Biology subjects

Kumar, S.

Publications and source records attributed to Kumar, S..

At least 343 records · Page 19Linked to original sources

bioRxiv2023

Multicomponent Ayurveda formulation Lodhrasavam ameliorates steatosis and lipotoxicity in HepG2 cell model of NAFLD

BackgroundMetabolic-dysfunction associated steatotic liver disease (MASLD) is a complex, multifactorial condition and a leading cause of liver-related morbidity worldwide. Due to its heterogeneous pathogenesis, conventional single-target therapies often show limited efficacy. Multicomponent ayurveda formulations offer a promising alternative through their multitargeted actions. This study explores the therapeutic potential and underlying mechanisms of Lodhrasavam (LS), a classical, polyherbal ayurveda formulation, in both in vitro and in vivo models of MASLD. MethodsLS was evaluated for its anti-steatotic and anti-obesogenic potential using in vitro (HepG2, 3T3-L1) and in vivo (HF-HFD-induced rat) models. Antioxidant, lipase inhibition, cytotoxicity, anti-steatotic, and anti-adipogenic activities were assessed via DPPH, MTT, ORO/BODIPY staining, triglyceride quantification, ROS assay, and qPCR. Lipidomic profiling was done in HepG2 cells. Biochemical analyses (GLP-1, insulin, lipid panel), histopathology and OGTT further validated efficacy. Data were statistically analyzed by one-way ANOVA (p<0.05). Results/discussion: In HepG2 cells, LS reduced PA-induced lipid accumulation, ROS, triglycerides, and restored viability, while downregulating lipogenic genes (PPAR{gamma}, SREBP-1c, FASN). Lipidomics confirmed lipid modulation. In 3T3-L1 cells, LS suppressed adipogenesis and key adipogenic genes. In HFD-fed rats, LS reduced weight gain, hepatic steatosis, serum lipids, and improved liver histology. LS enhanced insulin and GLP-1 secretion, improved glucose tolerance, and restored pancreatic islet structure. These findings highlight the multitargeted potential of LS in ameliorating MASLD by modulating lipid metabolism, oxidative stress, inflammation, and glucose homeostasis. ConclusionLodhrasavam ameliorates MASLD by modulating the liver-adipose-pancreas axis, improving lipid metabolism, reducing inflammation, and enhancing insulin and GLP-1 secretion through multitargeted mechanisms.

pharmacology and toxicology↗

bioRxiv2023

B cell repertoire sequencing of HIV-1 pediatric elite-neutralizers identifies multiple broadly neutralizing antibody clonotypes

A limited subset of HIV-1 infected adult individuals typically after at least 2-3 years of chronic infection, develop broadly neutralizing antibodies (bnAbs), suggesting that highly conserved neutralizing epitopes on the HIV-1 envelope glycoprotein are difficult for B cell receptors to effectively target, during natural infection. Recent studies have shown the evolution of bnAbs in HIV-1 infected infants. We used bulk BCR sequencing (BCR-seq) to profile the B cell receptors from longitudinal samples (3 time points) collected from a rare pair of antiretroviral-naive, HIV-1 infected pediatric monozygotic twins (AIIMS_329 and AIIMS_330) who displayed elite plasma neutralizing activity against HIV-1. BCR-seq of both twins revealed convergent antibody characteristics including V-gene use, CDRH3 lengths and somatic hypermutation (SHM). Further, antibody clonotypes with genetic features similar to highly potent bnAbs isolated from adults showed ongoing development in donor AIIMS_330 but not in AIIMS_329, corroborating our earlier findings based on plasma bnAbs responses. An increase in SHM was observed in sequences of the IgA isotype from AIIMS_330. This study suggests that children living with chronic HIV-1 can develop clonotypes of HIV-1 bnAbs against multiple envelope epitopes similar to those isolated from adults, highlighting that such B cells could be steered to elicit bnAbs responses through vaccines aimed to induce bnAbs against HIV-1 in a broad range of people including children.

