Biology subjects

Mukherjee, S.

Publications and source records attributed to Mukherjee, S..

At least 163 records · Page 9Linked to original sources

bioRxiv2022

High resolution cryo-EM structures of two potently SARS-CoV-2 neutralizing monoclonal antibodies of same donor origin that vary in neutralizing Omicron variants

While vaccines have by large been found to effective against the evolving SARS-CoV-2 variants, the profound and rapid effectivity of monoclonal antibodies (mAbs) in significantly reducing hospitalization to severe disease outcomes have also been demonstrated. In the present study, by high resolution cryo-electron microscopy (cryo-EM), we examined the structural insights of two trimeric spike (S) protein bound mAbs isolated from an Indian convalescent individual infected with ancestral SARS-CoV-2 which we recently reported to potently neutralize SARS-CoV-2 from its ancestral form through highly virulent Delta form however different in their ability to neutralize Omicron variants. Our findings showed binding and conformational heterogeneities of both the mAbs (THSC20.HVTR04 and THSC20.HVTR26) bound to S trimer in its apo and hACE-2 bound forms. Additionally, cryo-EM resolved structure assisted modeling highlighted key residues associated with the ability of these two mAbs to neutralize Omicron variants. Our findings highlighted key interacting features modulating antigen-antibody interacting that can further aid in structure guided antibody engineering to enhance their breadth and potency. HighlightsO_LITwo potent human mAbs obtained from a single donor differ binding to Omicron spikes C_LIO_LIPattern of binding and conformation of these mAbs bound to full length spike differs C_LIO_LIAntibody binding alters the conformational states of S trimer in its apo and hACE-2 bound forms. C_LIO_LICryo-EM structure guided modeling highlighted correlates of interacting residues associated with resistance and sensitivity of BA.1, BA.2, BA.4/BA.5 resistance and sensitivity against these mAbs. C_LI

biophysics↗

bioRxiv2022

Structural basis of sRNA RsmZ regulation of Pseudomonas aeruginosa virulence

Pseudomonas aeruginosa is an opportunistic human pathogen that poses threats to hospitalized immunocompromised patients 1. A non-coding small RNA (sRNA) from the repressor of secondary metabolites (Rsm) system, RsmZ, sequesters the global repressor protein RsmA to regulate downstream gene expressions that reprogram virulence repertoires associated with acute and chronic P. aeruginosa infections 2,3. Molecular insights into the full-length RsmZ architecture remain elusive, leading to the lack of understanding of RsmZ binding to RsmA and subsequent modulations of gene expressions. Here we use cryo-electron microscopy (cryo-EM) to resolve structures of the full-length RsmZ in complexes with RsmA, in which five stem-loops (SLs) and one single-stranded junction carrying the GGA binding sites in RsmZ form three pairs of clamps, each binding to a RsmA homodimer. Disruptions of the base-pairings in all stems of RsmZ significantly reduced the binding affinity to RsmA by 17-fold, which resulted in enhanced RsmA downregulation of gene expressions and phenotypes associated to both acute and chronic virulence of P. aeruginosa. Double mutations that rescued these stems of RsmZ restored the binding to RsmA by more than 5-fold, and recovered the corresponding phenotypes. Our results reveal the molecular mechanism of RsmZ regulation of P. aeruginosa virulence and suggest RsmZ as a potential target for the development of new antimicrobial agents.

microbiology↗

bioRxiv2022

Ancestral SARS-CoV-2 driven antibody repertoire diversity in an unvaccinated individual correlates with expanded neutralization breadth

