Biology subjects

Singh, B.

Publications and source records attributed to Singh, B..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Distinct region-specific neutralization profiles of contemporary HIV-1 clade C against best-in-class broadly neutralizing antibodies

Broadly neutralizing antibodies (bnAb) have been clinically proven to be an excellent choice for HIV-1 prevention. However, the relative effectiveness of best-in-class bnAbs against regionally relevant circulating HIV-1 forms is not clear. In the present study, we compared the degree of neutralization sensitivity of contemporary HIV-1 Indian clade C with that of South African origin. Phylogenetic analysis revealed that these clade C viruses continue to evolve distinctly from one another. Env-pseudotyped viruses prepared using contemporary HIV-1 clade C env genes (N=115) obtained from nine geographically distinct sites in India (between 2020-2023) were found to be most sensitive to V3-directed bnAbs 10-1074 and BG18, and second generation CD4 binding site (CD4bs) directed bnAbs (VRC07, N6 and 1-18), however they were found to be significantly resistant to V1/V2 apex directed bnAbs. Moreover, we observed that the degree of sensitivity varied between contemporary Indian and South African clade C viruses. Differences in degree of neutralization susceptibility were associated with differences observed in key residues that form bnAb contact sites, gp120 loop lengths and the number of N-linked glycans in the V4 hypervariable region. Interestingly, the second generation CD4bs bnAbs (VRC07, N6, 1-18) showed neutralization of VRC01 and 3BNC117 resistant viruses but with 2-7-fold reduced potency compared to the VRC01 sensitive counterparts, likely due to the enrichment of resistance associated residues observed in loop D. Predictive analysis indicated that combination of BG18, N6 and PGDM1400 can provide over 95% neutralization coverage at 1g/mL of contemporary India clade C, an observation found to be distinct to that reported for the Africa clade C viruses. Taken together, we found distinct neutralization patterns and env signatures associated with resistance to key bnAbs. Our study highlights that towards achieving clinical effectiveness, both the complementarity of bnAb classes and the regionally relevant HIV forms need to be considered. Author summaryWhile the development of vaccines to prevent HIV infection remains a global priority, their potential effectiveness is limited by the extraordinarily diversified circulating forms of HIV-1. The prospect of best-in-class bnAbs as potential prevention option has been demonstrated in several studies including the Phase II Antibody Mediated Prevention (AMP) trial; however, to be broadly applicable, bnAbs will need to overcome the substantial variability of HIV env. The present study highlights that the contemporary HIV-1 clade C viruses are evolving to be less sensitive to the best-in-class bnAbs and HIV-1 clade C that predominates in India and South Africa vary in their degree of susceptibility to best-in-class clinically relevant bnAbs. This indicates differences in the antigenic properties between globally circulating HIV-1 clade C at a population level. Overall, the outcome of this study highlights the need for periodic assessment of sequence and neutralization profiles of the circulating regionally relevant HIV-1 forms towards prioritizing the bnAb combination suitable for effective intervention.

immunology↗

bioRxiv2024

Intrinsic multiplication rate variation of Plasmodium falciparum (in clinical isolates prior to elimination in Malaysia

Replication rates and virulence of pathogens are hypothesised to evolve in response to varying intensity of transmission and competition among genotypes. Under exponential growth conditions in culture, clinical isolates of the malaria parasite Plasmodium falciparum have variable intrinsic multiplication rates, but comparisons of samples from different areas are needed. To analyse parasites from an area of low endemicity, Malaysian clinical isolates cryopreserved prior to malaria elimination were studied. The mean and range of P. falciparum multiplication rates in Malaysian isolates was no less than that seen among isolates from more highly endemic populations in Africa, which does not support a hypothesis of adaptation to prevailing levels of infection endemicity. Moreover, the distribution of multiplication rates was similar between isolates with single parasite genotypes and those containing multiple genotypes, which does not support a hypothesis of facultative adjustment to competing parasites. Based on clinical isolates, the findings indicate that parasites may not evolve lower multiplication rates under conditions of reduced transmission, and that the virulence potential is likely to be undiminished in pre-elimination settings. This encourages efforts to eliminate endemic infection completely, as has been achieved at the national level in Malaysia.

