Biology subjects

Kumar, D.

Publications and source records attributed to Kumar, D..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

Microglia ferroptosis is prevalent in neurodegenerative disease and regulated by SEC24B

Iron dysregulation has been implicated in multiple neurodegenerative diseases, including Parkinsons Disease (PD), Amyotrophic Lateral Sclerosis (ALS), and Multiple Sclerosis (MS). One prominent feature of affected brain regions are iron-loaded microglia, but how iron overload influences microglia physiology and disease response is poorly understood. Here we show that microglia are highly susceptible to ferroptosis, an iron-dependent form of cell death. In a tri-culture of human iPSC-derived neurons, astrocytes, and microglia, under ferroptosis-inducing conditions, microglia undergo a drastic shift in cell state, with increased ferritin levels, disrupted glutathione homeostasis, and altered cytokine signaling. Similar ferroptosis-associated signature (FAS) microglia were uncovered in PD, and the signature was also found in a large cohort of PD patient blood samples, raising the possibility that ferroptosis can be identified clinically. We performed a genome-wide CRISPR screen which revealed a novel regulator of ferroptosis, the vesicle trafficking gene SEC24B. A small molecule screen also nominated several candidates which blocked ferroptosis, some of which are already in clinical use. These data suggest that ferroptosis sits at the interface of cell death and inflammation, and inhibition of this process in microglia and other brain cells may provide new ways for treating neurodegenerative disease.

neuroscience↗

bioRxiv2021

Changes in microbial composition, diversity, and functionality in the Amblyomma maculatum microbiome following infection with Rickettsia parkeri

BackgroundTicks are the primary vectors for emerging and resurging pathogens of public health significance worldwide. Examining tick bacterial composition, diversity, and functionality across developmental stages and tissues is necessary for designing new strategies to control ticks and prevent tick-borne diseases. MethodsA high-throughput sequencing approach was used to determine the influence of blood meal and Rickettsia parkeri infection on changes in Amblyomma maculatum microbiome composition, diversity, and functionality across the developmental timeline and in different tissues. Quantitative insight into microbial ecology analysis allowed us to determine microbial population structure, composition, and diversity. A non-metric multidimensional scaling, the sparse correlations for compositional data (SparCC) module, and phylogenetic investigation of communities by reconstruction of unobserved states 2 (PICRUSt2) software were used in the assessment. ResultsThe Amblyomma maculatum microbiome comprises ten bacterial genera present across tick life cycle stages. Among the top ten bacterial genera (the core tick microbiome), Rickettsia, Francisella, and Candidatus Midichloria are the key players, with positive interactions within each developmental stage and adult tick organ tested. The bacterial abundances, based on the number of operational taxonomic units (OTUs), increase with blood meal in each stage, helping bacterial floral growth. The growth in bacterial numbers is related to highly abundant energy metabolism orthologs with blood meal, according to functional analysis. Whereas R. parkeri had a positive correlation with Candidatus Midichloria during the tick life cycle, based on the increased number of OTUs and network analysis, this was due to an increased level of metabolic activity. Interestingly, R. parkeri replaces Francisella, based on the lower level of OTUs representing Francisella in R. parkeri-infected ticks (in all stages/organs) and negatively correlated according to network and linear discriminant analysis effect size (LEfSe). ConclusionsWe found that Rickettsia and Francisella predominate in the core microbiome of the Gulf Coast tick, whereas Candidatus Midichloria and Cutibacterium levels increase with infection. Network analysis and functional annotation suggest that R. parkeri interacts positively with Candidatus Midichloria and negatively with Francisella and that metabolic profiles are upregulated with blood meal and R. parkeri infection. Overall, this is the first study to determine the combinatorial outcome of blood meal and pathogen interaction on microbiome composition over the developmental stages of Am. maculatum. This new study expands on our existing knowledge of the Am. maculatum microbiome and further highlights the need to investigate pathogen-symbiont interactions between R. parkeri and Francisella or Candidatus Midichloria to facilitate the development of strategies for controlling tick-transmitted diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/465777v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1c30a2aorg.highwire.dtl.DTLVardef@1d0713borg.highwire.dtl.DTLVardef@1b9eadborg.highwire.dtl.DTLVardef@e9f5ad_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

microbiology↗

bioRxiv2021

Machine learning-based modulation of Ca2+-binding affinity in EFhand proteins and comparative structural insights into site-specific cooperative binding