Understanding the quality of immune repertoire triggered during natural infection can provide vital clues that form the basis for development of humoral immune response in some individuals capable of broadly neutralizing pan SARS-CoV-2 variants. We assessed the diversity of neutralizing antibody responses developed in an unvaccinated individual infected with ancestral SARS-CoV-2 by examining the ability of the distinct B cell germline-derived monoclonal antibodies (mAbs) in neutralizing known and currently circulating Omicron variants by pseudovirus and authentic virus neutralization assays. The ability of the antibodies developed post vaccination in neutralizing Omicron variants was compared to that obtained at baseline of the same individual and to those obtained from Omicron breakthrough infected individuals by pseudovirus neutralization assay. Broadly SARS-CoV-2 neutralizing mAbs representing unique B cell lineages with non-overlapping epitope specificities isolated from a single donor varied in their ability to neutralize Omicron variants. Plasma antibodies developed post vaccination from this individual demonstrated neutralization of Omicron BA.1, BA.2 and BA.4 with increased magnitude and found to be comparable with those obtained from other vaccinated individuals who were infected with ancestral SARS-CoV-2. Development of B cell repertoire capable of producing antibodies with distinct affinity and specificities for the antigen immediately after infection capable of eliciting broadly neutralizing antibodies offers highest probability in protecting against evolving SARS-CoV-2 variants. ImportanceDevelopment of robust neutralizing antibodies in SARS-CoV-2 convalescent individuals is known, however varies at population level. We isolated monoclonal antibodies from an individual infected with ancestral SARS-CoV-2 in early 2020 that not only varied in their B cell lineage origin but also varied in their capability and potency to neutralize all the known VOC and currently circulating Omicron variants. This indicated establishment of unique lineages that contributed in forming B cell repertoire in this particular individual immediately following infection giving rise to diverse antibody responses that could compensate each other in providing broadly neutralizing polyclonal antibody response. Individuals who were able to produce such potent polyclonal antibody responses after infection have a higher chance of being protected from evolving SARS-CoV-2 variants.

immunology↗

bioRxiv2022

Combinatorial control of biofilm development by quorum-sensing and nutrient-sensing regulators.

The human pathogen Pseudomonas aeruginosa, a leading cause of hospital-acquired infections, inhabits and forms sessile antibiotic-resistant communities called biofilms in a wide range of biotic and abiotic environments. In this study, we examined how two global sensory signaling pathways - the RhlR quorum-sensing system and the CbrA/CbrB nutritional adaptation system - intersect to control biofilm development. Previous work has shown that individually these two systems repress biofilm formation. Here, we used biofilm analyses, RNA-seq, and reporter assays to explore the combined effect of information flow through RhlR and CbrA on biofilm development. We find that the {Delta}rhlR{Delta}cbrA double mutant exhibits a biofilm morphology and an associated transcriptional response distinct from wildtype and the parent {Delta}rhlR and {Delta}cbrA mutants indicating codominance of each signaling pathway. The {Delta}rhlR{Delta}cbrA mutant rapidly gains suppressor mutations that map to the carbon catabolite repression protein Crc. The combined absence of RhlR and CbrA leads to drastic reduction in the abundance of the Crc antagonist small RNA CrcZ. Thus, CrcZ acts as the molecular convergence point for quorum- and nutrient-sensing cues. Furthermore, in the absence of antagonism by CrcZ, Crc promotes the expression of biofilm matrix components - Pel exopolysaccharide, and CupB and CupC fimbriae. Therefore, this study uncovers a regulatory link between nutritional adaption and quorum sensing with potential implications for anti-biofilm targeting strategies. AUTHOR SUMMARYBacterial pathogens often form multicellular communities encased in an extra cytoplasmic matrix called biofilms as a virulence strategy. Biofilm development is controlled by various environmental stimuli that are decoded and converted into appropriate cellular responses. How information from two or more stimuli is integrated is poorly understood. Using Pseudomonas aeruginosa biofilm formation as a model, we studied the intersection of two global sensory signaling pathways - quorum sensing and nutritional adaptation. We find parallel regulation by each pathway that converges on the abundance of a small RNA. Thus, we describe a regulatory link between P. aeruginosa quorum-sensing and nutritional adaptation pathways that allows integration of information from each system into the control of biofilm development. These results expand our understanding of the genetic regulatory strategies that allow P. aeruginosa to successfully colonize host during chronic infections.

