Biology subjects

Kumar, V.

Publications and source records attributed to Kumar, V..

At least 91 records · Page 5Linked to original sources

bioRxiv2025

Metagenomic Profiling of Drinking Water Microbiomes: Insights into Microbial Diversity and Antimicrobial Resistance

Monitoring microbial components in drinking water is as essential as tracking its chemical composition. Although traditional culture-based methods provide valuable insight into microbial morphology and behaviour, their scope is restricted to culturable species. With the advent of high-throughput sequencing, we can now detect a wider range of microbes in any ecosystem, along with efficient insights into their functional potential and metabolic capabilities. In this study, metagenomic analyses were performed to fully understand the microbiome of drinking water supplied through public distribution systems in an Indian city. Our findings identified bacteria from the phyla Pseudomonadota, Planctomycetota, Bacteroidota, and Actinomycetota, consistent with previous studies of drinking water microbiomes of other countries. At the species level, Afipia carboxidovorans, Klebsiella pneumoniae, Pseudomonas aeruginosa, Sphingopyxis macrogoltabida, and Variovorax paradoxus were identified as members of the core microbiome. It was observed that the temperature of the water samples, even as little as a 5{o}C increase, influenced the composition and diversity of the microbial communities. No significant correlation was detected between the abundance of microbial species and the metal concentration in the sample. In addition, we traced the distribution of antibiotic resistance genes (ARGs), finding widespread resistance to aminoglycosides, tetracyclines, and macrolides in samples. In particular, ARGs such as adeF and ermR, which are known to be associated with multidrug resistance, were detected. Although this study did not directly assess the pathogenicity or mobility of these genes, their presence in potable water raises potential public health concerns due to the possibility of horizontal gene transfer (HGT) in environmental settings. Therefore, continuous monitoring of antibiotic resistance genes (ARGs) is imperative to accurately evaluate long-term risks and to guide evidence-based water quality management strategies. In summary, this study provides a comprehensive metagenomic overview of drinking water microbiota, ARGs, and water quality, offering a foundation for future surveillance and risk mitigation strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/636216v3_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@819c0org.highwire.dtl.DTLVardef@1d29372org.highwire.dtl.DTLVardef@1ce3cf9org.highwire.dtl.DTLVardef@1050997_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

FUS post-transcriptional splicing is autoregulated via RNA condensation with therapeutic potential for ALS-FUS

Mutations in the FUS gene cause aggressive and often juvenile forms of amyotrophic lateral sclerosis (ALS-FUS). In addition to mRNA, the FUS gene gives rise to a partially processed RNA with retained introns 6 and 7. We demonstrate that these FUSint6&7-RNAs form nuclear condensates scaffolded by the highly structured intron 7 and associated with nuclear speckles. Using hybridization-proximity labelling proteomics, we show that the FUSint6&7-RNA condensates are enriched in splicing factors and the m6A reader YTHDC1. These ribonucleoprotein structures facilitate post-transcriptional FUS splicing and depend on m6A/YTHDC1 for their maintenance. FUSint6&7-RNAs become hypermethylated in cells expressing mutant FUS, leading to their enhanced condensation and consequently, splicing. We further demonstrate that FUS protein is repelled by m6A. Thus, ALS-FUS mutations may cause an abnormal activation of FUS post-transcriptional splicing via altered RNA methylation. Strikingly, ectopic expression of FUS intron 6&7 sequences dissolves the endogenous FUSint6&7-RNA condensates, downregulating FUS mRNA and protein. Overall, we describe an RNA condensation-dependent mechanism regulating FUS splicing that can be harnessed for developing new therapies.

cell biology↗

bioRxiv2025

Polymerase Eta Recruits FANCD2 to Common Fragile Sites to Maintain Genome Stability

