Biology subjects

Deshmukh, A.

Publications and source records attributed to Deshmukh, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A 3D Soft X-Ray Structural Atlas of Multicellular Cable Bacteria

The multicellular filamentous cable bacteria perform centimeter-scale electron transport via a continuous periplasmic conductive network. Resolving how this electrical architecture is coordinated with intracellular organization requires an integrated approach coupling 3D ultrastructure with in situ biochemical composition. Here, we integrate soft X-ray tomography (SXT) with cryo-electron tomography, elemental mapping, and fluorescence microscopy to generate a volumetric 3D atlas of intact cable bacteria filaments. Whole-cell reconstructions reveal physiological heterogeneity and cell-cycle-dependent reorganization of intracellular vesicles and granules. X-ray absorption measurements resolve biochemical differences within the physically continuous conductive apparatus, where periplasmic conductive fibers possess higher biomolecular density than junction lamellae at cell junctions. Furthermore, cell elongation preceding division is associated with two high-density, ring-like nucleoid-associated domains, accompanied by asymmetric membrane constriction and localized FtsZ assemblies. These findings establish SXT as a powerful approach for studying multicellular bacterial organization and provide a multi-scale framework for understanding the conductive machinery of cable bacteria.

cell biology↗

bioRxiv2026

Septin inhibitors exert opposing effects on distinct higher-order septin structures in melanoma cells

Septins are cytoskeletal GTPases that assemble into diverse higher-order structures with context-dependent mechanical and signaling functions. Recent work identified a septin-dependent pro-survival pathway in metastatic cancer cells, termed bleb signaling, in which plasma membrane blebbing promotes the formation of septin signaling hubs that support survival during cellular detachment and dissemination. Disrupting these hubs may offer a novel therapeutic strategy targeting disseminated cancer cells. Here, we compare the effects of three septin-targeting compounds, forchlorfenuron (FCF), UR214-9, and REM127, on septin organization and viability in melanoma cells using confocal microscopy and quantitative computer vision analysis. FCF and UR214-9 both effectively destabilized septin hubs, while REM127 had no appreciable effect. FCF also reproduced the context-specific viability reduction previously seen in detached melanoma cells, while UR214-9 caused broad off-target toxicity and cell death in all growth conditions. As with hub disruption, REM127 produced no effect on viability. Surprisingly, though none of the compounds caused an increase in higher-order septin structures during detachment, all three increased end-on-end polymerization of filamentous septins at stress fibers when grown adhered to substrate. These findings demonstrate that septin-targeting compounds exert divergent effects on different classes of septin higher-order structures within the same cell type, promoting filamentous assembly while disrupting or failing to promote membrane-associated signaling hubs. This highlights not only the structural distinctiveness of such classes, but that pharmacological targeting can produce opposite effects on them. Moreover, this study emphasizes the need for next-generation septin inhibitors capable of selectively targeting disease-relevant septin assemblies.

cell biology↗

bioRxiv2026

Functional analysis of novel microneme proteins from Plasmodium vivax blood stages identifies vaccine candidates