Ca2+-binding proteins are present in almost all living organisms and different types display different levels of binding affinities for the cation. Here, we applied two new scoring schemes enabling the user to manipulate the binding affinities of such proteins. We specifically designed a unique EF-hand loop capable of binding calcium with high affinity by altering five residues of the loop based on the scoring scheme. We worked on the N-terminal domain of Entamoeba histolytica calcium-binding protein1 (NtEhCaBP1), and used site-directed mutagenesis to incorporate the designed loop sequence into the second EF hand motif of this protein. The binding isotherms calculated using ITC calorimetry showed a [~]500-fold greater association constant (Ka) for the mutant. The crystal structure of the mutant was also determined, and displayed more compact Ca2+-coordination spheres in both of its EF loops than did the structure of the wildtype protein, consistent with the greater calcium-binding affinities of the mutant. The NtEhCaBP1 mutant was also shown to form a hexamer rather than just a trimer, and this hexamer formation was attributed to the position of the last helix of the mutant having been changed as a result of the strong calcium coordination. Further dynamic correlation analysis revealed that the mutation in the second EF loop changed the entire residue network of the monomer, resulting in a stronger coordination of Ca2+ even in other EF hand loop.

biophysics↗

bioRxiv2021

Dual avatars of E. coli grxB encoded Glutaredoxin 2 perform ascorbate recycling and ion channel activities

Glutaredoxins (Grxs) are single-domain redox enzymes of the thioredoxin superfamily, and primarily function as glutathione (GSH) dependent disulphide reductases. Whereas, the E. coli Glutaredoxin 2 (EcGrx2) encoded by grxB has two conserved GST-fold domains, it still lacks a classical Grx-like functions. In this study, we show for the first time, that EcGrx2 exists in both soluble and membrane integrated forms. The soluble form associates with a previously unidentified GSH dependent dehydroascrobate (DHA) reductase, and the membrane integrated form possesses ion channel activities. Using enzyme kinetic data and structural data we unequivocally demonstrate that EcGrx2 recycles ascorbate (AsA) from DHA. This ability to recycle AsA is inhibited by Zinc (Zn2+). We also show that both wildtype and the E. coli grxB deletion mutant can be rescued from H2O2-induced oxidative stress using ascorbate as an antioxidant, which otherwise is only known as a carbon source in bacteria. Moreover, the grxB- mutant is susceptible to intracellular killing by ROS producing macrophages. We further discovered that EcGrx2 integrates into the native E. coli membrane and show that the purified soluble protein readily inserts into artificial lipid bilayer membrane and conducts ions in vitro. Our data demonstrates a highly conserved functional similarity among EcGrx2-orthologs and highlights that the utilization and subsequent recycling of ascorbate as an antioxidant by grxB harbouring gram-negative bacteria, including human pathogens, may provide a survival advantage under hostile oxidative environments.

biochemistry↗

bioRxiv2021

E156/G and Arg158, Phe-157/del mutation in NTD of spike protein in B.1.167.2 lineage of SARS-CoV-2 leads to immune evasion through antibody escape

Emerging variants of SARS-CoV-2 with better immune escape mechanisms and higher transmissibility remains a persistent threat across the globe. B.1.617.2 (Delta) variant was first emerged from Maharashtra, India in December, 2020. This variant is classified to be a major cause and concern of the second wave of COVID-19 in India. In the present study, we explored the genomic and structural basis of this variant through computational analysis, protein modelling and molecular dynamics (MD) simulations approach. B.1.617.2 variant carried E156G and Arg158, Phe-157/del mutations in NTD of spike protein. These mutations in N-terminal domain (NTD) of spike protein of B.1.617.2 variant revealed more rigidity and reduced flexibility compared to spike protein of Wuhan isolate. Further, docking and MD simulation study with 4A8 monoclonal antibody which was reported to bind NTD of spike protein suggested reduced binding of B.1.617.2 spike protein compared to that of spike protein of Wuhan isolate. The results of the present study demonstrate the possible case of immune escape and thereby fitness advantage of the new variant and further warrants demonstration through experimental evidence. Our study identified the probable mechanism through which B.1.617.2 variant is more pathogenically evolved with higher transmissibility as compared to the wild-type. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/447321v3_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@b92cborg.highwire.dtl.DTLVardef@1d261f7org.highwire.dtl.DTLVardef@11da73eorg.highwire.dtl.DTLVardef@1cef6ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2021