microbiology↗

bioRxiv2024

Editing cis-elements of OsPHO1;2 improved phosphate transport and yield in rice

Increasing grain yield is the main aim of crop improvement, which is globally impacted by the low availability of soil phosphate (Pi). Overexpressing Pi transporters for increasing Pi uptake often result in Pi toxicity and growth retardation. Despite advancements in genetic engineering, targeting cis-regulatory motifs of Pi transporters remains underexplored for understanding plant mechanisms and improving Pi status. Here, we demonstrated that the excision of the transcription inhibitor motif from the promoter of Pi transporter, OsPHO1;2, enhanced its expression and increased root-to-shoot Pi transport, leading to improved grain yield. Using, in-silico and DNA-protein interaction studies, we show the role of OsWRKY6 transcription factor in the negative regulation of OsPHO1;2 expression by binding to the cis-regulatory element (W-box) present in its promoter. oswrky6 knockout lines displayed a higher OsPHO1;2 expressions, and improved shoot Pi levels. Further, we engineered the OsPHO1;2 promoter for precisely removing the W-box and enhancing OsPHO1;2 expression. The phenotypic and physiological evaluation at the vegetative stage shows that OsPHO1;2 promoter edited (OsPHO1;2:PE) lines have increased shoot length, plant biomass and more root-shoot Pi export under low and normal P conditions. Notably, the 33Pi uptake assay reveals that OsPHO1;2:PE lines exhibited enhanced root Pi uptake, supported by higher root-associated Pi transporters (OsPHTs) expression. An extensive agronomical assessment revealed that OsPHO1;2:PE lines acquire increased seed and panicle numbers, thereby increasing yield without affecting seed quality. Our findings offer valuable insight into the potential of promoter editing to raise plants with better Pi use and improved crop yield.

plant biology↗

bioRxiv2024

Novel Role of Endothelial CD45 in Regulating Endothelial-to-Mesenchymal Transition in Atherosclerosis

BackgroundThe protein tyrosine phosphatase CD45 is expressed in all nucleated cells of the hematopoietic system and in mitral valve endothelial cells (ECs) undergoing endothelial-to-mesenchymal transition (EndoMT). Our recent work indicated that activation of endogenous CD45 in human endothelial colony-forming cells (ECFCs) induced expression of multiple EndoMT marker genes. We hypothesized that CD45 may contribute to atherosclerosis; however, detailed molecular mechanisms underlying how CD45 may contribute to EndoMT and the impact of therapeutic manipulation of CD45 expression in atherosclerosis are unknown. MethodsWe generated a tamoxifen-inducible EC-specific CD45-deficient mouse strain (EC-iCD45KO) on an ApoE-deficient (WT/ApoE-/-) background and fed them a Western diet (WD) to produce atherosclerosis. We enriched mouse aortic ECs with anti-CD31 beads to perform single-cell RNA sequencing. Cellular, biochemical and molecular approaches were used to investigate the effect of endothelial CD45-specific deletion on EndoMT and lesion development in an ApoE-/- mouse model of atherosclerosis. ResultsEC-iCD45KO mice showed reductions in lesion development, plaque macrophage infiltration, and expression of cell adhesion molecules when compared to WT/ApoE-/- controls. Single-cell RNA sequencing revealed that loss of endothelial CD45 decreases EndoMT marker expression and TGF-{beta} signaling in atherosclerotic mice, which is associated with reduction of lesions. Mechanistically, CD45 loss increases Fibroblast Growth Factor Receptor 2 (FGFR2) expression in mouse aortic ECs and Kruppel-like Factor 2 (KLF2) expression in the aortic root. Endothelial CD45-deficiency also inhibits EndoMT and TGF{beta} signaling in atherosclerosis. ConclusionsOur findings demonstrate that genetic depletion of endothelial CD45 protects against EndoMT-driven atherosclerosis by promoting FGFR2 and KLF2 expression while inhibiting Transforming Growth Factor beta (TGF{beta}) signaling and EndoMT. Consequently, targeting endothelial CD45 may represent a novel therapeutic strategy to reduce EndoMT in atherosclerosis.