immunology↗

bioRxiv2023

Detergent induced structural perturbations in peanut agglutinin: Insights from spectroscopic and molecular dynamic simulation studies

The three dimensional structure of a protein is very important for its structure. Studies relating to protein structure have been numerous and the effect of denaturants on proteins can help understand the process of protein folding and misfolding. Detergents are important denaturants and play important roles in various fields. Here we explored the effect of sodium dodecyl sulphate (SDS) and cetyltrimethylammonium bromide (CTAB) on the structure of peanut agglutinin (PNA). The protein was purified from its natural source and impact of SDS and CTAB was studied by circular dichroism, intrinsic fluorescence, 8-anilino-1-napthalenesulfonic acid, molecular docking and molecular dynamics simulation. Pure peanut agglutinin showed a trough at 220 nm and positive ellipticity peak at 195 nm, specific for lectins. Results from the experimental and simulation studies suggest how oppositely charged detergents can interact differently and lead to varied structural perturbations in PNA. Both the surfactants induce all protein-like circular dichroism in the protein, above its critical micelle concentrations, with significant change in accessible surface area that became more hydrophobic upon the treatment. Major interactions between the surfactants and protein, resulting in PNA conformational rearrangement, are electrostatic and van der Waals interactions. However, CTAB, like a cationic surfactant, has similar effects as anionic surfactant (SDS) but at significantly very low concentration. Though the effects followed same pattern in both the surfactant treatment, i.e. above respective CMC, the surfactants were inducing -helix/coil like conformation in PNA.

biochemistry↗

bioRxiv2023

Thin Microfluidic Chips with Active Valves

We report the fabrication of very thin microfluidic active and passive devices on rigid and flexible substrates for sample-space-restricted applications. Thin glass coverslips are commonly used substrates, but these being fragile often crack during experiments, leading to device failure. Here, we used PET as a flexible substrate to fabricate robust thin devices. We proposed a simpler process for PET-PDMS bonding without any silane, adhesive, and/or plasma treatment. We presented the compatibility of the thin devices with a digital in-line holographic microscope (DIHM) as a use case. The substitution of the conventional microscope with DIHM in microfluidic large-scale integrated systems renders simplicity, cost-effectiveness, portability, and miniaturization of the overall system. It also enables a customized and parallel multisite optical observation for a complex microfluidic circuit chip. These chips comprise various microfluidic components made of active microvalves, particularly Quake valves. We also successfully demonstrated the function of microvalves fabricated with our method to regulate the fluidic flow. Thus, are suited to making sophisticated microfluidic circuit chips to fit a variety of applications like organ-on-chip, cell culture, wearable biosensors, pressure sensors, etc.

bioengineering↗

bioRxiv2023

Using a P. falciparum genetic cross to dissect the relative contributions of pfcrt and plasmepsin II/III to piperaquine response-related traits

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin (DHA) as a first-line treatment against malaria parasites. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multi-drug resistant KEL1/PLA1 lineage isolate (KH004) and a drug susceptible parasite isolated in Malawi (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and four copies of pm2/3. We recovered 104 unique recombinant progeny and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3 for detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes, including PPQ survival assay (PSA), area under the dose-response curve (AUC), and a limited point IC50 (LP-IC50). We find that inheritance of the KH004 pfcrt allele is required for PPQ resistance, whereas copy number variation in pm2/3 further enhances resistance but does not confer resistance in the absence of PPQ-R-associated mutations in pfcrt. Deeper investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background, to a range of PPQ-related traits and confirm the critical role of the PfCRT G367C substitution in PPQ resistance. ImportanceResistance to PPQ used in combination with DHA has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention, and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and have the distinct advantage of assaying the effects of naturally evolved genetic variation. Our study was significantly strengthened because the full a range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multi-gene model suggests an important role for both loci in the evolution of this ACT resistant parasite lineage.