microbiology↗

bioRxiv2022

Supervised Machine Learning Enables Geospatial Microbial Provenance

The recent increase in publicly available metagenomic datasets with geospatial metadata has made it possible to determine location-specific, microbial fingerprints from around the world. Such fingerprints can be useful for comparing microbial niches for environmental research, as well as for applications within forensic science and public health. To determine the regional specificity for environmental metagenomes, we examined 4305 shotgun-sequenced samples from the MetaSUB Consortium dataset - the most extensive public collection of urban microbiomes, spanning 60 different cities, 30 countries, and 6 continents. We were able to identify city-specific microbial fingerprints using supervised machine learning (SML) on the taxonomic classifications, and we also compared the performance of ten SML classifiers. We then further evaluated the five algorithms with the highest accuracy, with the city and continental accuracy ranging from 85-89% to 90-94%, respectively. We then used these results to develop Cassandra, a random-forest-based classifier that identifies indicator species to aid in fingerprinting and can infer higher-order microbial interactions at each site. We further tested the Cassandra algorithm on the Tara Oceans dataset, the largest collection of marine-based microbial genomes, where it classified the oceanic sample locations with 83% accuracy. These results and code show the utility of SML methods and Cassandra to identify bioindicator species across both oceanic and urban environments, which can help guide ongoing efforts in biotracing, environmental monitoring, and microbial forensics (MF).

microbiology↗

bioRxiv2022

Structural insights into pore dynamics of human Pannexin isoforms

Pannexins are single-membrane large-pore ion channels that release ATP upon activation. Three isoforms of pannexins, 1, 2, and 3, perform diverse cellular roles, including inflammation, differentiation, neuropathic pain, and ATP release. In this study, we report the cryoEM structure of pannexin 3 at 3.9 [A] and characterize the structural differences with pannexin isoforms 1 and 2. We observe the organization of the Pannexin 3 vestibule into two distinct chambers with a wider pore radius in comparison to both PANX1 and 2 isoforms. We further report the structure of pannexin1 congenital mutant R217H in the resolution range of 3.9 [A]. The congenital mutant R217H in transmembrane helix3 (TM3), R217H induce structural changes that leads to a partially closed pore and altered ATP interaction propensities. The channel conductance of the congenital mutant displays weakened voltage sensitivity. The results showcase a complete comparison of the three pannexin isoform structures that along with the structure of Pannexin 1 congenital mutant highlight distinct structural features of pannexin isoforms and the allosteric role of distant substitutions in dictating channel behavior in Pannexin 1.

biochemistry↗

bioRxiv2022

A megaprotein-based molecular bridge critical for lipid trafficking and cold resilience

Cells adapt to cold by increasing levels of unsaturated phospholipids and membrane fluidity through homeostatic mechanisms conserved in nearly all forms of life. As most eukaryotic enzymes for lipid synthesis and desaturation localize on endoplasmic reticulum (ER) membranes, it remains unknown how ER-resident lipids rapidly distribute to plasma membranes (PM). Here we report an exceptionally large and evolutionarily conserved protein LPD-3 in C. elegans that plays critical roles in lipid trafficking and cold resilience. We identified lpd-3 mutants in a mutagenesis screen for genetic suppressors of the lipid desaturase FAT-7, and found that the 452 kDa megaprotein LPD-3 bridges ER and PM, consisting of a structurally predicted hydrophobic tunnel for lipid trafficking. Loss of LPD-3 caused abnormal cellular distribution of phospholipids, diminished FAT-7 abundance, and organismic vulnerability to cold. These phenotypic defects of lpd-3 mutants were rescued by Lecithin comprising unsaturated phospholipids. Importantly, we found that deficient lpd-3 homologues in Zebrafish and mammalian cells led to defects similar to those observed in C. elegans. As mutations in KIAA1109/BLTP1, the human orthologue of lpd-3, cause Alkuraya-Kucinskas syndrome, we propose that the LPD-3 family proteins may serve as evolutionarily conserved "highway bridges" critical for ER-associated non-vesicular trafficking of lipids and resilience to cold stress in eukaryotic cells.

cell biology↗

bioRxiv2022

Universal gut microbial relationships in the gut microbiome of wild baboons

Ecological relationships between bacteria mediate the services that gut microbiomes provide to their hosts. Knowing the overall direction and strength of these relationships within hosts, and their generalizability across hosts, is essential to learn how microbial ecology scales up to affect microbiome assembly, dynamics, and host health. Here we gain insight into these patterns by inferring thousands of correlations in bacterial abundance between pairs of gut microbiome taxa from extensive time series data (5,534 microbiome profiles from 56 wild baboon hosts over a 13-year period). We model these time series using a statistically robust, multinomial logistic-normal modeling framework and test the degree to which bacterial abundance correlations are consistent across hosts (i.e., "universal") or individualized to each host. We also compare these patterns to two publicly available human data sets. We find that baboon gut microbial relationships are largely universal: correlation patterns within each baboon host reflect a mixture of idiosyncratic and shared patterns, but the shared pattern dominates by almost 2-fold. Surprisingly, the strongest and most consistently correlated bacterial pairs across hosts were overwhelmingly positively correlated and typically belonged to the same family--a 3-fold enrichment compared to pairs drawn from the data set as a whole. The bias towards universal, positive bacterial correlations was also apparent in monthly samples from human infants, and bacterial families that had universal relationships in baboons also tended to be universal in human infants. Together, our results advance our understanding of the relationships that shape gut microbial ecosystems, with implications for microbiome personalization, community assembly and stability, and the feasibility of microbiome interventions to improve host health.