Host cell invasion by malaria parasites requires specific molecular interactions with host receptors. Plasmodium vivax merozoite invasion of reticulocytes is mediated by P. vivax Duffy binding protein (PvDBP) and its homolog, P. vivax erythrocyte binding protein (PvEBP). Here, we identify and characterize two novel P. vivax merozoite proteins, PvMP45 and PvMP36, which co- localize with PvDBP and PvEBP in the micronemes and bind reticulocyte receptors. PvMP45 and PvMP36 share high sequence identity with their P. knowlesi homologs, PkMP45 and PkMP36, which form a complex with other invasion related proteins. Field studies reveal that naturally acquired antibodies against PvMP36, PvEBP and PvDBP are associated with protection against clinical P. vivax malaria. We demonstrate that naturally acquired antibodies to PvEBP bind Fcy receptors and likely mediate protection by enabling opsonic phagocytosis. In addition, we show that combining antibodies against PvDBP and PvMP36 results in an additive invasion inhibitory effect against P. vivax blood stages. These results suggest that combining PvDBP, PvEBP and PvMP36 in a multivalent blood stage vaccine could elicit diverse immune mechanisms against P. vivax to achieve high efficacy. ImportanceAll the clinical symptoms of malaria are attributed to the blood stage of malaria parasites during which merozoites invade and multiply within red blood cells. A clear understanding of the host- parasite interactions that enable invasion can open paths for development of novel methods to block parasite growth and prevent malaria. Here, we identify and characterize two novel invasion related proteins from P. vivax merozoites that form an invasion complex and bind host RBC receptors. We demonstrate that antibodies targeting these proteins can block RBC invasion by P. vivax and naturally acquired antibodies that develop following P. vivax infection against one of these proteins are associated with protection against P. vivax malaria. These studies not only expand our understanding of the molecular mechanisms that enable host cell invasion by P. vivax but open new avenues for development of vaccines to protect against P. vivax malaria.

microbiology↗

bioRxiv2026

Api5 Regulates Genomic Stability and Chemotherapy Resistance in Cancer

Api5 is elevated in a number of cancers and is associated with many hallmarks of cancer, including resistance to apoptosis, immune escape, stemness, chemotherapy resistance, high proliferation, and cell-cycle dysregulation. In this study, we identified the DNA and chromatin-binding activities of Api5 in tumorigenic cells, as well as its association with genomic instability and chemotherapy resistance. Knockdown of Api5 resulted in reduced nuclear volume, DNA content, and chromosome number, and increased sensitivity to DNA damage. The survival of Api5-knockdown cells decreased following UV and cisplatin treatments due to the accumulation of damaged DNA and inefficient nucleotide excision repair. Interestingly, Api5 knockdown cells also exhibited low pChk1 levels following UV damage. Further, we confirmed the chemotherapy resistance phenotype in cancers with elevated Api5 levels, demonstrating that xenograft tumours with Api5 knockdown responded better to cisplatin, with significant tumour regression. SummaryApoptosis inhibitor 5 (Api5) contributes to chemotherapy resistance by conferring a survival advantage and promoting efficient DNA repair following genotoxic stress through regulation of Chk1 activation.

Cancer Biology↗

bioRxiv2026

Morphotype-Resolved 3D Morphometry Reveals a Structure-Density-Location Coupling in Mitochondrial Networks

Mitochondrial architecture plays a critical role in cellular function, yet how organelle structure, metabolic density, and subcellular position are jointly remodeled across whole cells remains poorly understood. We applied quantitative 3D soft X-ray tomography to analyze intact INS-1E cells in a native, cryo-hydrated state. By integrating morphometric profiling with voxel-level linear absorption coefficients (LAC) and contour-based radial mapping, we tracked the structural, biochemical, and spatial remodeling of fragmented, intermediate, and interconnected mitochondrial morphotypes under high glucose and Exendin-4 stimulation. High glucose induces morphotype-specific hypertrophy, fission, and perinuclear redistribution of low-density fragments. Co-stimulation with Exendin-4 stabilizes interconnected networks and increases metabolic density at the cell periphery. Morphotype-resolved analysis uncovers a structure-density-location coupling in which mitochondrial shape, macromolecular packing, and radial position shift in concert. These results offer a quantitative framework and high-resolution spatial constraints for whole-cell modeling of organelle dynamics. HighlightsO_LIMitochondrial morphotypes occupy distinct regions during metabolic stimulation C_LIO_LIHigh glucose drives early mitochondrial swelling, fission, and spatial dispersion C_LIO_LIExendin-4 prevents structural collapse and preserves dense interconnected networks C_LIO_LIMorphotype-resolved analysis uncovers a structure-density-location coupling in mitochondrial networks. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/712811v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1d495ecorg.highwire.dtl.DTLVardef@1bee80dorg.highwire.dtl.DTLVardef@74df9dorg.highwire.dtl.DTLVardef@104db6d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