genomics↗

bioRxiv2023

Multi-Scale Cortical Bone Traits Vary in Two Mouse Models of Genetic Diversity

Understanding the genetic basis of cortical bone traits can allow for the discovery of novel genes or biological pathways regulating bone health. Mice are the most widely used mammalian model for skeletal biology and allow for the quantification of traits that cant easily be evaluated in humans, such as osteocyte lacunar morphology. The goal of our study was to investigate the effect of genetic diversity on multi-scale cortical bone traits of three long bones in skeletally-mature mice. We measured bone morphology, mechanical properties, material properties, lacunar morphology, and mineral composition of mouse bones from two populations of genetic diversity. Additionally, we compared how intra-bone relationships varied in the two populations. Our first population of genetic diversity included 72 females and 72 males from the eight Inbred Founder strains used to create the Diversity Outbred (DO) population. These eight strains together span almost 90% of the genetic diversity found in mice (Mus musculus). Our second population of genetic diversity included 25 genetically unique, outbred females and 25 males from the DO population. We show that multi-scale cortical bone traits vary significantly with genetic background; heritability values range from 21% to 99% indicating genetic control of bone traits across length scales. We show for the first time that lacunar shape and number are highly heritable. Comparing the two populations of genetic diversity, we show each DO mouse does not resemble a single Inbred Founder but instead the outbred mice display hybrid phenotypes with the elimination of extreme values. Additionally, intra-bone relationships (e.g., ultimate force vs. cortical area) were mainly conserved in our two populations. Overall, this work supports future use of these genetically diverse populations to discover novel genes contributing to cortical bone traits, especially at the lacunar length scale. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/543484v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1e61c1org.highwire.dtl.DTLVardef@a3f941org.highwire.dtl.DTLVardef@1411dceorg.highwire.dtl.DTLVardef@bfc8c_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2023

Respiratory syncytial virus directly binds miR-26, miR-27, and let-7 to de-repress gene targets through canonical and non-canonical mechanisms

microRNAs (miRNAs) regulate nearly all physiological processes but our understanding of exactly how they function remains incomplete, particularly in the context of viral infections. Here we adapt a biochemical method (CLEAR-CLIP) and analysis pipeline to identify targets of miRNAs in lung cells infected with Respiratory syncytial virus (RSV). We show that RSV binds directly to miR-26 and miR-27 through seed pairing and demonstrate that these miRNAs target distinct gene networks associated with cell cycle and metabolism (miR-27) and antiviral immunity (miR-26). Many of the targets are de-repressed upon infection and we show that the miR-27 targets most sensitive to miRNA inhibition are those associated with cell cycle. Finally, we demonstrate that high confidence chimeras for miR-26 and miR-27 also map to regulatory regions. We validate that a proportion of miR-27 and Argonaute 2 (AGO2) is nuclear in infected cells and identify a long non-coding RNA (lncRNA) as a miR-27 target that is linked to transcriptional regulation of nearby genes. This work expands the target networks of miR-26 and miR-27 to include direct interactions with RSV and lncRNAs and implicate these miRNAs in regulation of key genes that impact the viral life cycle associated with cell cycle, metabolism, and antiviral immunity.

molecular biology↗

bioRxiv2023

Characterization of B646L (p72) gene and the resistance pattern of African swine fever Virus tolerant to indigenous Doom Pig breeds of India

African Swine Fever (ASF) has affected all pig breeds in North-East India since 2020, except Doom pigs, a unique indigenous breed from Assam and the closest progenitor to Indian wild pigs, resulting in significant economic losses for pig farmers in the region. Based on the complete sequences of the B646L (p72) gene, it has been determined that the virus responsible for the outbreak is ASFV genotype II. The further characterization of three complete sequences of the B646L (p72) gene established 100% identical with other existing sequences of different parts of the world as well as confirmed that there is no co-circulation of different genotypes of ASFV in India except genotype II. Present studies also corroborate that MYD88, LDHB and IFIT1 were important genes of the immune system involved in the pathogenesis of ASFV. The differential expression patterns of these genes in ASFV-infected survived, and healthy Doom breed pigs, compared to healthy control pigs, were studied to distinguish the expression pattern at different stages. The hardiness and ability of the Doom pig to withstand common pig diseases, along with its genetic resemblance to wild pigs, make it an ideal candidate for studying tolerance to ASFV infection. So, the present study investigates the natural resistance to ASF in Doom pigs from an endemic area in North-East India to support the proposition that Doom pigs can co-exist with virulent ASFVs recently break in North-East India. The results of this study also provide important molecular insights into the regulation of the ASFV-tolerant gene. ImportanceStudying the natural resistance to African Swine Fever (ASF) in Doom pigs from North-East India holds crucial importance. ASF has inflicted significant economic losses on pig farmers in the region, necessitating the identification and comprehension of factors contributing to resistance and tolerance in specific pig breeds such as Doom pigs. Understanding the molecular mechanisms and genetic factors associated with ASFV tolerance could help in breeding programs and the selection of resilient pig breeds, ultimately aiding in disease control efforts.

