Biology subjects

Kang, S.

Publications and source records attributed to Kang, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Importance of a heat snap in RT-PCR quantification of rotavirus double-stranded RNA in wastewater

Quantification of copies of double stranded RNA using RT-PCR methods may require denaturation of the double stranded structure using an initial high temperature incubation followed by rapid cooling, herein called "heat snap". Papers in the literature that report rotavirus RNA concentrations in fecal and environmental samples do not consistently report the use of such a "heat snap". In this study, we quantified rotavirus RNA in diverse environmental samples (wastewater solids, wastewater, and drainage samples) using digital RT-PCR methods with and without a heatsnap. Concentrations were higher in samples by a factor of 125 when a heat snap was applied. This was consistent across sample types, and across laboratories and PCR instrumentation. We recommend a heat snap be used when enumerating double stranded RNA from rotavirus and other viruses in environmental samples.

molecular biology↗

bioRxiv2025

FragDockRL: A Reinforcement Learning Framework for Fragment-Based Ligand Design via Building Block Assembly and Tethered Docking

Efficient exploration of combinatorial chemical space under synthetic constraints remains a central challenge in computational molecular design. Here, we present FragDock, a molecular design framework that combines building block (BB)-based virtual synthesis with tethered docking guided by a predefined core structure. FragDock defines a structured search space by assembling molecules from synthetically accessible BBs through known chemical reactions and evaluating candidates using tethered docking with a restrained core binding pose. Within this framework, we introduce FragDockRL, a reinforcement learning-based search method that uses docking-score-based rewards and a modified Deep Q-Network (DQN) to guide stepwise molecular growth. We evaluated FragDockRL on three protein targets, CSF1R, FA10, and VEGFR2, using training-cycle analysis and benchmark comparisons with One-Step Reaction, Random Search, Beam Search, and Monte Carlo Tree Search. FragDockRL progressively enriched molecules with favorable docking scores during learning and generated more cutoff-passing unique molecules than Random Search across all three targets, supporting the benefit of learning-guided prioritization. However, the best-performing search strategy was target-dependent: One-Step Reaction, FragDockRL, and Beam Search each showed advantages in different cases. Representative molecular case studies showed that selected compounds retained reference-like binding poses while introducing structural variation in peripheral regions. The reaction schemes used commercially available BBs and well-established medicinal chemistry transformations, supporting the synthetic plausibility of the selected compounds. Overall, FragDock provides a flexible framework for synthetically constrained, structure-guided molecular exploration, and FragDockRL offers a learning-guided search mode for productive candidate prioritization under limited generation budgets.

biochemistry↗

bioRxiv2025

Dicer is essential for proper maturation, composition, and function in the postnatal retina.

microRNAs (miRNAs) play a pivotal role during the early phases of retinal development, but their impact on late-phase retinogenesis is unknown. We depleted miRNAs in late retinal progenitor/precursor cells (RPCs/PCs) via a conditional Dicer knock-out. Optical coherence tomography (OCT), electroretinography (ERG), histological, and transcriptional analyses were conducted in young and adult mice. Alterations in gene expression of late-born cells were observed as early as postnatal day 7 (P7), resulting in impaired rod function, a significantly reduced number of rod bipolar cells and their associated function, and a decreased Muller glia population at adult age. These defects appear to be caused by a delay in differentiation/ incomplete maturation, as indicated by an enlarged progenitor/precursor population at young ages that persists into adulthood. Notably, an increased population of HuC/D+ amacrine cells was found. Luciferase assays led us to speculate that this increase may be due to the absence of Elavl3 suppression via RPC-miRNAs. This suggests that Dicer/miRNAs in late RPC/PCs are essential for the proper formation and maturation of late RPC progenies and may also play a role in regulating cell state. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/635135v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@9e0950org.highwire.dtl.DTLVardef@1757e2corg.highwire.dtl.DTLVardef@aeb5ecorg.highwire.dtl.DTLVardef@29accb_HPS_FORMAT_FIGEXP M_FIG C_FIG Summary statementLate-retinal progenitor microRNAs are essential for proper postnatal retinogenesis and retinal function.