Integrated biophysical and spatial remodeling during insulin secretory granule maturation at the mitochondrial network

Effective insulin secretion and blood glucose homeostasis depend on the multistep maturation of insulin secretory granules (ISGs), a process that includes lumen acidification, enzymatic insulin processing, and biophysical remodeling of the granule. An under studied aspect of ISG maturation is the role of inter-organelle contacts in organelle remodeling. While a correlation between ISG-mitochondria contacts and ISG maturation has been observed, many questions remain on how this interaction may impact maturation (1-5). We sought to address this gap in knowledge by using multi-scale imaging approaches (fluorescent microscopy, soft X-ray tomography, and cryo-electron tomography) to examine how the biophysical properties and spatial organization of ISGs change around the mitochondrial network. Our data suggests that ISGs in proximity to mitochondria exhibit lower pH, higher biomolecular density, and smaller vesicle diameter. Time-resolved imaging using a SNAP tag labelling system also shows that as ISGs age, their proximity to the mitochondria network is increased between 3-6 hours after biosynthesis, suggesting that ISG-mitochondria association is dynamically spatiotemporally regulated in pancreatic {beta}-cells. These data suggest that mitochondrial proximity contributes to the maturation and remodeling of ISGs in pancreatic beta cells.

cell biology↗

bioRxiv2026

Identification of low threshold off-target activation pathways during stimulation of carotid baroreceptor afferents in swine.

ObjectiveElectrical stimulation of the baroreceptors pathways at the carotid sinus bulb - known as baroreflex activation therapy (BAT) - is intended to change autonomic tone and ultimately reduce blood pressure and heart rate. BAT is pre-market approved by the United States Food and Drug Administration (FDA) for the treatment of heart failure and received an FDA humanitarian device exemption for drug resistant hypertension. However, responder rates are limited by side-effects including numbness in the head and neck, altered speech, respiratory constriction, dry cough, vomiting, and altered sensory and motor function of the tongue. We hypothesized that these side-effects are driven by activation of other nearby nerve fibers of similar or lower threshold than the carotid sinus nerve. In this study, we sought to identify the neural sources responsible for off-target muscle activation contributing to these side-effects. These sources would inform strategies mitigating off-target activation in BAT therapy. MethodsDomestic swine were used in this work as the diameter and thickness of the swine carotid artery are closer to human than those of canine models. A monopolar disk electrode mimicking the clinical CVRx(R) Neo electrode was surgically placed proximal to the carotid bifurcation with the position optimized for stimulation dose responsive changes in blood pressure. Evoked responses were recorded during dose response testing from multiple neck muscles, and the corresponding off-target nerve pathways were identified by sequential transection of nearby nerves. ResultsThe following activated off-target muscle groups and their corresponding nerve pathways were verified which included 1) the cricoarytenoid via recurrent laryngeal nerve, 2) cricothyroid via superior laryngeal nerve, 3) sternocleidomastoid via accessory nerve, and 4) the tongue via hypoglossal nerve. The constrictor muscle group was also activated through a more complex neural pathway. ConclusionWe identified multiple sources of therapy-limiting side-effects of BAT in the swine model. These results will help guide the design of improved stimulation electrodes, surgical placement, and parameter programming to reduce BAT-associated side-effects while increasing on-target activation in patients.

bioengineering↗

bioRxiv2025

Circuit-specific gene editing for precision modulation of neuronal activity with CRISPR-rabies virus