microbiology↗

bioRxiv2023

Genetic profiling and in silico sequence analysis of CSN2 (beta-casein) and CSN3 (κ-casein) genes in the indian buffalo (Bubalus bubalis)

Buffalo rank second for milk production in the world and play important role in Indian economy. There are four types of caseins -S1-casein, -S2-casein, {beta}-casein and {kappa}-casein in milk. The major function of the casein protein in milk is to chelate colloidal calcium phosphate and serves as a major source of amino acids, calcium and phosphate. In buffalo milk all four casein proteins (s1, s2, {beta} and {kappa}) are encoded by four closely linked autosomal genes (CSN1S1, CSN1S2, CSN2 and CSN3 respectively) that are present on chromosome 7. Bovine CSN2 ({beta}-casein) gene is 8.5 kb long and contains nine exons and CSN3 ({kappa}-casein) is [~] 13 kb long. The aim of the study was to characterize CSN2 and CSN3 genes, In-silico analysis of {beta}-casein and {kappa}-casein protein and evolutionary relationship with other species. Buffalo mammary gland tissue was collected from local slaughterhouse (New Delhi, India) and total RNA was isolated from Buffalo Mammary Epithelial Cells. The ORF region of CSN2 and CSN3 genes were amplified and sequenced for characterization. Physiochemical properties showed that both buffalo {beta}-casein (Bu_CSN2) and {kappa}-casein (Bu_CSN3) proteins are stable and hydrophobic in nature.The presence of high phosphorylated residues in both {beta}-casein and {kappa}-casein proteins residues suggested that they are involved in signal transduction processes, cell growth and metabolism. The N-glycosylation result showed that both proteins are less in foldable state. The presence of methylation and acetylation sites in both protein revealed that they are involved in different cellular process. The evolutionary analysis showed that both buffalo genes more closely to Bos grunniens (yak).

molecular biology↗

bioRxiv2023

Muscle-specific MEF2D alpha 2 isoform regulates mice exercise capacity, muscle ketolysis, and systemic ketone body availability in mice

Ketone bodies are an alternate fuel source generated by the liver in response to low carbohydrate availability in neonates and after starvation and exhausting exercise in adulthood. The postnatal alternative splicing generates a highly conserved muscle-specific MEF2D2 protein isoform of the transcription factor MEF2D. Here, we discovered that compared to WT mice, MEF2D2 exon knockout (Eko) mice displayed reduced running capacity and muscle expression of all three ketolytic genes, BDH1, OXCT1, and ACAT1. Consistent with reduced muscle utilization of ketone bodies, MEF2D2 Eko mice also showed increased ketone body levels in a tolerance test, after exercise, and upon feeding a ketogenic diet. Lastly, using mitochondria isolated from skeletal muscle, we showed reduced ketone body utilization and respiration in Eko compared to WT mice. Thus, we identified a new role of MEF2D2 protein isoform in regulating skeletal muscle ketone body oxidation, exercise capacity, and its effect on systemic ketone body levels.

molecular biology↗

bioRxiv2023

PLGA-based dual-loaded nanoformulation of DIM and TMZ - An advanced clinical strategy for brain cancer treatment in a combinatorial approach