developmental biology↗

bioRxiv2025

Enzyme-responsive Hemostatic Elastin-like Polypeptides for Fibrin Stabilization and Enhanced Coagulation in Thrombocytopenia

Hemorrhage remains a leading cause of mortality in trauma and surgery, and treatment options are limited for thrombocytopenic patients with impaired platelet function. Current plasma-derived hemostatic products face challenges including limited supply, storage requirements, and infectious risk. Here we report a recombinant protein-based hemostat designed to enhance clot mechanics through enzyme responsiveness and self-assembly, which integrates biophysical design principles with clot-targeted drug delivery. We rationally designed a library of enzyme-responsive glutamine (Q)-containing block elastin-like polypeptides (Q-block-ELPs) that reinforce fibrin clots through phase separation and covalent cross-linking. Q-block-ELPs incorporate glutamine residues within a peptide motif recognized by coagulation factor XIIIa, enabling site-specific grafting into fibrin networks during clot formation. By tuning polymer length, Q-block valency, and lower critical solution temperature (LCST) behavior, we engineered Q-block-ELPs to phase separate at body temperature and integrate into the fibrin architecture. In vitro, Q-block-ELPs increase fibrin network density and stiffness. In a thrombocytopenic mouse model, systemic administration reduced blood loss and accelerated clot formation. This strategy delivers a programmable, pathogen-free platform for systemic bleeding control, bridging biophysical protein design with translational hemostatic therapy, addressing an urgent need for platelet-deficient bleeding disorders.

bioengineering↗

bioRxiv2025

The constitutive oncogenic and signaling activities of phospha-tidylinositol 3-kinase (PI3K) isoforms p110β and p110δ

AbstractThe p110{beta} and p110{delta} isoforms of the catalytic subunit of phosphatidylinositol 3-kinase (PI3K) show enhanced oncogenic and signaling activities as compared with the p110 protein. The adapter binding domains (ABDs) of p110{beta} and p110{delta} contain an isoform-specific PXXP mo- tif. Mutations of this PXXP to AXXA diminish the oncogenic and signaling activities. This loss of function can be compensated by placing a gain-of-function mutation in the helical domain of the P/A mutants. The P/A mutants still associate with the regulatory p85 subunit, but the affinity of this interaction is decreased. p110 with the ABD of either p110{beta} or p110{delta} shows an increase of oncogenic and signaling activities, whereas p110{beta} or p110{delta} with the ABD of p110 have greatly reduced oncogenic and signaling activities. Introducing the PXXP motif in the ABD of p110 re- sults in a significant gain of function. We conclude that the PXXP motif in the ABD of p110{beta} and p110{delta} is essential for the elevated oncogenic and signaling activities of these isoforms. We pro- pose that the PXXP motif in the ABD of p110{beta} and p110{delta} affects the interaction with the iSH2 domain of p85, shifting the regulatory SH2 domains into less effective inhibitory conformations.

cancer biology↗

bioRxiv2025

The Role of Paraventricular Nucleus of Thalamus in Sleep Disturbance Induced by Withdrawal from Repeated Ethanol Exposure

Sleep disturbance is known to be comorbid with withdrawal from repeated ethanol exposure and could be a negative reinforcement for the majority of people with alcohol use disorder (AUD). The paraventricular nucleus of the thalamus (PVT) has been highlighted for its function in integrating arousal states and associated modulation in sleep homeostasis. However, there is limited understanding of the involvement of PVT neurons in regulating sleep patterns, especially during withdrawal from chronic ethanol exposure. In this study, we investigated the potential function of the PVT in sleep disturbance during ethanol withdrawal using electrophysiology, in vivo calcium imaging, biochemical, and chemogenetic approaches. At 24 hours post-withdrawal from chronic intermittent ethanol exposure (CIE) for four weeks, there is an increase in wake time and a decrease in non-rapid eye movement (NREM) sleep. The calcium transient levels in the PVT neurons are positively correlated with the transition from sleep to wakefulness. CIE elevates the PVT neuronal activity in a subregion-specific manner, resulting in a significant rise in cFos levels in the anterior PVT (aPVT). Temporal suppression of aPVT excitatory neurons via chemogenetics ameliorates the disturbance in sleep patterns generated by CIE. The aPVT has a notable distinction in the expression of the m-type potassium channel subunit, KCNQ2, with a higher expression level compared to the posterior PVT (pPVT). While the expression of KCNQ2 in the aPVT is reduced in CIE mice, the restoration of KCNQ2 expression using viral gene transfer within the aPVT alleviates the sleep disturbances produced by CIE. This data indicates a significant role of the PVT in sleep disturbance during ethanol withdrawal, which may partially be due to the downregulation of M-channels, hence underscoring M-channels in the PVT as a potential therapeutic target for sleep disturbance in alcohol use disorder.

neuroscience↗

bioRxiv2025

Reduced inhibition, bursting, and accelerated oscillations drive early hippocampal hyperactivity in Alzheimer's disease

Early hippocampal hyperactivity is a well-documented yet poorly defined phenomenon in Alzheimers disease (AD). While reported in both patients and animal models, its functional manifestations and underlying neurophysiological mechanisms in vivo remain unclear. Here, we address this gap using in vivo high-resolution patch-clamp, high-throughput single-unit, and local field recordings in young amyloidopathy mice, at a stage when A{beta} remains largely soluble. We uncover previously unidentified cellular mechanisms in vivo, characterised by reduced inhibitory synaptic input, hypoactivity of fast-spiking interneurons, and enhanced bursting in pyramidal neurons. At the network level, we reveal accelerated hippocampal oscillations, marked by increased theta and beta power, a departure from the conventional view of oscillation slowing in AD. Mechanistically, this acceleration stems from strengthened synchrony of excitatory currents at higher frequencies and an overall reduction in oscillation-associated inhibitory currents. Our findings provide the first direct in vivo evidence linking early hippocampal hyperactivity to specific synaptic transmission and network dysfunctions, resolving a long-standing ambiguity. Moreover, we propose accelerated oscillations in the hippocampus as a functional biomarker for early AD and a potential therapeutic target for restoring network stability before cognitive decline occurs.

neuroscience↗

bioRxiv2025

Glutamatergic Dysfunction of Astrocytes in Paraventricular Nucleus of Thalamus Contributes to Adult Anxiety Susceptibility in Adolescent Ethanol Exposed Mice

Repeated ethanol exposure during adolescence increases the risk for displaying anxiogenic phenotype in adulthood, but the underlying mechanisms are not fully understood. The paraventricular nucleus of thalamus (PVT) has been considered a hub brain area for controlling the anxiety network in the brain. Recent structural and functional investigations indicate that the PVT exhibits diverse neural signals aligned with early-life events, which are highly linked with anxiety-like behaviors. However, it remains unknown if repeated ethanol exposure during adolescence will affect the coordinated brain activities of the PVT in adulthood, and consequent behavioral adaptation. Here we show that adolescent repeated intermittent ethanol exposure (AIE) triggers anxiety-like behaviors and parallelly induces the glutamatergic adaptation in the PVT after four weeks withdrawal from the last ethanol exposure. The firing rates, along with the spatiotemporal calcium transients in the PVT neurons during behavior were increased in the AIE mice compared to those in the counterpart control mice. Importantly, with the chemogenetic inhibition of the PVT neurons, we found alleviated the anxiety-like behavior in the AIE mice. The increased neuronal activities in the PVT of AIE mice was, at least partly, via the reduction of GLT1 (an astrocyte dominant glutamate transporter, known as EAAT2, slc1a2). Our non-invasive magnetic resonance spectroscopy (MRS) measures also showed an increase in glutamate/GABA ratio in the thalamic area including the PVT of the GLT1 conditional knock-down mice, which exhibited the heightened anxiety-like behavior. In addition, while the selective knock-out of GLT1 in the astrocytes of PVT in the alcohol naive mice induces anxiogenic phenotypes, the selective upregulation of GLT1 in the PVT astrocytes of the mice that were treated with AIE paradigm alleviated the anxiety-like behaviors as well. These findings highlight the significant role of PVT astrocytic GLT1 in the anxiogenic phenotype in adulthood induced by withdrawal from adolescent repeated ethanol exposure, suggesting that GLT1 in the PVT could serve as a therapeutic target for alcohol use disorder and comorbid emotional disorders.

neuroscience↗

bioRxiv2025

Preconditioning Matters: Enhancing or Impairing Antitumor Immunity with DC Vaccines

Preconditioning regimens are essential for the success of adoptive cell therapies like CAR T-cells due to effects on the T-cell response, yet they are underexplored and generally absent from cancer vaccine clinical trials. To address this gap, we evaluated the impact of various preconditioning strategies on dendritic cell (DC) vaccine efficacy in a murine tumor model. Mice bearing syngeneic KP tumors expressing ovalbumin received preconditioning with low-dose radiation (LD RT; whole body or tumor only), cyclophosphamide, paclitaxel, LD RT plus cyclophosphamide, or no preconditioning, followed by administration of antigen-loaded DC1s. Tumor growth, survival, and antigen-specific CD8+ T-cell responses were assessed. LD RT preconditioning, whether whole body or tumor-directed, significantly enhanced vaccine-induced antitumor CD8+ T-cell responses and improved survival compared to DC vaccine alone and all other groups. Cyclophosphamide preconditioning reduced vaccine efficacy and negated the benefits of LD RT, while paclitaxel had no significant effect. Notably, whole-body LD RT induced the strongest antigen-specific T-cell response. These findings demonstrate that, similar to CAR T-cell therapy, preconditioning regimens can significantly influence cancer vaccine outcomes. Rational selection of preconditioning agents may either maximize or minimize the therapeutic potential of DC cancer vaccines, and should be considered carefully in clinical trials.

immunology↗

bioRxiv2025

From Transcripts to Cells: Dissecting Sensitivity, Signal Contamination, and Specificity in Xenium Spatial Transcriptomics

Spatial transcriptomics has transformed our ability to map gene expression within intact tissues at cellular and subcellular resolution. Among current platforms, Xenium is widely adopted for its reliability, accessibility, and high data quality. Yet, the properties and limitations of Xenium-derived data remain poorly characterized. Here, we present one of the most comprehensive Xenium datasets to date, encompassing over 40 breast and lung tumor sections profiled using a diverse set of gene panels. Leveraging this resource, we systematically dissect technical noise--including transcript diffusion--alongside assay specificity, panel performance, and segmentation strategies. Our comparison of targeted panels with the newer 5K panel reveals that although the latter captures more transcripts overall, it suffers from reduced per-gene sensitivity and persistent diffusion, even with enhanced chemistry. We demonstrate that single-nucleus RNA-seq (snRNA-seq) markedly improves cell type annotation and enables more precise quantification of diffusion. Building on this, we introduce SPLIT (Spatial Purification of Layered Intracellular Transcripts), a novel method that integrates snRNA-seq with RCTD deconvolution to enhance signal purity. SPLIT effectively resolves mixed transcriptomic signals, improving background correction and cell-type resolution. Together, our findings provide a critical benchmark for Xenium performance and introduce a scalable strategy for signal refinement--advancing the accuracy and utility of spatial transcriptomics.

cancer biology↗

bioRxiv2025

Altered Duodenal Mucosa-Associated Microbiota and Immune Profiles in Functional Dyspepsia: A Study of Host-Microbiome Homeostasis

Recent work suggests an altered duodenal mucosa-associated microbiota (MAM) in patients with functional dyspepsia (FD) when compared to outpatient controls, these differences may reflect alterations in host-microbiome homeostasis. Given that specific mucosal immune signatures have been identified in FD, we hypothesised that these changes would be associated with specific microbial changes. We aimed to profile the duodenal MAM to identify microbes associated with known changes in FD mucosal and peripheral immune homeostasis. Duodenal biopsies were collected from 11 asymptomatic outpatient controls and 17 FD patients. Separate biopsies were collected for MAM 16S rRNA amplicon sequencing, histological evaluation, and mucosal lamina propria mononuclear cell (LPMC) isolation. Where available peripheral blood mononuclear cells (PBMC) were isolated from whole blood. PBMC and LPMC populations were analysed for CD4 and CD8 T cell populations by flow cytometry. Initial comparisons of histological and immune measures revealed significant differences between FD and outpatient controls, with decreased villi goblet cells and increased LPMC CD4 Central Memory, LPMC CD8, and PBMC CD4+ Central Memory Th17 in FD compared to controls. While microbiome profiles varied between groups, specific associations with the histological and immunological scoring found that in controls, villi goblet cells correlated positively with Massilia and negatively with Exiguobacterium, while FD patients showed no significant correlations. Additionally, controls exhibited a negative correlation between LPMC CD4 Central Memory and Veillonella, with FD patients showing no significant correlation. Notably, FD patients demonstrated a significant negative correlation between LPMC CD8 and Sulfophobococcus, and a positive correlation between PBMC CD4+ Central Memory Th17 and both Gemella and Fusobacterium. ImportanceWhile several papers have reported the alteration in FD duodenal MAM, study numbers are low and consensus on specific biomarker signatures within the microbiome have not been reached. Our findings contribute to this growing body of evidence, indicating that patients with FD exhibit distinct alterations in duodenal MAM and immune profiles compared to outpatient controls. Furthermore, the immune-microbiome associations present in control populations were absent in FD patients suggesting a loss of host-microbiome homeostasis that may contribute to FD pathophysiology. Our work highlights potential microbial biomarkers which may be a consequence or driver of the mucosal micro-inflammation state which is a characteristic of FD. Future work is required to validate whether these specific microbes are responsible for driving inappropriate host immune responses to inform treatment strategies and assist with disorder diagnosis.

microbiology↗

bioRxiv2025

Scalable, high-throughput isolation of extracellular vesicles using electrokinetic-assisted mesh filtration

As extracellular vesicles (EVs) are increasingly recognized for their superior functions for therapeutics, the need for large-scale EV isolation technology is becoming more critical for clinical and industrial applications. Most existing EV isolation methods are optimized for small-scale laboratory samples, limiting their efficiency and scalability for large-scale production. Here, an electrokinetic-assisted filtration system (ExoFilter), which introduces charge interaction into physical mesh flow filtration, is proposed as a new candidate to address the challenges of scalable EV isolation. The hybrid filtration system demonstrates outstanding high-throughput EV isolation performance (a flux of [~]750 mL/min) using only a coarse physical filter, by electrokinetically arresting EVs flowing through the filter lattice. Furthermore, the recovery efficiency of ExoFilter, analyzed based on the ELISA results, was found to be approximately 98%, demonstrating the filters exceptional efficiency in EV isolation. Additionally, ExoFilter enables the rapid isolation of EVs from small samples as little as 200 {micro}L, facilitating quick and easy blood-based EV research. Furthermore, low-molecular-weight albumin from plasma samples was effectively removed. The high-throughput and high-efficiency characteristics of ExoFilter make it well-suited for scalable EV production, offering greater convenience for various clinical applications. HighlightsO_LIElectrokinetic-assisted mesh filtration (ExoFilter) enables scalable and rapid isolation of extracellular vesicles (EVs). C_LIO_LIA high throughput of [~]750 mL/min is demonstrated while maintaining high yield and purity. C_LIO_LIExoFilter effectively removes albumin contaminants from EVs through size-exclusive electrokinetic-assisted mesh filtration. C_LIO_LIEfficient EV isolation performance is achieved for human plasma, saliva, urine, and cell culture media. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/645682v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@7ac3d5org.highwire.dtl.DTLVardef@1ced638org.highwire.dtl.DTLVardef@521c63org.highwire.dtl.DTLVardef@c5bd49_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Precise, Specific, and Sensitive De Novo Antibody Design Across Multiple Cases

The precision design of antibodies, which naturally recognize diverse molecules through six variable loops, remains a critical challenge in therapeutic molecule discovery. In this study, we demonstrate that precise, sensitive, and specific antibody design can be achieved without prior antibody information across eight distinct target proteins. For each target, binders were identified from a yeast display scFv library of approximately 106 sequences, constructed by combining 102 designed light chain sequences with 104 designed heavy chain sequences. Binders with varying binding strengths were identified for all eight targets, including a case where no experimentally resolved target protein structure was available, demonstrating the highest level of precision compared to previous de novo antibody design reports. To further validate the designed antibodies, they were characterized in the IgG format for five distinct targets. The antibodies exhibited favorable developability properties, positioning them as promising hit or lead candidates. Notably, for one target in particular, the IgG-formatted antibodies exhibited affinity, activity, and developability, comparable to a commercial antibody, highlighting the sensitivity of the design method. Furthermore, binders capable of distinguishing closely related protein subtypes or mutants were identified, demonstrating that the method can achieve high molecular specificity. Cryo-EM analysis experimentally validated the designs accuracy by confirming that the key binding interactions were precisely as designed. These findings underscore the effectiveness of precision molecular design based on atomic-accuracy structure prediction. This study establishes computational antibody design as a viable approach for generating therapeutic molecules with tailored properties, with promising potential for achieving the efficacy and safety required for successful therapeutics.

bioengineering↗

bioRxiv2025

Combinatorial effects of Zingiber officinale and Citrus limon juices: Hypolipidemic and antioxidant insights from in vivo, in vitro, and in silico investigations

The combined use of Zingiber officinale (ginger) and Citrus limon (lemon) has long been valued in traditional medicine for their significant therapeutic properties, particularly in supporting cardiovascular health. However, their synergistic effects on hyperlipidemia and oxidative stress, both critical risk factors for cardiovascular diseases, remain underexplored. This study employed a multidisciplinary approach including in vivo (mice model with triton WR-1339-induced hyperlipidemia), in vitro (antioxidant assays such as DPPH and ABTS), and in silico (molecular docking targeting HMG-CoA reductase) to evaluate the hypolipidemic and antioxidant potential of ginger and lemon juices. Phytochemical analysis identified high concentrations of bioactive compounds, with ginger juice containing 6-gingerol (15.54% peak area) and lemon juice featuring hesperidin (13.85% peak area) and rutin (5.57% peak area). These compounds were instrumental in the observed pharmacological effects. The combined formulation significantly improved lipid profiles, reducing total cholesterol by 54.3%, triglycerides by 49.8%, and LDL by 58.1% while increasing HDL by 47.6% compared to hyperlipidemic controls (p < 0.05). Antioxidant assays revealed high free radical scavenging activity, with IC50 values of 30.92 {+/-} 2.00 {micro}g/mL for ABTS and 44.94 {+/-} 1.02 {micro}g/mL for DPPH in the combined formulation. Molecular docking confirmed strong binding affinities, with hesperidin and 6-gingerol showing binding energies of -10.4 kcal/mol and -9.6 kcal/mol, respectively, against HMG-CoA reductase. In conclusion, combining Zingiber officinale and Citrus limon juices demonstrates robust hypolipidemic and antioxidant effects, significantly outperforming the individual juices. These results support their potential as natural therapeutic agents for managing hyperlipidemia and oxidative stress. Further clinical studies are recommended to validate these promising findings and explore their application in nutraceutical development.

pharmacology and toxicology↗

bioRxiv2025

Pallidal prototypic neuron and astrocyte activities regulate flexible reward-seeking behaviors

Behavioral flexibility allows animals to adjust actions to changing environments. While the basal ganglia are critical for adaptation, the specific role of the external globus pallidus (GPe) is unclear. This study examined the contributions of two major GPe cell types--prototypic neurons projecting to the subthalamic nucleus (ProtoGPe[->]STN neurons) and astrocytes--to behavioral flexibility. Using longitudinal operant conditioning with context reversals, we found that ProtoGPe[->]STN neurons dynamically represent contextual information correlating with behavioral optimality. In contrast, GPe astrocytes exhibited gradual contextual encoding independent of performance. Deleting ProtoGPe[->]STN neurons impaired adaptive responses to changing action-outcome contingencies without altering initial reward-seeking acquisition, highlighting their specific role in enabling behavioral flexibility. Furthermore, we discovered that ProtoGPe[->]STN neurons integrate inhibitory striatal and excitatory subthalamic inputs, modulating downstream basal ganglia circuits to support flexible behavior. This research elucidates the complementary roles of ProtoGPe[->]STN neurons and astrocytes in cellular mechanisms of flexible reward-seeking behavior.

neuroscience↗

bioRxiv2025

Dicer loss in Muller glia leads to a defined sequence of pathological events beginning with cone dysfunction

PurposeThe loss of Dicer in Muller glia (MG) results in severe photoreceptor degeneration as it occurs in retinitis pigmentosa or AMD. However, the sequence of events leading to this severe degenerative state is unknown. The aim of this study was to conduct a chronological functional and structural characterization of the pathological events in MG-specific Dicer-cKO mice in vivo and histologically. MethodsTo delete Dicer and mature microRNAs (miRNAs) in MG, two conditional Dicer1 knock-out mouse strains namely RlbpCre:Dicer-cKOMG and GlastCre:Dicer-cKOMG, were created. Optical coherence tomography (OCT), electroretinograms (ERGs) as well as histological analyses were conducted to investigate structural and functional changes up to six months after Dicer deletion. ResultsDicer/miRNA loss in MG leads to 1) impairments of the external limiting membrane (ELM) - retinal pigment epithelium (RPE), 2) cone photoreceptor dysfunction and 3) retinal remodeling and functional loss of the inner retina, 1, 3 and 6 months after Dicer loss, respectively, in both strains. Furthermore, in the Rlbp:Dicer-cKOMG strain, rod photoreceptor impairment was found 4 months after Dicer depletion (4) accompanied by alteration of RPE integrity (5). ConclusionsMG Dicer loss in the adult mouse retina impacts cone function prior to any measurable changes in rod function, suggesting a pivotal role for MG Dicer and miRNAs in supporting cone health. A partially impaired RPE however seems to accelerate rod degeneration and overall degenerative events.

neuroscience↗

bioRxiv2025

The calcineurin pathway regulates extreme thermotolerance, cell membrane and wall integrity, antifungal resistance, and virulence in Candida auris

Candida auris, an emerging fungal pathogen characterized by its multidrug resistance and high mortality rates, poses a significant public health challenge. Despite its importance, the signaling pathways governing virulence and antifungal resistance in C. auris remain poorly understood. This study investigates the calcineurin pathway in C. auris, critical for virulence and antifungal resistance in other fungal pathogens. Calcineurin, a calcium/calmodulin-dependent protein phosphatase, comprises a catalytic subunit (Cna1) and a regulatory subunit (Cnb1) in C. auris. Our findings reveal that deletion of CNA1 or CNB1 disrupts extreme thermotolerance and cell membrane and wall integrity, leading to increased susceptibility to azoles and echinocandins. Moreover, we identified a downstream transcription factor, Crz1, which plays a central role in this pathway in other fungal species. Deletion of CRZ1 resulted in similar membrane integrity defects observed in the cna1{Delta} and cnb1{Delta} mutants and increased susceptibility to azole drugs. Supporting it, fluconazole treatment induced Crz1 nuclear translocation in a Cna1-dependent manner. However, unlike cna1{Delta} and cnb1{Delta} mutants, the crz1{Delta} mutant displayed increased resistance to echinocandins, suggesting the opposing roles for Crz1 in regulating cell wall integrity. Nevertheless, echinocandins also promoted Crz1 nuclear translocation via Cna1, underscoring the complex regulatory mechanisms at play. Cna1 was found to be required for virulence in both the Drosophila systemic infection model and the murine skin infection model. However, in a systemic murine infection model, both calcineurin and Crz1 appeared dispensable for C. auris virulence. Our findings highlight that the evolutionarily conserved calcineurin pathway employs distinct regulatory mechanisms to perform divergent roles in regulating cell wall and membrane integrity, antifungal drug resistance, and virulence in C. auris. Author SummaryThe fungal pathogen Candida auris presents a global health threat due to its multidrug resistance and high mortality rates. Despite its clinical significance, the molecular mechanisms underlying its virulence and antifungal resistance remain poorly understood. This study investigates the complex role of the calcineurin signaling pathway in C. auris pathogenicity. Deletion of the calcineurin complex impairs extreme thermotolerance and compromises cell membrane and wall integrity, leading to increased susceptibility to azoles and echinocandins, antifungal agents targeting the cell membrane and wall, respectively. We also identified the Crz1 transcription factor as a downstream target of calcineurin signaling. Interestingly, unlike calcineurin mutants, Crz1 mutants are susceptible only to cell-membrane-targeting azoles but surprisingly exhibit increased resistance to cell-wall-targeting echinocandins, suggesting that the calcineurin pathway may regulate multiple transcription factors, including Crz1. Additionally, calcineurin was found to be essential for virulence in vivo, using two different animal infection models, Drosophila and mice. These findings highlight the essential role of the calcineurin pathway in C. auris virulence, offering novel insights into its role in antifungal resistance and virulence, and paving the way for targeted therapies.

microbiology↗

bioRxiv2024

A PRDM16-CtBP1/2 Complex Interacts with HDAC1/2 to Regulate Transcriptional Programs of Neurogenesis and Guide Cortical Neuron Migration

Chromatin regulation of transcriptional enhancers plays a central role in cell fate specification and differentiation. However, how the coordinated activity of transcription factors and chromatin-modifying enzymes regulates enhancers in neural stem cells (NSCs) and dictates subsequent stages of neuronal differentiation and migration is not well understood. The histone methyltransferase PRDM16 is expressed in NSCs of the developing mouse and human cerebral cortex and is essential for determining the position of upper-layer cortical neurons. Here, we report that PRDM16 interacts with C-terminal binding protein 1 (CtBP1) and CtBP2 to control the transcriptional programs of cortical neurogenesis and regulate upper-layer neuron migration. PRDM16 and CtBP1/2 co-regulate enhancers by interacting with histone deacetylase 1 (HDAC1) and HDAC2, and lysine-specific demethylase 1 (LSD1). In addition, our results suggest that the CCCTC-binding factor CTCF plays a key role in recruiting CtBP1/2 to cortical enhancers. These findings underscore that reduced interactions between PRDM16 and ubiquitous chromatin regulators may contribute to neurodevelopmental deficits in patients with PRDM16 haploinsufficiency.

developmental biology↗