cell biology↗

bioRxiv2024

Targeting Liver Epsins Ameliorates Dyslipidemia in Atherosclerosis

BackgroundThe low-density lipoprotein receptor (LDLR) in the liver plays a crucial role in clearing low-density lipoprotein cholesterol (LDL-C) from the bloodstream. This process takes place mainly in the liver. Under atherogenic conditions, Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), secreted by the liver, binds to LDLR on hepatocytes, preventing its recycling and enhancing its lysosomal degradation. This process reduces LDL-C clearance, promoting hypercholesterolemia. Epsins, a family of ubiquitin-binding endocytic adaptors, are key regulators of atherogenesis in lesional cells, including endothelial cells and macrophages. Given epsins canonical role in regulating endocytosis of cell surface receptors, we aimed to determine whether and how liver epsins contribute to PCSK9-mediated LDLR endocytosis and degradation, thereby impairing LDL-C clearance and accelerating atherosclerosis. MethodsLiver-specific epsin knockout (Liver-DKO) atherosclerotic models were generated in ApoE-/- and PCSK9-AAV8-induced atheroprone mice fed on a Western diet. We utilized single-cell RNA sequencing, along with molecular, cellular, and biochemical analyses, to investigate the physiological role of liver epsins in PCSK9-mediated LDLR degradation. Additionally, we explored the therapeutic potential of nanoparticle-encapsulated siRNAs targeting epsins 1 and 2 in ApoE-/- mice with established atherosclerosis. ResultsWestern diet (WD)-induced atherosclerosis was significantly attenuated in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls, as well as in PCSK9-AAV8-induced Liver-DKO mice compared with PCSK9-AAV8-induced wild-type (WT) mice accompanied by reductions in blood cholesterol and triglyceride levels. Mechanistically, single-cell RNA sequencing of hepatocytes and aortas isolated from atherosclerotic ApoE-/- and ApoE-/-/Liver-DKO mice revealed epsin-deficient Ldlrhi hepatocytes with diminished lipogenic potential. Notably, pathway analysis of hepatocytes showed increased LDL particle clearance and enhanced LDLR-cholesterol interactions under WD treatment in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls, correlating with decreased plasma LDL-C levels. Furthermore, pathway analysis of the aortas showed attenuated inflammation and endothelial activation, coupled with reduced lipid uptake, and enhanced cholesterol efflux under WD treatment in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls. Moreover, the absence of liver epsins led to an upregulation of LDLR protein expression in hepatocytes. We further demonstrated that epsins bind LDLR via the ubiquitin-interacting motif (UIM), enabling PCSK9-mediated LDLR degradation. Depleting epsins abolished this degradation, thereby preventing atheroma progression. Lastly, targeting liver epsins with nanoparticle-encapsulated epsins siRNAs effectively ameliorates dyslipidemia and inhibits atherosclerosis progression. These results are consistent with findings showing an increased epsin1 and epsin2 expression in atherosclerotic cardiovascular disease patients. ConclusionsLiver epsins drive atherogenesis by promoting PCSK9-mediated LDLR degradation, thereby elevating circulating LDL-C levels and heightening lesional inflammation. As such, targeting epsins in the liver represents a promising therapeutic strategy to mitigate atherosclerosis by preserving LDLR and enhancing LDL-C clearance in the liver.

cell biology↗

bioRxiv2024

HCM-associated mutations in MYH6/7 drive pathologic expression of TGF-β1 in cardiomyocytes within weeks of developmental specification