Glioblastoma multiforme (GBM) is a highly aggressive form of primary brain tumor in adults, which unfortunately has an abysmal prognosis and poor survival rates. Even though several FDA-approved multimodal treatments for targeting GBM are available, the effectiveness in most patients are not satisfactory. The reason behind this poor success rate is mainly attributed to insufficient drug distribution to the tumor site across the blood-brain barrier (BBB) and induction of resistance for single-drug based therapies. Chemotherapy with Temozolomide (TMZ) having a median overall survival of around 12-15 months, envisages the urgent necessity for more effective treatment strategies. Based upon these facts, in this study, we have developed a novel approach for repurposing TMZ along with inhibition of EGFR, which overexpressed in GBM, to achieve our goal. PLGA-based nanoencapsulation of both TMZ and 3,3-diindoyl methane (DIM), an EGFR inhibitor, in a combinatorial approach enhances the delivery of them together. Their synergistic mode of actions, significantly enhances the cytotoxic effect of TMZ in vitro and in vivo. Moreover, the dual-loaded nanoformulation works more efficiently than their individually packed nanoparticles on DNA damage and apoptosis, resulting in a several-fold reduction in tumor burden, systemic drug toxicity, and increased survival. These findings suggest the preclinical potential of this new treatment strategy.

cancer biology↗

bioRxiv2023

Phase variation in LPS O-antigen biosynthetic gene cluster of the rice pathogen Xanthomonas oryzaepv. oryzae

Bacteria respond to environmental cues in different ways. Phase variation is one such adaptation where heritable and reversible changes in DNA aid bacteria to alter the expression of specific genes. The bacterial plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) causes the serious bacterial blight disease of rice. The mucoid phenotype of Xoo colonies is attributed to the secreted exopolysaccharide (EPS), xanthan gum. Spontaneous non-mucoid variants of Xoo which are deficient in EPS production and virulence were observed to accumulate in long-term stationary phase cultures. This phenomenon was termed stationary phase variation and variant colonies as stationary phase variants (SPV). Several but not all of these SPVs have been earlier described to carry spontaneous insertions of endogenous insertion sequence elements in the gum operon which encodes genes involved in EPS biosynthesis. In this study, we show that a number of SPVs harbour variations in the lipopolysaccharide (LPS) outer antigen (O-antigen) biosynthetic gene cluster. The data revealed that the vast majority of variations are due to either insertion of endogenous insertion sequence (IS) elements or slipped strand mispairing (SSM). Also, it was observed that many of these SPVs exhibited reversion to wild type mucoid phenotype via restoration of the wild type genotype. The results indicate that the phenomenon of phase variation is occurring in the LPS O-antigen biosynthetic gene cluster of Xoo.

microbiology↗

bioRxiv2023

A broadly protective CHO cell expressed recombinant spike protein subunit based vaccine (IMT-CVAX) against SARS-CoV-2

Protective immunity induced by COVID-19 vaccines is mediated mainly by spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report the development of a recombinant prefusion stabilized SARS-CoV-2 spike protein-subunit-based COVID-19 vaccine produced in the mammalian cell line. The gene encoding ectodomain (ECD) of the spike protein was engineered and cloned into Freedom pCHO 1.0, a mammalian expression vector, and subsequently expressed in the Chinese Hamster Ovary suspension cell line (CHO-S). The recombinant S protein ectodomain (hereafter referred to as IMT-CVAX) was purified using a combination of tangential flow filtration and liquid chromatography. Biochemical and biophysical characterization of IMT-CVAX was done to ensure its vital quality attributes. Intramuscular immunization of mice with two doses of adjuvanted IMT-CVAX elicited a strong anti-Spike IgG response. In pseudovirus-based assays, IMT-CVAX- immune mice sera exhibited a broad-spectrum neutralization of several SARS-CoV-2 variants of concern (VoCs). Golden Syrian Hamster immunized with IMT-CVAX provided excellent protection against SARS-CoV-2 infection, and, hamster immune sera neutralized the live SARS-CoV-2 virus. The adjuvanted IMT-CVAX induced robust Tfh-cells response and germinal center (GC) reaction in human ACE2 receptor-expressing transgenic mice. The findings of this study may pave the way for developing next-generation protein subunit-based vaccines to combat the existing SARS-CoV-2 and its emerging VoCs. The IMT-CVAX is produced using a scalable process and can be used for large-scale vaccine production in an industrial setup.