The replicative polymerase delta is inefficient copying repetitive DNA sequences. Error-prone translesion polymerases have been shown to switch with high-fidelity replicative polymerases to help navigate repetitive DNA. We and others have demonstrated the importance of one such translesion polymerase, polymerase Eta (pol eta), in facilitating replication at genomic regions called common fragile sites (CFS), which are difficult-to-replicate genomic regions that are hypersensitive to replication stress. However, the mechanistic basis for pol etas role in facilitating DNA replication at CFS and(or) at other genomic regions is currently unclear. Importantly, the functional importance of three non-catalytic domains of pol eta, the Ubiquitin-binding Zinc finger (UBZ), PCNA interacting protein (PIP) domain, and the F1 domain which mediates its switch with replicative DNA polymerases in mediating replication stress, especially at CFS loci is not clear. Here, we report that the PIP and UBZ domains of Pol Eta are both critical for its role in mediating cellular replication stress, especially at CFS. The absence of either domain induced elevated replication stress, replication stalling and DNA damage accumulation genome wide. This effect was even more pronounced at CFS loci leading to the accumulation of under replication DNA in G2/M. Importantly, while the inactivation of the UBZ domain resulted in a robust FANCD2 monoubiquitylation (a prominent marker of FANCD2 activation), FANCD2 recruitment genome wide was significantly impacted, especially at CFSs such as FRA16D. These S-phase phenotypes result in ssDNA gap formation and the persistence of under-replicated genomic regions upon transition to G2/M. While post-replicative gap filing/ repair by Mitotic DNA synthesis is activated in the mutants, it only effectively resolves UFBs in the F1* cells. The PIP*, UBZ* and pol eta-/- cells unfortunately manifest excessive toxic cytosolic DNA that instigates a strong innate immune response. These results collectively show that translesion polymerase Eta functions in a common pathway with FANCD2 to prevent replication perturbation and instability at CFS loci.

molecular biology↗

bioRxiv2025

NF-κB mediates transactivation of HNRNPD, resulting in PTEN destabilization and constitutive activation of the PI3K-AKT pathway in oral cancer cells

Heterogeneous Ribonucleoprotein D (hnRNPD), an RNA binding protein transcriptionally upregulated by NF-{kappa}B transcription factor, is associated with poor outcome of Oral Squamous Cell Carcinoma (OSCC). However, the role of hnRNPD in OSCC remains elusive. This study reveals that hnRNPD positively affects the proliferation, migration, invasion, and survival of OSCC cells. Transcriptome profiling in hnRNPD knockout cells identified significant upregulation of PTEN and inhibition of the PI3K/AKT/mTOR axis. HnRNPD mediates the destabilization of PTEN mRNA by binding to the class II AU-Rich Element (ARE) in 3UTR of PTEN. The expression of hnRNPD and PTEN are strongly negatively correlated in OSCC tissue specimens, further corroborating hnRNPD-mediated PTEN destabilization. The hnRNPD knockout inhibited autophagy, evident by an accumulation of autophagic vesicles and decreased autophagic flux. Mechanistically, the hnRNPD knockout reduced the expression of NF-{kappa}B, eventually downregulating its transcriptional target LC3b, a key mediator of autophagy. SA-{beta}-Galactosidase staining in hnRNPD KO cells conclusively demonstrated the onset of cellular senescence. The present study demonstrates hnRNPD-driven positive modulation of autophagy via NF-{kappa}B, independent of the PI3K/AKT/mTOR axis, highlighting it as a novel therapeutic target for treating oral cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/631360v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@11f6844org.highwire.dtl.DTLVardef@f65124org.highwire.dtl.DTLVardef@1492571org.highwire.dtl.DTLVardef@13931dd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIHnRNPD mediates oral cancer cell proliferation, migration, invasion, and survival. C_LIO_LIHnRNPD acts as a novel regulator of the PI3K/AKT/mTOR axis by destabilization of PTEN. C_LIO_LINF-{kappa}B/RelA downregulated on knockout of HNRNPD, inhibiting autophagy through downregulating its transcriptional target LCB-II. C_LIO_LIHnRNPD mediates cellular senescence in oral cancer cells. C_LI

cancer biology↗

bioRxiv2024

Engineering Multiplexed Synthetic Breath Biomarkers as Diagnostic Probes

Breath biopsy is emerging as a rapid and non-invasive diagnostic tool that links exhaled chemical signatures with specific medical conditions. Despite its potential, clinical translation remains limited by the challenge of reliably detecting endogenous, disease-specific biomarkers in breath. Synthetic biomarkers represent an emerging paradigm for precision diagnostics such that they amplify activity-based biochemical signals associated with disease fingerprints. However, their adaptation to breath biopsy has been constrained by the limited availability of orthogonal volatile reporters that are detectable in exhaled breath. Here, we engineer multiplexed breath biomarkers that couple aberrant protease activities to exogenous volatile reporters. We designed novel intramolecular reactions that leverage protease-mediated aminolysis, enabling the sensing of a broad spectrum of proteases, and that each release a unique reporter in breath. This approach was validated in a mouse model of influenza to establish baseline sensitivity and specificity in a controlled inflammatory setting and subsequently applied to diagnose lung cancer using an autochthonous Alk-mutant model. We show that combining multiplexed reporter signals with machine learning algorithms enables tumor progression tracking, treatment response monitoring, and detection of relapse after 30 minutes. Our multiplexed breath biopsy platform highlights a promising avenue for rapid, point-of-care diagnostics across diverse disease states.