Single Cell Atlas of Human Putamen Reveals Disease Specific Changes in Synucleinopathies: Parkinson's Disease and Multiple System Atrophy

Understanding disease biology at a cellular level from disease specific tissues is imperative for effective drug development for complex neurodegenerative diseases. We profiled 87,086 nuclei from putamen tissue of healthy controls, Parkinsons Disease (PD), and Multiple System Atrophy (MSA) subjects to construct a comprehensive single cell atlas. Although both PD and MSA are manifestations of -synuclein protein aggregation, we observed that both the diseases have distinct cell-type specific changes. We see a possible expansion and activation of microglia and astrocytes in PD compared to MSA and controls. Contrary to PD microglia, we found absence of upregulated unfolded protein response in MSA microglia compared to controls. Differentially expressed genes in major cell types are enriched for genes associated with PD-GWAS loci. We found altered expression of major neurodegeneration associated genes -- SNCA, MAPT, LRRK2, and APP -- at cell-type resolution. We also identified disease associated gene modules using a network biology approach. Overall, this study creates an interactive atlas from synucleinopathies and provides major cell-type specific disease insights. Link to interactive atlas will be made available at the time of publication.

genomics↗

bioRxiv2021

Single Nuclei Sequencing of Human Putamen Oligodendrocytes Reveals Altered Heterogeneity and Disease-Associated Changes in Parkinson's Disease and Multiple System Atrophy

The role of oligodendrocytes in neurodegenerative diseases remains incompletely understood and largely unexplored at the single cell level. We profiled 87,086 single nuclei from human brain putamen region for healthy control, Parkinsons Disease (PD), and Multiple System Atrophy (MSA). Oligodendrocyte lineage cells were the dominant cell-type in the putamen with oligodendrocyte subpopulations clustered by transcriptomic variation found to exhibit diverse functional enrichment patterns, and this oligodendrocyte heterogeneity was altered in a disease-specific way. Among profiled oligodendrocyte subpopulations, differences in expression of SNCA, HAPLN2, MAPT, APP, and OPALIN were observed for PD and MSA compared with healthy controls. Intriguingly, greater activation of unfolded protein response pathway gene expression was observed in PD nuclei versus MSA. Using network analysis, we then identified specific PD- and MSA-correlated gene co-expression modules enriched with disease relevant pathways; the PD-correlated module was significantly enriched for Parkinsons Disease GWAS loci (p = 0.01046). Our analysis provides a broader understanding of oligodendrocyte heterogeneity and reveals distinctive oligodendrocyte pathological alterations associated with PD and MSA which may suggest potential novel therapeutic targets and new strategies for disease modification.

genomics↗

bioRxiv2021

Transcriptional Inactivation of TP53 and the BMP Pathway Mediates Therapy-induced Dedifferentiation and Metastasis in Prostate Cancer

Unsupervised clustering and deconvolution analysis identifies a novel subtype of M-CRPC endowed with hybrid epithelial/mesenchymal (E/M) and luminal progenitor-like traits (Mesenchymal and Stem-like PC, MSPC). Analysis of patient datasets and mechanistic studies indicate that MSPC arises as a consequence of therapy-induced lineage plasticity. AR blockade instigates two separate and complementary processes: 1) transcriptional silencing of TP53 and hence acquisition of hybrid E/M and stem-like traits; and 2) inhibition of the BMP signaling, which promotes resistance to the pro-apoptotic and anti-proliferative effects of AR inhibition. The drug-tolerant prostate cancer cells generated through reprogramming are rescued by neuregulin and generate metastases in mice. Combined inhibition of HER2/3 and AR or mTORC1 exhibit efficacy in preclinical models of mixed ARPC/MSPC or MSPC, respectively. These results identify a novel subtype of M-CRPC, trace its origin to therapy-induced lineage plasticity, and reveal its dependency on HER2/3 signaling.