CRISPR gene editing has revolutionized our ability to study and manipulate specific genes, enabling novel insights into gene function and potential therapies for brain disorders. Recent advances in cell-type-specific regulatory elements and viral delivery systems have made precise in vivo gene editing possible. However, neurons with similar molecular profiles can belong to different circuits, complicating efforts to manipulate circuit function and behavior. To address this, we developed CRISPR-rabies virus (CRV), which leverages the trans-synaptic spread of rabies virus to enable gene editing within anatomically defined neural circuits. By pairing CRV with cell type-specific Cas9 expression, we achieved targeted gene modifications in specific circuits. We demonstrate that CRV can modulate sodium and potassium channel expression in parvalbumin interneurons, thereby effectively regulating synaptic transmission of pyramidal neurons in the CA3 region of the hippocampus. Its compatibility with 3'-capture single-cell RNA-seq allows simultaneous circuit perturbation and molecular profiling. In summary, CRV allows precise circuit-level gene modulation, providing a platform for studying gene function in neural circuits and developing novel gene therapies for brain disorders.

neuroscience↗

bioRxiv2025

Dielectrophoresis Reveals Stimulus-Induced Remodeling of Insulin Granule Subpopulations

The pancreatic {beta}-cell contains several functional subpopulations of insulin secretory granules (ISGs). These subpopulations vary in maturity, age, and secretory capacity. Differences in protein and lipid composition of ISGs are correlated with disease but require further study to understand how ISG remodeling regulates normal biology. Due to limitations in traditional separation methods, the extent of these subpopulations, any overlap between them, and how they are affected by insulinotropic signals have not been determined. In this work, we adapted direct current insulator-based dielectrophoresis (DC-iDEP) to separate ISGs isolated from INS-1E cells, an immortalized rat insulinoma cell line model, according to their electrokinetic mobility ratio (EKMr). We were able to separate ISG subpopulations from unstimulated cells to determine a baseline distribution and identify characteristic profiles for immature, young, and old ISGs. We then analyzed distributions of subpopulations from cells stimulated with insulin secretion signals known to induce biophysical remodeling and maturation. We found significant changes in each subpopulation studied in response to stimulation, consistent with the increases in maturation, crystallization, and changes in size reported in the literature. This work provides new insights into how the cell controls ISG remodeling and may drive future development of more effective therapies. SIGNIFICANCEUnderstanding insulin secretory granule (ISG) heterogeneity and the functional role of subpopulations is a critical step towards unraveling the mechanisms of insulin secretion. We adapted direct current insulator-based dielectrophoresis (DC-iDEP) to resolve immature, young, and old ISG subpopulations based on their biophysical properties. We also explored the biophysical remodeling of ISGs under insulinotropic stimuli to further probe how the cell controls remodeling and secretion. Our work provides a new framework for quantifying granule heterogeneity by linking biophysical features to functional subtypes and assessing how environmental stimuli remodel ISGs. This methodology establishes a broadly applicable platform to interrogate organelle and vesicle diversity in complex biological systems.

biophysics↗

bioRxiv2025

MitoXRNet: Deep Learning-enabled 3D Segmentation of Mitochondria and Nucleus in Soft X-ray Tomograms

Mitochondrial morphology is crucial for cellular function, but large-scale analysis is limited by challenges in high-resolution imaging and segmentation. MitoXRNet, a compact 3D deep-learning model, efficiently segments mitochondria and nuclei from Soft X-ray Tomography data using multi-axis slicing, Sobel-based boundary enhancement, and combined BCE-Robust Dice loss. With 1.4M parameters, it achieves a Dice score of 73.8% on INS-1E cells, outperforming existing models. Automated analysis indicated that glucose induced larger mitochondria and higher matrix density, and that GIP and GKA induced smaller and denser mitochondria, highlighting previously unreported {beta}-cell mitochondrial remodeling. MitoXRNet allows for scalable profiling of organelle-level morpho-biophysical data. HighlightsO_LIA data-efficient method for 3D segmentation of mitochondria and nucleus from Soft X-ray tomograms. C_LIO_LIIncorporates domain-specific Sobel filter-based preprocessing to improve segmentation accuracy and quality under imperfect or noisy labels. C_LIO_LIEnables rapid and automated analysis of mitochondrial morphology, facilitating quantitative assessment of pharmacological effects on cellular ultrastructure. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/682919v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@8600f6org.highwire.dtl.DTLVardef@1a9230eorg.highwire.dtl.DTLVardef@13c6f09org.highwire.dtl.DTLVardef@9dc9f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Classifying Biophysical Subpopulations of Insulin Secretory Granules using Quantitative Whole Cell Structure Analysis

Pancreatic {beta}-cells contain insulin secretory granules (ISGs), organelles where proinsulin is converted into insulin. As ISGs mature, they undergo extensive biophysical remodeling, producing a spectrum of subpopulations with heterogeneous molecular and spatial characteristics. However, systematic methods to define ISG subpopulations remain underdeveloped. To address this gap in knowledge, we employed soft x-ray tomography (SXT), which can quantitatively measure the biochemical density of ISGs within whole {beta}-cells. Using unsupervised clustering, we classified subpopulations based on molecular density, size, and spatial positioning. Across different insulin secretory stimuli, we observed shifts towards mature and releasable subtypes, demonstrating that exogenous signals can dynamically remodel ISG subpopulation distributions. We extended this methodology to primary {beta}-cells characterized using volume electron microscopy (vEM). Integrating subpopulations from SXT and vEM uncovered insights inaccessible by a single method in isolation. This strategy establishes a framework for defining therapeutic approaches aimed at enriching physiologically beneficial ISG subpopulations.

biophysics↗

bioRxiv2025

Modeling Atrial Fibrillation through Intermittent Tachypacing-Induced Remodeling in hiPSC-Derived Atrial Cardiomyocytes and Atrial Fibroblast

BackgroundHuman in vitro models for atrial fibrillation (AF) are limited. Human-induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) provide a valuable tool to study AF pathophysiology by facilitating in vitro modeling. ObjectivesTo investigate the effects of an intermittent tachypacing protocol (ITPP) in matured hiPSC-aCMs co-cultured with human atrial cardiac fibroblast (haCF) to mimic AF-associated electroanatomical phenotypes. Methods and ResultshiPSC-aCMs were cultured alone or co-cultured with haCFs at 90/10 and 70/30 ratios. ITPP was applied through field stimulation, and optical mapping assessed action potentials (APs) and calcium transients (CaTs). Immunostaining was performed to quantify pro-fibrotic biomarkers (Collagen III and TGF{beta}1). ITPP led to increased spontaneous AP frequency ({Delta}=+31{+/-}7%, P<0.0001) and reduced AP duration at 80% repolarization (APD80%; {Delta}=-15{+/-}4%, P=0.001). Additionally, the upstroke slope ({Delta}=-41{+/-}11%, P=0.001) and amplitude (dF/F0; {Delta}=-51{+/-}13%, P<0.001) of intracellular CaT were significantly reduced. Co-culture at the 70/30 hiPSC-aCM/haCF ratio, showed a >100-fold increase in Collagen III expression (P<0.0001), diminished excitability ({Delta}Hz=-61{+/-}6%, P<0.0001), prolonged {Delta}APD80% ({Delta}=+130{+/-}10%, P<0.0001), prolonged AP triangulation ({Delta}APDTri=+143{+/-}13%, P<0.0001), reduced upstroke slope ({Delta}=-66{+/-}6%, P<0.0001), conduction block ({Delta}=-52{+/-}18%, P=0.0260), and diminished intracellular calcium handling (upstroke slope {Delta}=-50{+/-}8%, P<0.0001; {Delta}dF/F0=-34{+/-}9%, P=0.0003). Finally, the application of ITPP to the 70/30 co-culture model recapitulated an AF-mediated phenotype ({Delta}Hz=+25{+/-}8%, P=0.02; {Delta}APD80%=-16{+/-}6%, P=0.01) while introducing conduction block ({Delta}CV100/0 vs 70/30= -27{+/-}15%; P=0.0005). ConclusionsCo-cultures of matured hiPSC-aCMs and haCFs exhibited structural and electrophysiological remodeling, including conduction abnormalities, mirroring key AF mechanisms. This model holds potential for patient-specific therapies and drug discovery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/655123v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@1c56760org.highwire.dtl.DTLVardef@105d4a2org.highwire.dtl.DTLVardef@1cb18d4org.highwire.dtl.DTLVardef@cb3353_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Human iPSC-Model for Atrial Fibrillation C_FIG

bioengineering↗

bioRxiv2025

Prevalence of sympathetic fibers within the rat cervical vagus, and functional consequence on physiological effects mediated by vagus nerve stimulation (VNS).

IntroductionElectrical stimulation of the vagus nerve (VNS) is an FDA approved therapy for epilepsy, depression and rehabilitation after stroke, with recent clinical trials to treat heart failure and inflammation. VNS is often assumed to activate either parasympathetic efferents projecting to visceral organs, and/or sensory afferents projecting from these organs, for its therapeutic effects. Recent studies in humans, swine and dogs have shown that sympathetic nerve fibers from the sympathetic trunk (ST) can frequently be found within the cervical vagus nerve (VN). However, the prevalence and functional consequence of sympathetic fibers on VNS have yet to be elucidated in the most common high throughput animal model to study disease, the rodent. MethodsWe carefully traced ST from sympathetic cervical ganglion (SCG) to find its location in the carotid sheath with reference to the VN in a cohort of Long Evans rats. We then assessed the prevalence of ST fibers with the cervical VN across the cohort using microCT and immunohistochemistry. Finally, we stimulated the VN and the ST in isolation, and where they were conjoined, to evaluate the ST contribution to changes in heart rate. VNS induced heart rate changes are a commonly used surrogate for changes in sympathetic/parasympathetic tone. ResultsThe ST frequently runs in very close proximity to the VN in rats when traced caudally from the SCG. The ST is even conjoined with the VN for stretches within the carotid sheathe at the most common location to place an epineural cuff. Cross-connecting branches were found between the ST and the VN. VNS performed at locations where there was minimal ST crossover induced dose-dependent bradycardia (decrease in heart rate) across the cohort, with detectable bradycardia across the cohort beginning at 50 A (n=8 right, n=3 left). Conversely, stimulation of the isolated ST induced tachycardia (increase in heart rate) across the cohort beginning at [~]200 A (n=7 right, n=3 left). ConclusionThese data suggest that studies of VNS in the rodent model may also be stimulating sympathetic fibers from the ST in addition to canonical VN pathways. Concurrent sympathetic activation has profound implications for dissecting mechanisms of VNS for a host of diseases/disorders. As such, careful post-mortem assessment of the presence of hitchhiking sympathetic fibers within the VN is critical for understanding sources of variability in VNS outcomes.

neuroscience↗

bioRxiv2025

Secretory stimuli distinctly regulate insulin secretory granule maturation through structural remodeling

Insulin secretory granule (ISG) maturation is a crucial aspect of insulin secretion and glucose homeostasis. The regulation of this maturation remains poorly understood, especially how secretory stimuli affect ISG maturity and subcellular localization. In this study, we used soft X-tomography (SXT) to quantitatively map ISG morphology, density, and location in single INS-1E and mouse pancreatic {beta}-cells under the effect of various secretory stimuli. We found that the activation of glucokinase (GK), gastric inhibitory polypeptide receptor (GIPR), glucagon-like peptide-1 receptor (GLP-1R), and G-protein coupled receptor 40 (GPR40) promote ISG maturation. Each stimulus induces unique structural remodeling in ISGs, by altering size and density, depending on the specific signaling cascades activated. These distinct ISG subpopulations mobilize and redistribute in the cell altering overall cellular structural organization. Our results provide insight into how current diabetes and obesity therapies impact ISG maturation and may inform the development of future treatments that target maturation specifically.

biophysics↗

bioRxiv2025

Mechanisms of chromatin remodeling by a Snf2-type ATPase

Chromatin remodeling enzymes play a crucial role in the organization of chromatin, enabling both stability and plasticity of genome regulation. These enzymes use a Snf2-type ATPase motor to move nucleosomes, but how they translocate DNA around the histone octamer is unclear. Here we use cryo-EM to visualize the continuous motion of nucleosomal DNA induced by human chromatin remodeler SNF2H, an ISWI family member. Our work reveals conformational changes in SNF2H, DNA and histones during nucleosome sliding and provides the structural basis for DNA translocation. ATP hydrolysis induces conformational changes in SNF2H that pull the DNA tracking strand, distorting DNA and histones at SHL2. This is followed by SNF2H rotation on the nucleosome, which first pulls the DNA guide strand and creates one-base pair bulge at SHL2, and then releases the pulled DNA. Given the high conservation of the catalytic motors among ATP-dependent chromatin remodelers, the mechanisms we describe likely apply to other families.

biophysics↗

bioRxiv2024

Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

RationaleHyper IgE syndrome (STAT3-HIES), also known as Jobs syndrome, is a rare immunodeficiency disease typically caused by dominant-negative STAT3 mutations. STAT3-HIES syndrome is characterized by chronic pulmonary infection and inflammation, suggesting impairment of pulmonary innate host defense. ObjectivesTo identify airway epithelial host defense defects consequent to STAT3 mutations that, in addition to reported mutant STAT3 immunologic abnormalities, produce pulmonary infection. MethodsSTAT3-HIES sputum was evaluated for biochemical/biophysical properties. STAT3-HIES excised lungs were harvested for histology; bronchial brush samples were collected for RNA sequencing and in vitro culture. A STAT3-HIES-specific mutation (R382W), expressed by lentiviruses, and a STAT3 knockout, generated by CRISPR/Cas9, were maintained in normal human bronchial epithelia under basal or inflammatory (IL1{beta}) conditions. Effects of STAT3 deficiency on transcriptomics, and epithelial ion channel, secretory, antimicrobial, and ciliary functions were assessed. Measurements and Main ResultsMucus concentrations and viscoelasticity were increased in STAT3-HIES sputum. STAT3-HIES excised lungs exhibited mucus obstruction and elevated IL1{beta} expression. STAT3 deficiency impaired CFTR-dependent fluid and mucin secretion, inhibited expression of antimicrobial peptides, cytokines, and chemokines, and acidified airway surface liquid at baseline and post-IL1{beta} exposure in vitro. Notably, mutant STAT3 suppressed IL1R1 expression. STAT3 mutations also inhibited ciliogenesis in vivo and impaired mucociliary transport in vitro, a process mediated via HES6 suppression. Administration of a {gamma}-secretase inhibitor increased HES6 expression and improved ciliogenesis in STAT3 R382W mutant cells. ConclusionsSTAT3 dysfunction leads to multi-component defects in airway epithelial innate defense, which, in conjunction with STAT3-HIES immune deficiency, contributes to chronic pulmonary infection.

cell biology↗

bioRxiv2024

Epidural Spinal Cord Recordings (ESRs): Sources of Artifact in Stimulation Evoked Compound Action Potentials

IntroductionEvoked compound action potentials (ECAPs) measured using epidural spinal recordings (ESRs) during epidural spinal cord stimulation (SCS) can help elucidate fundamental mechanisms for the treatment of pain, as well as inform closed-loop control of SCS. Previous studies have used ECAPs to characterize the neural response to various neuromodulation therapies and have demonstrated that ECAPs are highly prone to multiple sources of artifact, including post-stimulus pulse capacitive artifact, electromyography (EMG) bleed-through, and motion artifact resulting from disturbance of the electrode/tissue interface during normal behavior. However, a thorough characterization has yet to be performed for how these sources of artifact may contaminate recordings within the temporal window commonly used to determine activation of A-beta fibers in a large animal model. MethodsWe characterized the sources of artifacts that can contaminate the recording of ECAPs in an epidural SCS swine model using the Abbott Octrode lead. Muscle paralytics were administered to block muscle activation preventing EMG from contaminating the recorded ECAPs. Concurrent EMG recordings of the longissimus, a long muscle of the back, were used to confirm a 2-4 millisecond (ms) latency source of EMG bleed-through that frequently contaminated the A-beta temporal window. Additionally, we obtained recordings approximately 5-10 minutes post-mortem after clear evoked A-beta and associated EMG responses ceased to characterize the representation of stimulation artifact across the array. ResultsSpinal ECAP recordings can be contaminated by capacitive artifact, short latency EMG from nearby long muscles of the back, and motion artifact from multiple sources. In many cases, the capacitive artifact can appear nearly identical in duration and waveshape to evoked A-beta responses. These sources of EMG can have phase shifts across the electrode array, very similar to the phase shift anticipated by propagation of an evoked A-beta fiber response across the array. This short latency EMG is often evident at currents similar to those needed to activate A-beta fibers associated with the treatment of pain. Changes in cerebrospinal fluid between the cord and dura, and motion induced during breathing created a cyclic oscillation in all evoked components of the recorded ECAP signal. ConclusionCareful controls must be implemented to accurately separate neural signal from the sources of artifact in spinal cord ECAPs. To address this, we suggest experimental procedures and associated reporting requirements necessary to disambiguate the underlying neural response from these confounds. These data are important to better understand the conceptual framework for recorded ESRs, with components such as ECAPs, EMG responses and artifacts, and have important implications for closed-loop control algorithms to account for transient motion such as postural changes and cough.

neuroscience↗

bioRxiv2024

Pyruvate Kinase M2 regulates Japanese encephalitis virus replication by interacting with NS1 protein

Pyruvate kinase isoform M2 (PKM2) is a key modulator of glucose metabolism. While the major role of PKM2 is to facilitate the breakdown of glucose, it is potentially associated with other additional non-glycolytic functions as well. The role of PKM2 in the autoimmune response and inflammatory process is increasingly being acknowledged as a crucial modulator of cellular pathophysiological activity. However, its role in modulating viral replication has not been explored in detail. In the present study, we have shown a significant increase in endogenous PKM2 expression in JEV-infected mouse neuroblastoma cells. Furthermore, overexpression of PKM2 significantly reduced JEV replication, suggesting a negative effect of PKM2 on JEV replication. This was further confirmed by siRNA-mediated downregulation of endogenous PKM2 expression, which resulted in enhanced JEV replication. In silico studies revealed the potential interaction between PKM2 and NS1 protein of JEV. The microscopic studies also showed cellular colocalization of PKM2 and NS1 in the ER of infected cells. The interaction was further validated in vitro by co-immunoprecipitation assay. The present study suggests that PKM2 negatively regulates the JEV replication by its possible interaction with NS1. ImportanceJapanese encephalitis (JE) is a neuroinflammatory disease caused by the Japanese encephalitis virus (JEV). JE is a major threat to public health not only because it causes many deaths but also for its permanent neuropsychiatric sequelae in children. Out of all non-structural proteins of JEV, NS1 is highly immunogenic. A wide range of possible interactive partners has been identified for the NS1, many of those have been linked to immune evasion and regulating viral replication. In the current study, we have described a novel host cell factor, PKM2 modulating JEV replication by interacting with NS1 protein. Considering PKM2s central role in regulating host cell metabolism, our findings suggest a previously unrecognized role for PKM2 in JEV neuropathogenesis. The identification and characterization of previously unknown host factors, as well as the elucidation of their regulatory mechanisms, are of utmost importance in the development of innovative treatments and antivirals against JEV.

microbiology↗