microbiology↗

bioRxiv2023

Enhanced protective efficacy of a novel, thermostable, RBD-S2 fusion immunogen against SARS-CoV-2 and its variants

With the rapid emergence of variants of concern (VOC), the efficacy of currently licensed vaccines has reduced drastically. VOC mutations largely occur in the S1 subunit of Spike. The S2 subunit of SARS-CoV-2 is conserved and thus more likely to elicit broadly protective immune responses. However, the contribution of the S2 subunit in improving the overall efficacy of vaccines remains unclear. Therefore, we designed, characterized, and evaluated the immunogenicity and protective potential of a stabilized SARS-CoV-2 Receptor Binding Domain (RBD) fused to a stabilized S2. Designed immunogens were expressed as soluble proteins with approximately fivefold higher purified yield than the Spike ectodomain and formulated along with Squalene-in-water emulsion (SWE) adjuvant. S2 immunization failed to elicit a neutralizing immune response but significantly reduced lung viral titers in mice challenged with the heterologous Beta variant. In hamsters, SWE-formulated RS2 showed enhanced immunogenicity and efficacy relative to corresponding RBD and Spike formulations. Despite being based on the ancestral Wuhan strain of SARS-CoV-2, RS2 exhibited broad neutralization, including against Omicron variants (BA.1, BA.5 and BF.7), as well as the clade 1a WIV-1 and SARS-CoV-1 strains. RS2 sera also showed enhanced competition with both S2 directed and RBD Class 4 directed broadly neutralizing antibodies, relative to RBD and Spike elicited sera. When lyophilized, RS2 retained antigenicity and immunogenicity even after incubation at 37 {degrees}C for a month. The data collectively suggest that the RS2 immunogen is a promising modality to combat SARS-CoV-2 variants.

biophysics↗

bioRxiv2023

Identification of chemotherapy targets reveals a nucleus-to-mitochondria ROS sensing pathway

Multiple chemotherapies are proposed to cause cell death in part by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs exactly how the resultant ROS function and are sensed is poorly understood. In particular, its unclear which proteins the ROS modify and their roles in chemotherapy sensitivity/resistance. To answer these questions, we examined 11 chemotherapies with an integrated proteogenomic approach identifying many unique targets for these drugs but also shared ones including ribosomal components, suggesting one mechanism by which chemotherapies regulate translation. We focus on CHK1 which we find is a nuclear H2O2 sensor that promotes an anti-ROS cellular program. CHK1 acts by phosphorylating the mitochondrial-DNA binding protein SSBP1, preventing its mitochondrial localization, which in turn decreases nuclear H2O2. Our results reveal a druggable nucleus-to-mitochondria ROS sensing pathway required to resolve nuclear H2O2 accumulation, which mediates resistance to platinum-based chemotherapies in ovarian cancers.

molecular biology↗

bioRxiv2023

Mutations in S2 subunit of SARS-CoV-2 Omicron spike strongly influence its conformation, fusogenicity and neutralization sensitivity

SARS-CoV-2 has remarkable ability to respond to and evolve against the selection pressure by host immunity exemplified by emergence of Omicron lineage. Here, we characterized the functional significance of mutations in Omicron spike. By systematic transfer of mutations in WT spike we assessed neutralization sensitivity, fusogenicity, and TMPRSS2-dependence for entry. The data revealed that the mutations in both S1 and S2 complement to make Omicron highly resistant. Strikingly, the mutations in Omicron S2 modulated the neutralization sensitivity to NTD- and RBD-antibodies, but not to S2 specific neutralizing antibodies, suggesting that the mutations in S2 were primarily acquired to gain resistance to S1-antibodies. Although all six mutations in S2 appeared to act in concert, D796Y showed greatest impact on neutralization sensitivity and rendered WT virus >100-fold resistant to S309, COVA2-17, and 4A8. S2 mutations greatly reduced the antigenicity for NAbs due to reduced exposure of epitopes. In terms of the entry pathway, S1 or S2 mutations only partially altered the entry phenotype of WT and required both sets of mutations for complete switch to endosomal route and loss of syncytia formation. In particular, N856K and L981F in Omicron reduced fusion capacity and explain why subsequent Omicron variants lost them to regain fusogenicity.

microbiology↗

bioRxiv2023

Split-Transformer Impute (STI): Genotype Imputation Using a Transformer-Based Model

MotivationDespite recent advances in sequencing technologies, genome-scale datasets continue to have missing bases and genomic segments. Such incomplete datasets can undermine downstream analyses, such as disease risk prediction and association studies. Consequently, the imputation of missing information is a common pre-processing step for which many methodologies have been developed. However, the imputation of genotypes of certain genomic regions and variants, including large structural variants, remains a challenging problem. ResultsHere, we present a transformer-based deep learning framework, called a split-transformer impute (STI) model, for accurate genome-scale genotype imputation. Empowered by the attention-based transformer model, STI can be trained for any collection of genomes automatically using self-supervision. STI handles multi-allelic genotypes naturally, unlike other models that need special treatments. STI models automatically learned genome-wide patterns of linkage disequilibrium (LD), evidenced by much higher imputation accuracy in high LD regions. Also, STI models trained through sporadic masking for self-supervision performed well in imputing systematically missing information. Our imputation results on the human 1000 Genomes Project show that STI can achieve high imputation accuracy, comparable to the state-of-the-art genotype imputation methods, with the additional capability to impute multi-allelic structural variants and other types of genetic variants. Moreover, STI showed excellent performance without needing any special presuppositions about the patterns in the underlying data when applied to a collection of yeast genomes, pointing to easy adaptability and application of STI to impute missing genotypes in any species.

genomics↗

bioRxiv2023

Musashi-2 causes cardiac hypertrophy and heart failure by inducing mitochondrial dysfunction through destabilizing Cluh and Smyd1 mRNA

Regulation of RNA stability and translation by RNA-binding proteins (RBPs) is a crucial process altering gene expression. Musashi family of RBPs comprising Msi1 and Msi2 are known to control RNA stability and translation. However, despite the presence of MSI2 in the heart, its function remains entirely unknown. Here, we aim to explore the cardiac functions of MSI2. We confirmed the presence of MSI2 in the adult mouse, rat heart, and neonatal rat cardiomyocytes. Furthermore, Msi2 was significantly enriched in the hearts cardiomyocyte fraction. Next, using RNA-seq data and isoform-specific PCR primers, we identified, Msi2 isoforms 1, 4, and 5 and two novel putative isoforms labeled as Msi2 isoforms 6 and 7 to be expressed in the heart. Overexpression of Msi2 isoforms led to cardiac hypertrophy in cultured cardiomyocytes. Additionally, Msi2 was also found to be significantly increased in a pressure-overload model of cardiac hypertrophy. To validate the hypertrophic effects, we selected isoforms 4 and 7 due to their unique alternative splicing patterns. AAV9-mediated overexpression of Msi2 isoforms 4 and 7 in murine hearts led to cardiac hypertrophy, dilation, heart failure, and eventually early death, confirming a pathological function for Msi2. Using global proteomics, gene ontology, transmission electron microscopy, and transmembrane potential measurement assays increased MSI2 was found to cause mitochondrial dysfunction in the heart. Mechanistically, we identified Cluh and Smyd1 as direct downstream targets of Msi2. Overexpression of Cluh or Smyd1 inhibited Msi2-induced hypertrophy and mitochondrial dysfunction in cardiomyocytes. Collectively, we show that Msi2 induces hypertrophy, mitochondrial dysfunction, and heart failure.

cell biology↗