cancer biology↗

bioRxiv2021

A ciliopathy complex builds distal appendages to initiate ciliogenesis

Cells inherit two centrioles, the older of which is uniquely capable of generating a cilium. Using proteomics and super-resolved imaging, we identified a module which we term DISCO (DIStal centriole COmplex). DISCO components CEP90, MNR and OFD1 underlie human ciliopathies. This complex localized to both distal centrioles and centriolar satellites, proteinaceous granules surrounding centrioles. Cells and mice lacking CEP90 or MNR did not generate cilia, failed to assemble distal appendages, and did not transduce Hedgehog signals. Disrupting the satellite pools did not affect distal appendage assembly, indicating that it is the centriolar populations of MNR and CEP90 that are critical for ciliogenesis. CEP90 recruited the most proximal known distal appendage component, CEP83, to root distal appendages formation, an early step in ciliogenesis. In addition, MNR, but not CEP90, restricted centriolar length by recruiting OFD1. We conclude that DISCO acts at the distal centriole to support ciliogenesis by restraining centriole length and assembling distal appendages, defects in which cause human ciliopathies. eTOC summaryKumar et al. identifies a multi-protein complex called DISCO (DIStal centriole COmplex) required to nucleate distal appendages and restrain centriole elongation, essential for the initiation of cilium assembly. Without DISCO, cells fail to ciliate and transduce Hedgehog signals, critical for mammalian development.

cell biology↗

bioRxiv2021

Host-virus chimeric events in SARS-CoV2 infected cells are infrequent and artifactual

Pathogenic mechanisms underlying severe SARS-CoV2 infection remain largely unelucidated. High throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in RNA-seq data from SARS-CoV2 infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV2 is a positive sense RNA virus that replicates in the cytoplasm it does not have a nuclear phase in its life cycle, it is biologically unlikely to be in a location where splicing events could result in genome integration. Here, we investigated the biological authenticity of HVC events. In contrast to true biological events such as mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with COVID-19 and infected cell lines, were highly irreproducible. RNA-seq library preparation is inherently error-prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spike-in RNA from an unrelated species, such as fruit-fly, we estimated that ~1% of RNA-seq reads are artifactually chimeric. In SARS-CoV2 RNA-seq we found that the frequency of HVC events was, in fact, not greater than this background "noise". Finally, we developed a novel experimental approach to enrich SARS-CoV2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich for HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV2 infected cells are extremely rare and are likely artifacts arising from either random template switching of reverse-transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes.

microbiology↗

bioRxiv2021

Hepatocyte nuclear factor 4a and glucocorticoid receptor coordinately regulate lipid metabolism in mice fed a high-fat-high-sugar diet

Hepatocyte nuclear factor 4 (HNF4) and glucocorticoid receptor (GR), master regulators of liver metabolism, are down-regulated in fatty liver diseases. The present study was aimed to elucidate the role of down-regulation of HNF4 and GR in fatty liver and hyperlipidemia. Adult mice with liver-specific heterozygote and knockout (knockout) of HNF4 were fed a low-fat diet (LFD) or a high-fat-high-sugar diet (HFHS) for 15 days. Compared to LFD-fed mice, HFHS-fed wildtype mice had hepatic induction of lipid catabolic genes and down-regulation of lipogenic genes. Compared to HFHS-fed wildtype mice, HNF4 heterozygote mice had down-regulation of lipid catabolic genes, induction of lipogenic genes, and increased hepatic and blood levels of lipids, whereas HNF4 knockout mice had mild hypolipidemia, down-regulation of lipid-efflux genes, but induction of genes for uptake/storage of lipids. Sterol-regulatory-element-binding protein-1c (SREBP-1C), a master lipogenic regulator, was induced in HFHS-fed HNF4 heterozygote mice. In reporter assays, HNF4 potently inhibited the transactivation of mouse and human SREBP-1C promoter by liver X receptor. Surprisingly, nuclear GR proteins were gene-dosage-dependently decreased in HNF4 heterozygote and knockout mice. HFHS-fed mice with liver-specific knockout of GR had increased hepatic lipids and induction of SREBP-1C and PPAR{gamma}. In reporter assays, GR and HNF4 synergistically/additively induced lipid catabolic genes. Phosphorylation of AMP-activated protein kinase (AMPK), a key GR modulator, was dramatically decreased in HNF4 knockout mice. Thus, cooperative induction of lipid catabolic genes and suppression of lipogenic genes by HNF4 and GR, modulated by AMPK, may mediate the early resistance to HFHS-induced fatty liver and hyperlipidemia.

molecular biology↗

bioRxiv2021

Lysosomal-Immune Axis Is Associated with COVID 19 Disease Severity: Insights from Patient Single Cell Data

SARS-COV-2 has become a leading cause of illness, hospitalizations, and deaths worldwide yet heterogeneity in disease morbidity remains a conundrum. In this study, we analyzed publicly available single-cell RNA-seq data from 75076 cells sequenced from clinically staged COVID-19 patients using a network approach and identified lysosomal-immune axis as a factor significantly associated with disease severity. Our results suggest modulation of lysosomal-immune pathways may present a novel drug-targeting strategy to attenuate SARS-Cov-2 infections.

genomics↗

bioRxiv2021

Lipophilic Nanocrystal Prodrug-Release Defines the Extended Pharmacokinetic Profiles of a Year-Long Cabotegravir

A single, once every eight-week cabotegravir (CAB) long-acting parenteral is more effective than daily oral emtricitabine and tenofovir disoproxil fumarate in preventing human immunodeficiency virus type one (HIV-1) transmission. Extending CAB dosing to a yearly injectable can advance efforts leading to the elimination of viral transmission. The current submission adds rigor, reproducibility and mechanistic insights for the extended apparent half-life of a yearlong antiretroviral injectable. Pharmacokinetic (PK) profiles of a nanoformulated fatty acid ester CAB prodrug (named NM2CAB) were affirmed at two academic and one contract research laboratory. PK profiles showed plasma CAB levels at or above the protein-adjusted 90% inhibitory concentration for up to one year after a single dose. Measures of drug biodistribution demonstrated sustained native drug at the muscle injection site and in lymphoid tissues (spleen and lymph nodes). The results paralleled NM2CAB uptake and retention in human macrophages. NM2CAB nanocrystals were stable in blood, tissue and liver homogenates. The long apparent drug half-life followed pH-dependent slow prodrug release in weeks from the nanocrystal. In contrast, solubilized prodrug was hydrolyzed in hours in plasma and tissues recorded from multiple mammalian species at basic pH. No measurable toxicities were recorded. These results, taken together, affirm the pharmacological mechanistic properties of a year-long nanoformulated CAB prodrug supporting the established protocol design for formulation safety, rigor and reproducibility.

pharmacology and toxicology↗

bioRxiv2020

Bivalent chromatin protects reversibly repressed genes from irreversible silencing

Bivalent chromatin is characterized by the simultaneous presence of H3K4me3 and H3K27me3, histone modifications generally associated with transcriptionally active and repressed chromatin, respectively. Prevalent in embryonic stem cells, bivalency is postulated to poise lineage-controlling developmental genes for rapid activation during embryogenesis while maintaining a transcriptionally repressed state in the absence of activation cues, but its function in development and disease remains a mystery. Here we show that bivalency does not poise genes for rapid activation but protects reversibly repressed genes from irreversible silencing. We find that H3K4me3 at bivalent gene promoters--a product of the underlying DNA sequence--persists in nearly all cell types irrespective of gene expression and confers protection from de novo DNA methylation. Accordingly, loss of H3K4me3 at bivalent promoters is strongly associated with aberrant hypermethylation and irreversible silencing in adult human cancers. Bivalency may thus represent a distinct regulatory mechanism for maintaining epigenetic plasticity. HIGHLIGHTSO_LIBivalent chromatin does not poise genes for rapid activation C_LIO_LIH3K4me3 at bivalent promoters is not instructive for transcription activation C_LIO_LIH3K4me3 at bivalent promoters protects reversibly repressed genes from de novo DNA methylation C_LIO_LILoss of H3K4me3/bivalency is associated with aberrant DNA hypermethylation in cancer C_LI

genomics↗

bioRxiv2020

PRAK-03202: A triple antigen VLP vaccine candidate against SARS CoV-2

The rapid development of safe and effective vaccines against SARS CoV-2 is the need of the hour for the coronavirus outbreak. Here, we have developed PRAK-03202, the worlds first triple antigen VLP vaccine candidate in a highly characterized S. cerevisiae-based D-CryptTM platform, which induced SARS CoV-2 specific neutralizing antibodies in BALB/c mice. Immunizations using three different doses of PRAK-03202 induces antigen specific (Spike, envelope and membrane proteins) humoral response and neutralizing potential. PBMCs from convalescent patients, when exposed to PRAK-03202, showed lymphocyte proliferation and elevated IFN-{gamma} levels suggestive of conservation of epitopes and induction of T helper 1 (Th1)-biased cellular immune responses. These data support the clinical development and testing of PRAK-03202 for use in humans.