ecology↗

bioRxiv2022

Macrophage differentiation is marked by increased abundance of the mRNA 3' end processing machinery, altered poly(A) site usage, and sensitivity to the level of CstF64

Regulation of mRNA polyadenylation is important for response to external signals and differentiation in several cell types, and results in mRNA isoforms that vary in the amount of coding sequence or 3 UTR regulatory elements. However, its role in differentiation of monocytes to macrophages has not been investigated. Macrophages are key effectors of the innate immune system that help control infection and promote tissue-repair. However, overactivity of macrophages contributes to pathogenesis of many diseases. In this study, we show that macrophage differentiation is characterized by shortening and lengthening of mRNAs in relevant cellular pathways. The cleavage/polyadenylation (C/P) proteins increase during differentiation, suggesting a possible mechanism for the observed changes in poly(A) site usage. This was surprising since higher C/P protein levels correlate with higher proliferation rates in other systems, but monocytes stop dividing after induction of differentiation. Depletion of CstF64, a C/P protein and known regulator of polyadenylation efficiency, delayed macrophage marker expression, cell cycle exit, attachment, and acquisition of structural complexity, and impeded shortening of mRNAs with functions relevant to macrophage biology. Conversely, CstF64 overexpression increased use of promoter-proximal poly(A) sites and caused the appearance of differentiated phenotypes in the absence of induction. Our findings indicate that regulation of polyadenylation plays an important role in macrophage differentiation.

molecular biology↗

bioRxiv2022

Structural basis for assembly and lipid-mediated gating of LRRC8A:C volume-regulated anion channels

Leucine-rich repeat-containing protein 8 (LRRC8) family members form volume regulated anion channels activated by hypoosmotic cell swelling. LRRC8 channels are ubiquitously expressed in vertebrate cells as heteromeric assemblies of LRRC8A (Swell1) and LRRC8B-E subunits. Channels of different subunit composition have distinct properties that explain the functional diversity of LRRC8 currents implicated in a broad range of physiology. However, the basis for heteromeric LRRC8 channel assembly and function is unknown. Here, we leverage a fiducial-tagging strategy to determine single-particle cryo-electron microscopy structures of heterohexameric LRRC8A:C channels in detergent micelles and lipid nanodiscs in three conformations. LRRC8A:C channels show pronounced changes in channel architecture compared to homomeric channels due to heterotypic cytoplasmic LRR interactions that displace LRRs and the LRRC8C subunit away from the conduction axis and poise the channel for activation. The structures and associated functional studies further reveal that lipids embedded in the channel pore block ion conduction in the closed state. Together, our results provide insight into determinants for heteromeric LRRC8 channel assembly, activity, and gating by lipids.

biophysics↗

bioRxiv2022

Oxonium Ion-Guided Ion Mobility-Assisted Glycoproteomics on the timsTOF Pro

Spatial separation of ions in the gas-phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro series combines a trapped ion mobility device with a quadrupole, collision-cell and a time-of-flight analyser to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from non-modified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared to non-modified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and non-modified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. This new approach was applied to selected glycoproteins, human plasma- and neutrophil-derived glycopeptides. We show that the achieved physical separation, combined with the focussing of the mass spectrometer, allows for improved extraction of information from the samples, even at shorter LC gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known make-up, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories.

biochemistry↗

bioRxiv2022

A Legionella toxin mimics tRNA and glycosylates the translation machinery to trigger a ribotoxic stress response.

Pathogens often secrete proteins or nucleic acids that mimic the structure and/or function of molecules expressed in their hosts. Molecular mimicry empowers pathogens to subvert critical host processes and establish infection. We report that the intracellular bacterium Legionella pneumophila secretes the toxin SidI (substrate of icm/dot transporter I), which possesses a transfer RNA (tRNA)-like shape and functions as a mannosyl transferase. The 3.1 [A] cryo-EM structure of SidI reveals an N-terminal domain that exhibits a characteristic inverted L-shape and charge distribution that is present in two other known protein mimics of tRNAs, the bacterial elongation factor EF-G and the mammalian release factor eRF1. In addition, we show that SidIs C-terminal domain adopts a glycosyl transferase B fold similar to a mannosyl transferase. This molecular coupling of the proteins fold and enzymatic function allows SidI to bind and glycosylate components of the host translation apparatus, including the ribosome, resulting in a robust block of protein synthesis that is comparable in potency to ricin, one of the most powerful toxins known. Additionally, we find that translational pausing activated by SidI elicits a stress response signature reminiscent of the ribotoxic stress response that is activated by elongation inhibitors that induce ribosome collisions. SidI-mediated effects on the ribosome activate the stress kinases ZAK and p38, which in turn drive an accumulation of the protein activating transcription factor 3 (ATF3). Intriguingly, ATF3 escapes the translation block imposed by SidI, translocates to the nucleus, and orchestrates the transcription of stress-inducible genes that promote cell death. Thus, using Legionella and its effectors as tools, we have unravelled the role of a ribosome-to-nuclear signalling pathway that regulates cell fate.

cell biology↗

bioRxiv2022

Genome-wide CRISPR screens identify novel regulators of wild-type and mutant p53 stability

Tumour suppressor p53 (TP53) is the most frequently mutated gene in cancer. Several hotspot p53 mutants not only lose tumour suppressive capabilities, but also function in a dominant-negative manner, suppressing canonical wild-type p53 function. Furthermore, some hotspot p53 mutants promote oncogenesis by gain-of-function mechanisms. Levels of p53 are regulated predominantly through regulation of protein stability and while wild-type p53 is normally kept at very low levels at steady-state, p53 mutants are often stabilized in tumours, which may be vital for their oncogenic properties. Here, we systematically profiled the factors that regulate protein stability of wild-type and mutant p53 using marker-based genome-wide CRISPR screens. We found that most proteins that regulate wild-type p53 also regulate a subset of p53 mutants with the exception of p53 R337H regulators, which are largely private to this mutant. Mechanistically, we identified FBXO42 as a novel positive regulator of a subset of p53 mutants comprising R273H, R248Q and R248W. We show that FBXO42 acts together with CCDC6 to regulate USP28-mediated p53 stabilization. Our work also identifies C16orf72 as a negative regulator of the stability of wild-type p53 and of all p53 mutants tested. C16orf72 is amplified in breast cancer, and we show that C16orf72 regulates p53 levels in mammary epithelium of mice and its overexpression results in accelerated breast cancer with reduced p53 levels. Together, this work provides a network view of the processes that regulate p53 stability, which might provide clues for reinforcing wild-type p53 or targeting mutant p53 in cancer.

cancer biology↗

bioRxiv2022

Decoding mechanism of action and susceptibility to drug candidates from integrated transcriptome and chromatin state

Omics-based technologies are driving major advances in precision medicine but efforts are still required to consolidate their use in drug discovery. In this work, we exemplify the use of multi-omics to support the development of 3-chloropiperidines (3-CePs), a new class of candidate anticancer agents. Combined analyses of transcriptome and chromatin accessibility elucidated the mechanisms underlying sensitivity to test agents. Further, we implemented a new versatile strategy for the integration of RNA-seq and ATAC-seq data, able to accelerate and extend the standalone analyses of distinct omic layers. This platform guided the construction of a perturbation-informed basal signature able to predict cancer cell lines sensitivity and to further direct compound development against specific tumor types. Overall, this approach offered a scalable pipeline to support the early phases of drug discovery, understanding of mechanism and potentially inform the positioning of therapeutics in the clinic.

systems biology↗

bioRxiv2022

Systematic Single Cell Pathway Analysis (SCPA) reveals novel pathways engaged during early T cell activation

Next generation sequencing technologies have revolutionized the study of T cell biology, capturing previously unrecognized diversity in cellular states and functions. