated Ca2+ signal. Results: Upon EPO stimulation, SOCE measured as area under the curve (AUC) was significantly increased in 32D JAK2V617F cells (p=0.035) as compared to 32D-WT cells. Conversely, under unstimulated conditions 32D-WT cells (p=0.001) showed significantly higher basal levels of cytosolic Ca2+ compared to 32D JAK2-V617F cells. Furthermore, we measured the Ca2+ levels following store depletion and SOCE in TPO stimulated CALR cells. The Ca2+ concentration during store depletion (Max peak: p=0.04; AUC: p=0.05) and SOCE (Max peak: p=0.06; AUC: p=0.07) was higher in WT CALR cells in comparison with CALR-ins5 and del52 cells. To dissect the underlying molecular mechanisms, we analyzed the key downstream components of EPO/JAK2 and TPO/MPL signaling pathways. Upon stimulation, essential elements of the Ca2+ signaling pathways such as PLCγ1 and IP3, were found to be activated in these cell lines. A substantial change in the dynamics of phosphorylated states of PLCγ1, IP3, STAT3, STAT5, AKT, MEK, and ERK was found in 32D JAK2-V617F and CALR mutated cells. Summary/Conclusion: The mutations in JAK2 and CALR differentially affect store-operated calcium entry. We here provide novel evidence that the JAK2-V617F mutation induces an essential change in the regulatory mechanism of EpoR/JAK2-dependent intracellular Ca2+ homeostasis upon EPO stimulation. This affects both basal and SOCE induced Ca2+ levels, cytokine dependent PLCγ1 signalling pathways, and cellular proliferation. These findings explain the clinically observed differential response of JAK2V617F vs. WT progenitor cells to cytokines in MPN. Keywords: Calcium, Intracellular signaling, Myeloproliferative disorder

T-Zellen sind ein wichtiger Bestandteil des adaptiven Immunsystems und spielen auch bei überschießenden Immun­antworten wie allergischen oder autoimmunen Erkrankungen eine Rolle. Der Energiestoffwechsel von T-Zellen unterscheidet sich abhängig von ihrem Aktivierungs- und Differenzierungsstatus. Ein besseres Verständnis des T-Zell-Metabolismus kann zur Weiterentwicklung von Diagnostik und Therapie bestimmter Erkrankungen beitragen.

T helper (Th) cells provide immunity to pathogens but also contribute to detrimental immune responses during allergy and autoimmunity. Th2 cells mediate asthmatic airway inflammation and Th1 cells are involved in the pathogenesis of multiple sclerosis. T cell activation involves complex transcriptional networks and metabolic reprogramming, which enable proliferation and differentiation into Th1 and Th2 cells. The essential trace element zinc has reported immunomodulatory capacity and high zinc concentrations interfere with T cell function. However, how high doses of zinc affect T cell gene networks and metabolism remained so far elusive. Herein, we demonstrate by means of transcriptomic analysis that zinc aspartate (UNIZINK), a registered pharmaceutical infusion solution with high bioavailability, negatively regulates gene networks controlling DNA replication and the energy metabolism of murine CD3/CD28-activated CD4 + T cells. Specifically, in the presence of zinc, CD4 + T cells show impaired expression of cell cycle, glycolytic and tricarboxylic acid cycle genes, which functionally cumulates in reduced glycolysis, oxidative phosphorylation, metabolic fitness and viability. Moreover, high zinc concentrations impaired nuclear expression of the metabolic transcription factor MYC, prevented Th1 and Th2 differentiation in vitro and reduced Th1 autoimmune central nervous system (CNS) inflammation and Th2 asthmatic airway inflammation induced by house dust mites in vivo. Together, we find that higher zinc doses impair the metabolic fitness of CD4 + T cells and prevent Th1 CNS autoimmunity and Th2 allergy.

T cells orchestrate adaptive and innate immune responses against pathogens and transformed cells. However, T cells are also the main adaptive effector cells that mediate allergic and autoimmune reactions. Within the last few years, it has become abundantly clear that activation, differentiation, effector function, and environmental adaptation of T cells is closely linked to their energy metabolism. Beyond the provision of energy equivalents, metabolic pathways in T cells generate building blocks required for clonal expansion. Furthermore, metabolic intermediates directly serve as a source for epigenetic gene regulation by histone and DNA modification mechanisms. To date, several antibiotics were demonstrated to modulate the metabolism of T cells especially by altering mitochondrial function. Here, we set out to systematically review current evidence about how beta-lactam antibiotics, macrolides, fluoroquinolones, tetracyclines, oxazolidinones, nitroimidazoles, and amphenicols alter the metabolism and effector functions of CD4+ T helper cell populations and CD8+ T cells in vitro and in vivo. Based on this evidence, we have developed an overview on how the use of these antibiotics may be beneficial or detrimental in T cell-mediated physiological and pathogenic immune responses, such as allergic and autoimmune diseases, by altering the metabolism of different T cell populations.

Recently, Li and colleagues provided in Nature immunology 1 a novel mechanism of immunosurveillance in epithelial tumors, in which cytotoxic innate lymphoid cells (ILCs) sense IL-15 secreted by the tumor itself.Both the adaptive immune system, including CD8 + cytotoxic lymphocytes (CTLs), and innate immune cells such as Natural Killer (NK) cells are capable of providing antitumor immunity through the release of perforins and granzymes.In addition, also cytotoxic group 1 innate lymphoid cells (ILC1s) were reported to exert NK cell-like antitumor cell cytotoxicity. 2ansler et al. now reveal that tumor-derived IL-15 is an important prerequisite for the cytotoxic function of ILC1s during renal cell carcinoma (RCC), which histologically can be subdivided into chromophobe RCC (chRCC) and clear cell RCC (ccRCC).By performing single-cell RNA sequencing (scRNAseq) of all CD45 + cells from ccRCC and chRCC the authors first identified distinct CD8 + T cell clusters between both RCC subtypes.Validation experiments using flow cytometry showed that ccRCC tumors are especially characterized by an infiltration of an exhausted PD1 + CD8 + T cell population, whereas this population was not significantly increased in chRCC.Further, the authors detected circulating NK cells and tissue-resident ILC1s in both types of RCC.However, the frequencies of these innate lymphocyte populations were increased only in chRCC but not in ccRCC.In addition, ILC1s in chRCC, but not in ccRCC, expressed higher levels of CD56, a marker enriched in tissue-resident ILCs.Together, these findings indicate that the immune cell landscape and, consecutively, the antitumor response in chRCC compared to ccRCC appear to be different.Importantly, using The Cancer Genome Atlas (TCGA) the authors showed that an immune cell composition in the tumor dominated by PD1 + CD8 + T cells is negatively correlated with patient survival in both ccRCC and chRCC.However, ILC1 gene signatures correlated with either better (chRCC) or worse (ccRCC) outcomes.Further, ILC1s in chRCC, in contrast to ccRCC, selectively expressed high levels of granzyme A. In addition, NK cells and ILC1s showed an increased expression of the IL-2/IL-15 receptor β chain and chRCC tumors from TCGA cohort exhibited increased expression of IL-15, which is capable of enhancing anti-tumor responses. 1o prove whether IL-15 directly affects ILC1s, the authors cultured ILC1s from RCC tumors in the presence of IL-15/IL-15Rα complexes: Here, IL-15/IL-15Rα complexes increased granzyme A