Hypertrophic cardiomyopathy (HCM) is characterized by myocyte hypertrophy, sarcomere disarray, and myocardial fibrosis, leading to significant morbidity and mortality. As the most common inherited cardiomyopathy, HCM largely results from mutations in sarcomeric protein genes. Current treatments for HCM primarily focus on alleviating late-stage symptoms, with a critical gap in the detailed understanding of early-stage deficiencies that drive disease progression. We recently showed, in monolayers of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) with MYH7 R723C and MYH6 R725C mutations, altered expression of several extracellular matrix (ECM)-related genes with associated defects in cardiomyocyte-ECM adhesion. To better evaluate the cardiomyocyte-ECM interface and pathological ECM dynamics in early-stage HCM, here we adopted a 3D engineered heart tissue (EHT) model containing both cardiomyocytes and fibroblasts, the primary contributor to ECM remodeling. Mutant EHTs showed aberrant cardiomyocyte distribution, augmented calcium handling, and force generation compared to controls. Altered proteoglycan deposition and increased phosphorylated focal adhesion kinase (pFAK) further indicated changes in ECM composition and connectivity. Elevated transforming growth factor beta-1 (TGF-{beta}1) secretion and a higher proportion of activated fibroblasts were identified in mutant EHTs, along with sustained TGF-{beta}1 transcription specifically in mutant cardiomyocytes. Remarkably, blocking TGF-{beta}1 receptor signaling reduced fibroblast activation and contraction force to control levels. This study underscores the early interplay of mutant hiPSC-CMs with fibroblasts, wherein mutant cardiomyocytes initiate fibroblast activation via TGF-{beta}1 overexpression, independent of the immune system. These findings provide a promising foundation for developing and implementing novel strategies to treat HCM well before the manifestation of clinically detectable fibrosis and cardiac dysfunction.

bioengineering↗

bioRxiv2024

Mesolimbic local field potentials are modulated by motor control

Background and ObjectivesWhile historically cortico-basal ganglia-thalamocortical loops were believed to process limbic and sensorimotor data in parallel, there is now evidence to suggest that the two information streams can be processed in a single open loop. However, the limbic-motor interface remains insufficiently characterized. We sought to further investigate how extrastriatal regions may regulate motor output by examining electrophysiological activity in these areas during a response inhibition paradigm. MethodsWe recorded local field potentials (LFPs) from epilepsy patients implanted with intracranial depth electrodes for seizure localization purposes. Participants performed the stop-signal task, during which they made speeded choice reactions to "go" stimuli and occasionally inhibited their reactions in the incident of a "stop" signal. To compare power during movement and the absence of movement, we applied a Wilcoxon signed-rank test. Additionally, we performed a linear mixed-effects model to relate power in limbic regions to power in the motor cortex. Finally, we implemented exploratory analyses to identify power differences for correct go versus correct stop trials and for correct stop versus incorrect stop trials using cluster-based permutation testing. Results14 patients participated. A comparison between movement and baseline fixation revealed that motor response is associated with reduced beta (15-35 Hz) power in the amygdala, hippocampus, and motor cortex and reduced gamma (35-100 Hz) power in the amygdala and hippocampus. Moreover, average beta and gamma power in the amygdala and hippocampus during motor execution were positively associated with average beta and gamma power in the motor cortex. Additionally, we identified significant differences between correct go and stop trials in delta (1-4 Hz) power for all three regions and in theta (4-8 Hz) power for the amygdala and motor cortex. Likewise, we identified significant differences between correct and incorrect stop trials in delta power for the hippocampus and motor cortex, in theta power for the motor cortex, in alpha (8-15 Hz) and beta power for the amygdala and motor cortex, and in gamma power for all three areas. DiscussionThese correlations between neural oscillations in the hippocampus and amygdala and movement strengthen the notion of mesolimbic modulation of motor activity.

neuroscience↗

bioRxiv2024

MELD-Adapt: On-the-Fly Belief Updating in Integrative Molecular Dynamics

Integrative structural biology synergizes experimental data with computational methods to elucidate the structures and interactions within biomolecules, a task that becomes critical in the absence of high-resolution structural data. A critical step in integrating the data, is knowing the expected accuracy or belief in the dataset. We previously showed that the Modeling Employing Limited Data (MELD) approach succeeds at predicting structures and finding the best interpretation of the data when the initial belief is equal or slightly lower than the real value. But, this initial belief is not known for many data sources. Here we introduce MELD-Adapt, designed to dynamically evaluate and infer the reliability of input data, thereby enhancing the accuracy of structure predictions. We demonstrate the utility of this method across different systems, particularly emphasizing its capability to correct initial assumptions and identify the correct fraction of data to produce reliable structural models. The approach is tested with two benchmark sets: the folding of twelve proteins with coarse physical insights and the binding of peptides with varying affinities to the Extrateterminal (ET) domain. Our findings also show the importance of how data is distributed in the system (locally clustered or globally distributed) and the subtle balance between data, beliefs, and force fields.

biophysics↗

bioRxiv2024

Human Breast Milk EVs Mitigate Endothelial Dysfunction: Preliminary Study

BackgroundEndothelial cell (EC) dysfunction is an early indicator of failing vascular integrity, leading to various cardiovascular diseases. Toll-like receptor 4 (TLR4) activation is a key mechanism. Milk-derived extracellular vesicles (EVs) are known for their anti-inflammatory properties, particularly in suppressing TLR4 activation in damaged intestinal epithelial cells. This study explores the therapeutic potential of human breast milk EVs (bEVs) in EC dysfunction related to cardiovascular diseases. MethodsHuman breast milk EVs (bEVs) were isolated from healthy nursing mothers using ultracentrifugation. bEVs were applied to LPS-treated HUVECs, and the expression of inflammatory markers was measured using qPCR and western blotting. Angiogenesis was assessed via a wound assay. Additionally, bEVs were orally administered weekly for six weeks to high-fat diet-induced obese mice and lean mice. Metabolic phenotype characteristics and EC-dependent vasorelaxation were evaluated. ResultsbEV pre-treatment inhibited LPS-induced expression of inflammatory genes, including IL-6, IL-1b and VCAM-1. It also suppressed phospho-NFkB and TLR4 protein expression. bEVs enhanced EC migration, significantly increasing wound closure. Oral administration of bEVs restored impaired EC-dependent vasorelaxation in the mesenteric artery of obese mice, though metabolic parameters remained unchanged. ConclusionOur findings demonstrate the beneficial effects of bEVs on EC dysfunction, highlighting their potential as novel therapeutics for cardiovascular diseases. Future studies will focus on identifying specific bEV cargos and further evaluating their therapeutic effects on EC dysfunction.

cell biology↗

bioRxiv2024

Pred-AHCP: Robust feature selection enabled Sequence Specific Prediction of Anti-Hepatitis C Peptides via Machine Learning

Every year, an estimated 1.5 million people worldwide contract Hepatitis C (HepC), a significant contributor to liver disease. Although many studies have explored machine learnings potential to predict antiviral peptides, very few have addressed predicting peptides against specific viruses such as Hepatitis C. In this study, we demonstrate the use of machine learning (ML) algorithms to predict peptides that are effective against HepC. We developed an explainable ML model that harnesses the amino acid sequence of a peptide to predict its potential as an anti-HepC (AHC) agent. Specifically, features were computed based on sequence and physicochemical properties, with feature selection performed utilizing a combined scheme of mutual information and variance inflation factor. This facilitated the removal of redundant and multicollinear features from the sequence data, enhancing the models generalizability in predicting AHCPs. The model using the random forest algorithm produced the best performance with an accuracy of about 90%. The feature selection analysis highlights that the distribution of hydrophobicity and polarizability, as well as the frequencies of glycine residues and di-peptide motifs--YXL, LXK, VXXXF, VL, LV, CC, RR, TXXXV, VXXA, CXXXC--emerged as the key predictors for identifying AHCPs targeting different components of the HepC virus. The model developed can be accessed through the Pred-AHCP web server, provided at http://tinyurl.com/web-Pred-AHCP. This resource facilitates the prediction and re-engineering of AHCPs for designing peptide-based therapeutics while also proposing an exploration of similar strategies for designing peptide inhibitors effective against other viruses.