bioengineering↗

bioRxiv2024

An integrated and scalable rodent cage system enabling continuous computer vision-based behavioral analysis and AI-enhanced digital biomarker development

1Home cage monitoring enables continuous observation of animals in familiar environments. It has large utility in preclinical testing, mechanistic studies, animal husbandry, and the general practice of the Replacement, Reduction, Refinement (3R) principles. Despite its acknowledged utility, home cage monitoring has not been broadly adopted. This is mainly due to the complexity of the tasks that must be solved to have a successful system that includes hardware and sensor development, data management, machine vision expertise, behavioral expertise, support, and user training. Here, we describe the Digital In Vivo System (DIV Sys), a modern end-to-end system for video-based rodent home cage monitoring. The DIV Sys consists of a cloud-based study design, monitoring, display, and visualization app (DIV App), local hardware for data acquisition cages (DAX), a machine learning model for tracking mice (mHydraNet) optimized for speed and accuracy, a study display and visualization app, and an advanced behavior quantification workbench (DIV Data). The platform seamlessly manages terabytes of video data in the cloud and is built around enterprise-level security and data standards. Collaborative tools enable teams across geographical locations to work together. As a demonstration of its utility, we used DIV Sys to analyze over a century of mouse videos across multiple geographic locations. We also characterized home cage behavior of 8 mouse strains and carried out customized video analysis. Together, we present a scalable home cage monitoring system for advanced behavior quantification for the rodent research community.

animal behavior and cognition↗

bioRxiv2024

A single-cell map of hypertension

Hypertension is a leading risk factor for disease burden and death worldwide. Several organ systems are involved in the development of hypertension, which contributes to stroke, heart disease, and kidney disease. Despite the broad health relevance, our understanding of the molecular landscape in hypertension is limited and lags other major diseases. Here we report an extensive analysis of the molecular landscape in hypertension and its end-organ damage and uncover novel mechanisms linking human genetic variants to the development of these diseases. We obtained single-nucleus RNA-seq (612,984 nuclei), single-nucleus ATAC-seq (179,637 nuclei), or spatial transcriptome data from five organs (hypothalamus, kidney, heart, 3rd order mesenteric artery, middle cerebral artery) in three mouse and rat models under twelve experimental conditions. More than one third of all hypertension research in animal models involves these three models. We identified both model-specific and convergent responses in cell types, genes, and pathways. By integrating our data with human genomic data, we partitioned the blood pressure and end-organ damage traits into cell type-specific transcriptional contributions and cell types common across multiple traits. Using genomic editing in animal models and human induced pluripotent stem cells, we extended key findings and identified new mechanisms linking human genetic variants to the development of hypertension and related renal injury. We anticipate that our rich data sets and findings will broadly drive forward the research of hypertension and hypertensive end-organ damage. Our approach of integrating multi-model and multi-tissue single-cell analysis with human genetic data and in vivo and in vitro genome editing can be applied to investigate other complex traits.

physiology↗

bioRxiv2024

Erythrocytic plasma membrane calcium ATPase (PMCA4b) variations mediate redox imbalance to determine artemisinin sensitivity in malaria

Artemisinin resistance in Plasmodium falciparum remains a major public health challenge for the ongoing malaria elimination programs. Till now, artemisinin resistance is linked with the variations in the genome of P. falciparum which are mainly clustered in the Pfkelch13 gene. Several studies reported artemisinin resistance in the malaria endemic areas without finding any evidence of genetic variation in parasite for this occurrence. This study interrogated whether host genetic variations have any association with artemisinin sensitivity of malaria parasite. We investigated the relationship of 1) genetic variations in the human ATP2B4 gene and 2) the resultant surface expression of plasma membrane calcium ATPase (PMCA4b) with the intraerythrocytic levels of calcium, reactive oxygen species (ROS) and the resistance towards artemisinin in the intraerythrocytic parasite. This study found negative correlation of PMCA4b expression level with intraerythrocytic calcium and ROS levels. Further in-vitro growth assays revealed that artemisinin sensitivity is reduced in the parasites growing within the RBCs having low PMCA4b and high oxidative stress. Overall, this study highlights the strong association of host PMCA4b in cellular calcium mediated redox imbalance, which significantly contributes to artemisinin resistance in malaria parasite. Hence, this is the first study to document the effect of host variations on artemisinin sensitivity of the parasite. We further emphasize that many redox modulating RBC polymorphisms that are prevalent in malaria endemic areas could influence artemisinin resistance and can serve as potential biomarkers for predicting therapeutic response. Thus, detailed population specific research may provide new insights for personalized malaria treatment and may inform future drug development strategies. SignificanceThe human host and malaria parasite share a closely interconnected relationship hence host variations could influence parasite drug resistance. This study demonstrates that reduced PMCA4b expression, the primary calcium efflux pump in RBCs, significantly increases the erythrocytic calcium and reactive oxygen species (ROS) levels, thereby decreasing the P. falciparum growth as well as artemisinin sensitivity. Many ROS inducing RBC polymorphisms, such as sickle cell, thalassemia, G6PD deficiency, PMCA4b variations etc. have emerged as strong malaria protective traits in several studies in malaria endemic areas. But these traits could also influence artemisinin resistance and can serve as potential biomarkers for predicting therapeutic response and highlights the importance of host erythrocytes oxidative microenvironment surveillance in monitoring antimalarial drug resistance.

microbiology↗

bioRxiv2024

Caloric restriction: A potential approach for mitigating neuronal damage: Lesson from cellular model of Alzheimer Disease

Alzheimers disease is a neurodegenerative disorder and characterized by amyloid beta accumulation, synaptic dysfunction, and oxidative stress, lacks effective therapies. Caloric restriction mimetics such as fisetin and chlorogenic acid, natural polyphenols with antioxidant and autophagy-inducing properties, show promise in mitigating age-related diseases. This study investigates their neuroprotective effects against amyloid beta induced toxicity in differentiated human neuroblastoma SHSY5Y cells. Amyloid beta exposure disrupted redox homeostasis, impaired autophagy, induced mitochondrial dysfunction, and exacerbating neuronal degeneration. Fisetin and chlorogenic acid treatments reversed these deleterious effects by restoring redox balance, suppressing reactive oxygen species and upregulating critical antioxidant enzymes like SOD1, GSR, and catalase. These compounds also attenuated amyloid beta induced mitophagy via reduced PINK1 expression and restored mitochondrial fusion by upregulating Mfn2. Autophagy-related pathways were significantly modulated, evidenced by increased AMPK and decreased mTOR mRNA levels, alongside elevated expression of ATG101, ATG13, ULK1, P62 and reduced ATG5 levels. Docking studies also revealed binding of fisetin and CGA within the binding pockets of AMPK and FKBP12 supporting their interaction. Furthermore, fisetin and CGA improved synaptic integrity by upregulating PSD95 and synaptophysin and reducing acetylcholinesterase expression. These findings highlight their potential in ameliorating amyloid beta induced neuronal toxicity through autophagy activation, synaptic preservation, and mitochondrial function enhancement. While this study demonstrates the transcriptional impact and binding affinities of these caloric restriction mimetics further translational and biophysical analyses are required to elucidate their mechanisms and confirm their therapeutic viability. This research underscores the potential of fisetin and CGA as neuroprotective agents, offering promising therapeutic avenues for combating related Alzheimers disease neuropathies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/628076v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1253dd0org.highwire.dtl.DTLVardef@1fd3ffforg.highwire.dtl.DTLVardef@1749d1dorg.highwire.dtl.DTLVardef@f71415_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

The CoREST complex inhibitor, corin, leads to decreased tumor growth, increased cellular differentiation and extended lifespan in atypical teratoid rhabdoid tumor xenograft models

BackgroundAtypical teratoid rhabdoid tumor (ATRT) is the most common malignant brain tumor in infants, and more than 60% of children with ATRT die from their tumor. ATRT is associated with mutational inactivation/deletion of SMARCB1, a member of the SWI/SNF chromatin remodeling complex, suggesting that epigenetic events play a critical role in tumor development and progression. Moreover, disruption of SWI/SNF allows unopposed activity of epigenetic repressors, which contribute to tumorigenicity. We therefore explored the role of the CoREST repressor complex in ATRT. MethodsWe evaluated the effects of the bifunctional LSD1/HDAC1/2 small molecule CoREST inhibitor, corin, on ATRT tumor cell growth, apoptosis, differentiation, gene expression and chromatin accessibility. ResultsWe found that corin inhibited the growth of ATRT cells regardless of their epigenetic subgroup, and was associated with increased tumor cell apoptosis and differentiation. ATAC-seq showed increases in chromatin accessibility in corin-treated ATRT cells, with changes seen at genes associated with neuronal differentiation and synaptic function. RNA-seq confirmed increased expression of neuronal differentiation genes and decreased DNA replication/cell cycle-associated genes in ATRT cells treated with corin. Corin suppressed orthotopic ATRT tumor growth, leading to significant extension of lifespan. In addition, increased histone acetylation (H3K9ac, H3K27ac) and methylation (H3K4Me1) was seen in corin-treated ATRT orthotopic xenografts, consistent with on-target pharmacodynamics. ConclusionThe CoREST inhibitor, corin, suppresses tumor growth, induces differentiation, and promotes apoptosis in ATRT, leading to significantly increased survival of mice bearing ATRT orthotopic xenografts. Our results suggest a potential application of CoREST complex inhibitors in patients with ATRT. Key PointsO_LICoREST complex inhibition by corin leads to decreased cell growth and increased apoptosis in ATRT C_LIO_LICorin promotes chromatin accessibility and neuronal differentiation in ATRT C_LIO_LICorin inhibits tumor growth and extends lifespan in ATRT animal models C_LI Importance of the StudyLoss of function of SMARCB1 is a hallmark of ATRT which leads to dysfunction of the mammalian SWI/SNF complex and an inability to counteract epigenetic repressor complexes. The CoREST complex functions as a chromatin remodeling complex that represses neuronal differentiation genes during development. Inhibition of the CoREST complex by corin in ATRT leads to decreased tumor cell growth, induction of apoptosis and increased survival of mice bearing ATRT orthotopic xenografts. These changes are associated with increased chromatin accessibility and expression of genes associated with neuronal differentiation. Corin therefore reverses the primary block of differentiation that maintains a stem cell state in ATRT, which contributes to tumorigenesis. These studies significantly improve our understanding of how to therapeutically address the underlying epigenetic drivers of ATRT and support further development of corin for ATRT.

cancer biology↗

bioRxiv2024

Aging disrupts spatiotemporal coordination in the cycling ovary

Throughout the female reproductive lifespan, the ovary completes hundreds of cycles of follicle development, ovulation, and tissue regeneration1-3. These processes rely on the precisely coordinated intricate multicellular interactions across time and space4. How aging disrupts these interactions, leading to an overall decline in reproductive and endocrine functions, remains understudied. To understand the multicellular dynamics that underlie ovarian function and their changes with age, here we use Slide-seq, a near-cellular spatial transcriptomics method, to profile 22 mouse ovaries across the reproductive cycle and chronological age, representing 610,620 near-cellular spots across 69 spatial transcriptomic profiles5,6. We develop a segmentation analysis to identify spatial niches that capture different states of folliculogenesis from static snapshots in situ, allowing us to examine the multicellular dynamics of 358 oocytes, 668 follicles, and 236 corpora lutea. We find that aging disrupts both the spatial organization and temporal coordination of folliculogenesis before the cessation of cycling, which may contribute to the dysregulation of hormone production and signaling. These disruptions are marked by altered immune cell dynamics, inflammatory signaling, and global tissue disorganization that impair the cyclic remodeling required for ovarian function. Our findings reveal how multicellular niches orchestrate ovarian function and demonstrate how age-related breakdown of tissue organization across time and space precedes reproductive decline.

genomics↗

bioRxiv2024

DIYNAFLUOR: An Affordable DIY Plug-and-Play Nucleic Acid Fluorometer for eDNA Quantification in Resource Limited Settings

Nucleic Acid (NA) fluorometry is widely employed for quantifying environmental DNA (eDNA) samples and their downstream DNA sequencing libraries, owing to its sensitivity, accuracy, and speed. However, the high cost of NA fluorometers presents a barrier to eDNA sequencing in resource limited settings (RLSs). For instance, at [~]$1.5-3.3k USD, current NA fluorometers present a greater capital cost than ONTs $1k USD portable MinION third-generation Nanopore sequencing platform. The collapse of international scientific device and consumable supply chains during the COVID-19 pandemic also highlighted the need for distributed manufacturing of molecular research tools to mitigate the impact of increased pricing to RLSs. To address these challenges, we have developed the "DIYNAFLUOR" (DIY Nucleic Acid FLUORometer), a portable, open-source, <$40 USD NA fluorometer, designed using readily available off-the-shelf components, simple 3D-printed parts, and plug-and-play, solder-free assembly. The DIYNAFLUOR was primarily designed to be compatible with the popular DNA-centric Qubit High Sensitivity (HS) and Broad Range (BR) assay kits. Notably, the DIYNAFLUOR demonstrated an in-assay Limit of Detection with the Qubit HS kit of 0.0028 ng/L, and an average absolute bias of 0.018 ng/L across a 0-10 ng/L working range using a 2-point linear calibration methodology. Device verification was performed by comparative measurements with a Qubit 4 fluorometer in a busy biotechnology laboratory, and build instructions were validated through assembly and qualification of three DIYNAFLUOR devices by researchers outside the primary design team. We also describe a custom "extreme"low-cost assay, <13{cents} USD per-measurement, that uses SYBR Safe dye to quantify DNA across a working range of 0-0.5 ng/L. This assay reports a lower sensitivity and accuracy than commercial kits but may be of use to RLSs in times of extreme resource constraints or as a teaching tool for STEM educators. To demonstrate its practical application for field-based eDNA analysis in RLSs, the DIYNAFLUOR was used to perform all quality control measurements throughout the preparation of a 16S and 18S metabarcoding library generated from eDNA extracted from Australian lake water, leading to the successful identification of Australian fauna via Nanopore sequencing. Finally, configurations of the DIYNAFLUOR for RNA and Protein quantification are briefly described. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/626200v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@28244dorg.highwire.dtl.DTLVardef@16749acorg.highwire.dtl.DTLVardef@15c31aorg.highwire.dtl.DTLVardef@bc03e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Recognizing amino acid sidechains in a medium resolution cryo-electron density map

Building an accurate atomic structure model of a protein into a cryo-electron microscopy (cryo-EM) map at worse than 3 [A] resolution is difficult. To facilitate this task, we devised a method for assigning the amino acid residue sequence to the backbone fragments traced in an input cryo-EM map (EMSequenceFinder). EMSequenceFinder relies on a Bayesian scoring function for ranking 20 standard amino acid residue types at a given backbone position, based on the fit to a density map, map resolution, and secondary structure propensity. The fit to a density is quantified by a convolutional neural network that was trained on [~]5.56 million amino acid residue densities extracted from cryo-EM maps at 3-10 [A] resolution and corresponding atomic structure models deposited in the Electron Microscopy Data Bank (EMDB). We benchmarked EMSequenceFinder by predicting the sequences of 58,044 distinct L-helix and {beta}-strand fragments, given the fragment backbone coordinates fitted in their density maps. EMSequenceFinder identifies the correct sequence as the best-scoring sequence in 77.8% of these cases. We also assessed EMSequenceFinder on separate datasets of cryo-EM maps at resolutions from 4 to 6 L. The accuracy of EMSequenceFinder (63.5%) was better than that of three tested state-of-the-art methods, including findMysequence (45%), ModelAngelo (27%), and sequence_from_map in Phenix (12.9%). We further illustrate EMSequenceFinder by threading the SARS-CoV-2 NSP2 sequence into eight cryo-EM maps at resolutions from 3.7 to 7.0 [A]. EMSequenceFinder is implemented in our open-source Integrative Modeling Platform (IMP) program. Thus, it is expected to be helpful for integrative structure modeling based on a cryo-EM map and other information, such as models of protein complex components and chemical crosslinks between them. EMSequenceFinder is available as part of our open source IMP distribution at https://integrativemodeling.org/.

biophysics↗

bioRxiv2024

Machine Learning Identifies Genes Linked to Neurological Disorders Induced by Equine Encephalitis viruses (EEV), Traumatic Brain Injuries (TBI), and Organophosphorus nerve agents (OPNA)

Venezuelan, eastern, and western equine encephalitis viruses (collectively referred to as equine encephalitis viruses---EEV) cause serious neurological diseases and are a significant threat to the civilian population and the warfighter. Likewise, organophosphorus nerve agents (OPNA) are highly toxic chemicals that pose serious health threats of neurological deficits to both military and civilian personnel around the world. Consequently, only a select few approved research groups are permitted to study these dangerous chemical and biological warfare agents. This has created a significant gap in our scientific understanding of the mechanisms underlying neurological diseases. Valuable insights may be gleaned by drawing parallels to other extensively researched neuropathologies, such as traumatic brain injuries (TBI). By examining combined gene expression profiles, common and unique molecular characteristics may be discovered, providing new insights into medical countermeasures (MCMs) for TBI, EEV infection and OPNA neuropathologies and sequelae. In this study, we collected transcriptomic datasets for neurological disorders caused by TBI, EEV, and OPNA injury, and implemented a framework to normalize and integrate gene expression datasets derived from various platforms. Effective machine learning approaches were developed to identify critical genes that are associated, either shared among the three neuropathologies or to either TBI, EEV, and OPNA. With the aid of deep neural networks, we were able to extract important association signals for accurate prediction of different neurological disorders by using integrated gene expression datasets of VEEV, OPNA, and TBI samples. Gene ontology and pathway analyses further identified neuropathologic features with specific gene product attributes and functions, shedding light on the fundamental biology of these neurological disorders. Collectively, we highlight a workflow to analyze published transcriptomic data using machine learning, which can be used for both identification of gene biomarkers that are unique to specific neurological conditions, as well as genes shared across multiple neuropathologies. These shared genes could serve as potential neuroprotective drug targets for conditions like EEV, TBI, and OPNA.

neuroscience↗

bioRxiv2024

ATF4 regulates mitochondrial dysfunction, mitophagy, and autophagy, contributing to corneal endothelial apoptosis under chronic ER stress in Fuchs dystrophy

PURPOSEEndoplasmic reticulum (ER) stress, mitochondrial dysfunction, and mitophagy are known to contribute independently to corneal endothelial cell (CEnC) apoptosis in Fuchs endothelial corneal dystrophy (FECD). However, the role of a specific ER stress pathway (PERK-ATF4-CHOP) in regulating these events is unknown. This study aims to investigate the role of ATF4 in regulating mitochondrial dysfunction and mitophagy, which ultimately leads to CEnC apoptosis in FECD. METHODSHuman corneal endothelial cell line (21T), Fuchs corneal endothelial cell line (F35T), and primary human corneal endothelial cells were treated with ER stressor tunicamycin (Tun). ATF4 siRNA was used to knock down ATF4 in 21T cell line and primary corneal endothelial cells. Mitophagy and apoptotic proteins were analyzed using Western blotting. ATF4+/- and ATF4 +/+ mice were irradiated with UVA to assess ER stress and corneal endothelial apoptosis. RESULTSF35T cell line had significantly increased expression of the ER stress pathway as well as caspase-mediated apoptotic molecules compared to 21T at baseline, which further increased after tunicamycin treatment. F35T cells exhibited significantly decreased ATP and MMP, and increased mitochondrial fragmentation, which was further exacerbated after Tunicamycin. F35T cell line also demonstrated inhibition of mitophagy, similar to 21T, after treatment with Tunicamycin, despite the upregulation of mitophagy initiators. ATF4 knockdown attenuated ER and mitochondrial stress proteins, rescued mitochondrial membrane potential (MMP) loss, downregulated mitochondrial fragmentation, activated mitophagy, and prevented cell death under chronic ER stress. ATF4+/-mice had increased CE numbers, with improved cellular morphology and decreased ER stress CHOP expression, compared to ATF4+/+ mice post-UVA. CONCLUSIONSPro-apoptotic ATF4 induction following ER stress disrupts mitochondrial function, leading to mitophagy inhibition and CEnC apoptosis. This study highlights the importance of ATF4 in ER-mitochondrial crosstalk and its contribution to CEnC apoptosis in FECD.

cell biology↗

bioRxiv2024

Endocytosis restricts synapse growth by attenuating Highwire/PHR1-dependent JNK signaling in a pathway parallel to the BMP signaling

Synaptic growth and organization are orchestrated by pre - and post-synaptic signaling, neuronal activity, and environmental cues. Although endocytosis is known to attenuate synaptic growth, the underlying signaling mechanisms have remained elusive. Here, we uncover a previously unrecognized mechanism by which endocytosis constrains synaptic growth through regulation of the neuronal E3 ubiquitin ligase Highwire (Hiw/Phr1). We show that loss of endocytosis causes Hiw to accumulate in neuronal cell bodies, leading to elevated MAP3K Wallenda (Wnd)/DLK levels and hyperactivation of JNK signaling. The accumulated Hiw assembles into dynamic liquid-liquid phase-separated condensates, as revealed by their rapid and reversible dissolution with 1,6-hexanediol. Acute blockade of endocytosis using a temperature-sensitive dynamin mutant Shibiretssimilarly triggered robust Hiw phase separation. We further demonstrate that Rab11-positive recycling endosomes are essential for proper Hiw localization and turnover, directly linking endosomal trafficking to the control of JNK signaling. Finally, we show that both BMP and JNK signaling are necessary and sufficient to guide synaptic morphogenesis at the Drosophila NMJ, thereby integrating endocytic trafficking with synaptic growth signaling. Our findings establish endocytosis as a critical regulator of Hiw/Phr1-dependent JNK signaling via liquid-liquid phase separation, with implications that extend beyond synaptic morphogenesis to axon injury and degeneration pathways.

neuroscience↗

bioRxiv2024

Single-Cell Transcriptomics Unveils Skin Cell Specific Antifungal Immune Responses and IL-1Ra- IL-1R Immune Evasion Strategies of Emerging Fungal Pathogen Candida auris

Candida auris is an emerging multidrug-resistant fungal pathogen that preferentially colonizes and persists in skin tissue, yet the host immune factors that regulate the skin colonization of C. auris in vivo are unknown. In this study, we employed unbiased single-cell transcriptomics of murine skin infected with C. auris to understand the cell type-specific immune response to C. auris. C. auris skin infection results in the accumulation of immune cells such as neutrophils, inflammatory monocytes, macrophages, dendritic cells, T cells, and NK cells at the site of infection. We identified fibroblasts as a major non-immune cell accumulated in the C. auris infected skin tissue. The comprehensive single-cell profiling revealed the transcriptomic signatures in cytokines, chemokines, host receptors (TLRs, C-type lectin receptors, NOD receptors), antimicrobial peptides, and immune signaling pathways in individual immune and non-immune cells during C. auris skin infection. Our analysis revealed that C. auris infection upregulates the expression of the IL-1RN gene (encoding IL-1R antagonist protein) in different cell types. We found IL-1Ra produced by macrophages during C. auris skin infection decreases the killing activity of neutrophils. Furthermore, C. auris uses a unique cell wall mannan outer layer to evade IL-1R-signaling mediated host defense. Collectively, our single-cell RNA seq profiling identified the transcriptomic signatures in immune and non-immune cells during C. auris skin infection. Our results demonstrate the IL-1Ra and IL-1R-mediated immune evasion mechanisms employed by C. auris to persist in the skin. These results enhance our understanding of host defense and immune evasion mechanisms during C. auris skin infection and identify potential targets for novel antifungal therapeutics.

microbiology↗

bioRxiv2024

Prefoldin 5 is a microtubule-associated protein that suppresses Tau-aggregation and neurotoxicity

Tauopathies represent a major class of neurodegenerative disorders associated with intracellular aggregates of the microtubule-associated protein Tau. To identify molecular modulators of Tau toxicity, we used a genetic screen to identify protein chaperones whose RNAi-mediated knockdown could modulate hTauV337M-induced eye-ommatidial degeneration in Drosophila. This screen identified the Prefoldins Pfdn5 and Pfdn6 as strong modifiers of hTauV337M cytotoxicity. Consistent with the known function of Pfdn as a cotranslational chaperone for tubulin, Pfdn5 mutants showed substantially reduced levels of tubulin monomer. However, additional microtubule-related functions were indicated by the robust unexpected association of Pfdn5 with axonal microtubules in vivo, as well as binding with stabilized microtubules in biochemical assays. Loss of Pfdn5 resulted in neuromuscular junctions (NMJ) defects similar to those previously described in hTau-expressing flies: namely, increased supernumerary boutons and fewer microtubule loops within mature presynaptic boutons. Significantly, synaptic phenotypes caused by hTauV337M overexpression were also strongly enhanced in a Pfdn5 mutant background. Consistent with a role in modulating Tau toxicity, not only did loss of Pfdn5 result in increased accumulations of Tau-aggregates in hTauV337M expressing neurons, but also neuronal overexpression of Prefoldin strikingly ameliorated age-dependent neurodegeneration and memory deficits induced by pathological hTau. Together, these and other observations described herein: (a) provide new insight into Prefoldin-microtubule interactions; (b) point to essential posttranslational roles for Pfdn5 in controlling Tau-toxicity in vivo; and (c) demonstrate that Pfdn5 overexpression is sufficient to restrict Tau-induced neurodegeneration.

neuroscience↗