immunology↗

bioRxiv2020

Benchmarking ensemble docking methods as a scientific outreach project

The discovery of new drugs is a time consuming and expensive process. Methods such as virtual screening, which can filter out ineffective compounds from drug libraries prior to expensive experimental study, have become popular research topics. As the computational drug discovery community has grown, in order to benchmark the various advances in methodology, organizations such as the Drug Design Data Resource have begun hosting blinded grand challenges seeking to identify the best methods for ligand pose-prediction, ligand affinity ranking, and free energy calculations. Such open challenges offer a unique opportunity for researchers to partner with junior students (e.g., high school and undergraduate) to validate basic yet fundamental hypotheses considered to be uninteresting to domain experts. Here, we, a group of high school-aged students and their mentors, present the results of our participation in Grand Challenge 4 where we predicted ligand affinity rankings for the Cathepsin S protease, an important protein target for autoimmune diseases. To investigate the effect of incorporating receptor dynamics on ligand affinity rankings, we employed the Relaxed Complex Scheme, a molecular docking method paired with molecular dynamics-generated receptor conformations. We found that CatS is a difficult target for molecular docking and we explore some advanced methods such as distance-restrained docking to try to improve the correlation with experiments. This project has exemplified the capabilities of high school students when supported with a rigorous curriculum, and demonstrates the value of community-driven competitions for beginners in computational drug discovery.

scientific communication and education↗

bioRxiv2020

Evaluation of Safety and Immunogenicity of an Adjuvanted, TH-1 Skewed, Whole Virion Inactivated SARS-CoV-2 Vaccine - BBV152

We report the development and evaluation of safety and immunogenicity of a whole virion inactivated SARS-COV-2 vaccine (BBV152), adjuvanted with aluminium hydroxide gel (Algel), or a novel TLR7/8 agonist adsorbed Algel. We used a well-characterized SARS-CoV-2 strain and an established vero cell platform to produce large-scale GMP grade highly purified inactivated antigen, BBV152. Product development and manufacturing were carried out in a BSL-3 facility. Immunogenicity was determined at two antigen concentrations (3g and 6g), with two different adjuvants, in mice, rats, and rabbits. Our results show that BBV152 vaccine formulations generated significantly high antigen-binding and neutralizing antibody titers, at both concentrations, in all three species with excellent safety profiles. The inactivated vaccine formulation containing TLR7/8 agonist adjuvant-induced Th1 biased antibody responses with elevated IgG2a/IgG1 ratio and increased levels of SARS-CoV-2 specific IFN-{gamma}+ CD4 T lymphocyte response. Our results support further development for Phase I/II clinical trials in humans.

immunology↗

bioRxiv2020

Outcomes of the 2019 EMDataResource model challenge: validation of cryo-EM models at near-atomic resolution

This paper describes outcomes of the 2019 Cryo-EM Map-based Model Metrics Challenge sponsored by EMDataResource (www.emdataresource.org). The goals of this challenge were (1) to assess the quality of models that can be produced using current modeling software, (2) to check the reproducibility of modeling results from different software developers and users, and (3) compare the performance of current metrics used for evaluation of models. The focus was on near-atomic resolution maps with an innovative twist: three of four target maps formed a resolution series (1.8 to 3.1 [A]) from the same specimen and imaging experiment. Tools developed in previous challenges were expanded for managing, visualizing and analyzing the 63 submitted coordinate models, and several novel metrics were introduced. The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual laboratory experiments and holdings of structure data archives such as the Protein Data Bank. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived from these benchmark maps by 13 participating teams, representing both widely used and novel modeling approaches. We also evaluate the pros and cons of the commonly used metrics to assess model quality and recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed density in the cryo-EM map.

biochemistry↗