bioinformatics↗

bioRxiv2024

Reward Circuit Local Field Potential Modulations Precede Risk Taking

Risk taking behavior is a symptom of multiple neuropsychiatric disorders and often lacks effective treatments. Reward circuitry regions including the amygdala, orbitofrontal cortex, insula, and anterior cingulate have been implicated in risk-taking by neuroimaging studies. Electrophysiological activity associated with risk taking in these regions is not well understood in humans. Further characterizing the neural signalling that underlies risk-taking may provide therapeutic insight into disorders associated with risk-taking. Eleven patients with pharmacoresistant epilepsy who underwent stereotactic electroencephalography with electrodes in the amygdala, orbitofrontal cortex, insula, and/or anterior cingulate participated. Patients participated in a gambling task where they wagered on a visible playing card being higher than a hidden card, betting $5 or $20 on this outcome, while local field potentials were recorded from implanted electrodes. We used cluster-based permutation testing to identify reward prediction error signals by comparing oscillatory power following unexpected and expected rewards. We also used cluster-based permutation testing to compare power preceding high and low bets in high-risk (<50% chance of winning) trials and two-way ANOVA with bet and risk level to identify signals associated with risky, risk averse, and optimized decisions. We used linear mixed effects models to evaluate the relationship between reward prediction error and risky decision signals across trials, and a linear regression model for associations between risky decision signal power and Barratt Impulsiveness Scale scores for each patient. Reward prediction error signals were identified in the amygdala (p=0.0066), anterior cingulate (p=0.0092), and orbitofrontal cortex (p=6.0E-4, p=4.0E-4). Risky decisions were predicted by increased oscillatory power in high-gamma frequency range during card presentation in the orbitofrontal cortex (p=0.0022), and by increased power following bet cue presentation across the theta-to-beta range in the orbitofrontal cortex (p=0.0022), high-gamma in the anterior cingulate (p=0.0004), and high-gamma in the insula (p=0.0014). Risk averse decisions were predicted by decreased orbitofrontal cortex gamma power (p=2.0E-4). Optimized decisions that maximized earnings were preceded by decreases within the theta to beta range in orbitofrontal cortex (p=2.0E-4), broad frequencies in amygdala (p=2.0E-4), and theta to low-gamma in insula (p=4.0E-4). Insula risky decision power was associated with orbitofrontal cortex high-gamma reward prediction error signal (p=0.0048) and with patient impulsivity (p=0.00478). Our findings identify and help characterize reward circuitry activity predictive of risk-taking in humans. These findings may serve as potential biomarkers to inform the development of novel treatment strategies such as closed loop neuromodulation for disorders of risk taking.

neuroscience↗

bioRxiv2024

Exosomes are vehicles for the stress-regulated secretion of histones

Histones are conserved nuclear proteins that function as part of the nucleosome in the regulation of chromatin structure and gene expression. Interestingly, extracellular histones populate biofluids from healthy individuals and when elevated may contribute to various acute and chronic diseases. It is generally assumed that most extracellular histones exist as nucleosomes, as components of extracellular chromatin. We analysed cell culture models under normal and stressed conditions to identify pathways of histone secretion. We report that core and linker histones localize to extracellular vesicles (EVs) and are secreted via the multivesicular body/exosome pathway. Upregulation of histone EV secretion occurs in response to cellular stress, with enhanced vesicle secretion and a shift towards a population of smaller EVs. Most histones were membrane associated with the outer surface of EVs. Degradation of EV-DNA did not impact significantly on EV-histone association. Individual histones or histone octamers bound strongly to liposomes and EVs, but nucleosomes did not, showing histones do not require DNA for EV binding. EV histones colocalized most frequently with the tetraspanin CD63 but using genetic or pharmacological intervention, we found that all known pathways of exosome biogenesis acted positively on histone secretion. Inhibition of autophagy and lysosomal degradation had a strong positive effect on EV histone release. Unexpectedly, EV-associated histones lacked the extensive post-translational modification of their nuclear counterparts, suggesting loss of PTMs may be involved in their trafficking or secretion. Our data does not support a significant role for EV-histones existing as nucleosomes. We show for the first time that histones are secreted from cells as membrane proteins via EVs/exosomes. This fundamental discovery provides support for further investigation of the biological activity of exosome associated histones and their role in disease.

cell biology↗

bioRxiv2024

Sifting Through the Noise: A Computational Pipeline for Accurate Prioritization of Protein- Protein Binding Candidates in High-Throughput Protein Libraries

Identifying the interactome for a protein of interest is challenging due to the large number of possible binders. High-throughput experimental approaches narrow down possible binding partners, but often include false positives. Furthermore, they provide no information about what the binding region is (e.g. the binding epitope). We introduce a novel computational pipeline based on an AlphaFold2 (AF) Competition Assay (AF-CBA) to identify proteins that bind a target of interest from a pull-down experiment, along with the binding epitope. Our focus is on proteins that bind the Extraterminal (ET) domain of Bromo and Extraterminal domain (BET) proteins, but we also introduce nine additional systems to show transferability to other peptide-protein systems. We describe a series of limitations to the methodology based on intrinsic deficiencies to AF and AF-CBA, to help users identify scenarios where the approach will be most useful. Given the speed and accuracy of the methodology, we expect it to be generally applicable to facilitate target selection for experimental verification starting from high-throughput protein libraries. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/576374v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@b90071org.highwire.dtl.DTLVardef@1cc2f66org.highwire.dtl.DTLVardef@3c1109org.highwire.dtl.DTLVardef@1827353_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Nitrogen Management on Improving Resilience of Flood-Tolerant (Sub1) Rice Varieties in Flood-Prone Rainfed Lowlands

Flash flood causing submergence adversely affects rice production in vast areas of rainfed lowlands of South and Southeast Asia. Introgression at the Sub1 locus by molecularly assisted backcrossing incorporated tolerance into varieties traditional/modern high-yielding varieties and has released some varieties for commercial planting by Indian farmers. In the present study, we investigated the application of fractional N to further improve the survival and productivity potential of two Sub1 introgression varieties, Sambha Mahsuri-Sub1, and BR11-Sub1.Thirty days (d) old seedlings were transplanted, and 28-d after transplanting i.e. 58 days old seedlings were completely submerged for 18-d. Full doses of phosphorus and potassium were applied as a basal, while nitrogen was applied in split doses. Plant survival was recorded at 0, 5 and 20 days after de-submergence (AS) to study the recovery dynamics of cultivar Sub1. Results evidently established that varieties with higher biomass, tiller number, total chlorophyll and soluble sugars concentrations before flooding have higher survival rates after water drainage. They also had faster growth and better recovery, which was reflected in yield characteristics and grain yield. The 20-day and 40- day AS N application replenished flood-disturbed soil N pools, resulting in higher N uptake and N-use efficiency. These results may contribute to better nitrogen fertilization programs for rice crops and improve stress tolerance in newly developed tolerant rice varieties. It is evident from perusal of results that treatment T2 i.e.1/4 N applied as a basal and rest of residual N applied in three split doses respectively after de-submergence on days 5, 20 and 40 resulted in higher survival thanT1 and T3 respectively minimising mortality rate. This method of nitrogen fertilization (T2) also significantly affected plant-1 stem biomass, plant-1 tiller number, total chlorophyll, and soluble sugar concentrations before and after flooding. Based on these findings we proposed that application of lower dose of N (30kg/ha) as basal and rest amount of N in three split doses along with P and K (40kg/ha) in the field might be exploited to improve submergence tolerance and to obtain higher yield under flood prone ecosystem due to higher survival after de submergence corresponding to less post oxidative damage through proper N management during before and post submergence period.

plant biology↗

bioRxiv2024

Epsins promote atherosclerosis through VSMC phenotypic modulation

Smooth muscle cells in major arteries play a crucial role in regulating coronary artery disease. Conversion of smooth muscle cells into other adverse cell types in the artery propels the pathogenesis of the disease. Curtailing artery plaque buildup by modulating smooth muscle cell reprograming presents us a new opportunity to thwart coronary artery disease. Here, our report how Epsins, a family of endocytic adaptor proteins oversee the smooth muscle cell reprograming by influencing master regulators OCT4 and KLF4. Using single-cell RNA sequencing, we characterized the phenotype of modulated smooth muscle cells in mouse atherosclerotic plaque and found that smooth muscle cells lacking epsins undergo profound reprogramming into not only beneficial myofibroblasts but also endothelial cells for injury repair of diseased endothelium. Our work lays concrete groundwork to explore an uncharted territory as we show that depleting Epsins bolsters smooth muscle cells reprograming to endothelial cells by augmenting OCT4 activity but restrain them from reprograming to harmful foam cells by destabilizing KLF4, a master regulator of adverse reprograming of smooth muscle cells. Moreover, the expression of Epsins in smooth muscle cells positively correlates with the severity of both human and mouse coronary artery disease. Integrating our scRNA- seq data with human Genome-Wide Association Studies (GWAS) identifies pivotal roles Epsins play in smooth muscle cells in the pathological process leading to coronary artery disease. Our findings reveal a previously unexplored direction for smooth muscle cell phenotypic modulation in the development and progression of coronary artery disease and unveil Epsins and their downstream new targets as promising novel therapeutic targets for mitigating metabolic disorders.

cell biology↗

bioRxiv2023

A single-cell transposable element atlas of human cell identity

Single cell RNA sequencing (scRNA-seq) is revolutionizing the study of complex biological systems. However, most sequencing studies overlook the contribution of transposable element (TE) expression to the transcriptome. In both scRNA-seq and bulk tissue RNA sequencing (RNA-seq), quantification of TE expression is challenging due to repetitive sequence content and poorly characterized TE gene models. Here, we developed a tool and analysis pipeline for Single cell Transposable Element Locus Level Analysis of scRNA Sequencing (Stellarscope) that reassigns multi-mapped reads to specific genomic loci using an expectation-maximization algorithm. Using Stellarscope, we built an atlas of TE expression in human PBMCs. We found that locus-specific TEs delineate cell types and define new cell subsets not identified by standard mRNA expression profiles. Altogether, this study provides comprehensive insights into the influence of transposable elements in human biology.

genomics↗

bioRxiv2023

Targeting endothelial FOXO1 protects diabetic β-cells and improves wound healing

The forkhead box O1 (FOXO1) transcription factor plays critical roles in regulating not only metabolic activity but also angiogenesis in the vascular endothelium1-4. Our previous studies show that epsin endocytic adaptors can regulate both angiogenesis and lymphangiogenesis5-7. Endothelial cells (ECs) lining the inside of blood vessels are continuously exposed to circulating insulin and insulin-like growth factors (IGFs). Emerging evidences suggest that ECs can affect {beta}-cell function8-11. Excessive IGF2, especially elevated local IGF2 levels in islets, may represent a risk factor for developing diabetes12-15; however, the underlying molecular mechanisms by which aberrant angiogenesis and endothelium-derived factors regulate pancreatic {beta}-cell function in diabetes remain unclear. Here, we report that the pancreas of diabetic patients as well as the pancreas, skin, and plasma of streptozotocin/high fat diet (STZ/HFD)-induced diabetic mice and db/db mice contains excess IGF2, which can lead to {beta}-cell dysfunction and apoptosis. Single-cell transcriptomics combined with mass spectrometry analysis reveal that endothelial-specific knockout of FOXO1 increases circulating soluble and cell-membrane or intracellular expression levels of IGF type 2 receptor (IGF2R) and CCCTC-binding factor (CTCF), while decreasing IGF2 levels in diabetes. Both IGFR215-17 and CTCF18-21 can reduce IGF2 levels and may ameliorate {beta}-cell decline associated with excess IGF2 in diabetes. Furthermore, depletion of FOXO1, epsins, or knockdown of ULK1 inhibits autophagy formation in ECs, preventing degradation of vascular endothelial growth factor receptor 2 (VEGFR2) to promote angiogenesis and improve wound healing in diabetes. Our findings reveal that endothelial FOXO1 regulates epsin-dependent angiogenesis and affects {beta}-cell function and fate through CTCF and IGF2-IGF2R, providing a potential strategy for ameliorating diabetes and accelerating cutaneous wound healing.

cell biology↗

bioRxiv2023

High-throughput single-cell phenotypic profiling and backtracing exposes and predicts clinically relevant subpopulations in isogenic Staphylococcus aureus communities.

Isogenic bacterial cell populations are phenotypically heterogenous and may include subpopulations of antibiotic tolerant or heteroresistant cells. The reversible nature of these phenotypes and lack of biomarkers to differentiate functionally different, but morphologically identical cells is a challenge for research and clinical detection. To overcome this, we present Cellular Phenotypic Profiling and backTracing (CPPT), a flexible fluorescence-activated cell sorting platform, that uses optical probes to visualize and quantify cellular traits and connects the resulting phenotypic profile with a cells experimentally determined fate in single cell-derived growth and antibiotic susceptibility analysis. By applying CPPT on Staphylococcus aureus populations we recorded phenotypic signatures for dormant cells, exposed microanatomy-independent bimodal growth patterns in colony-derived cells, and revealed different culturability of single cells on solid compared to liquid media. We demonstrate that vancomycin-bodipyFL marks cellular subpopulations with increased likelihood to survive antibiotic exposure, showcasing the value of CPPT for discovery of clinically relevant biomarkers.

microbiology↗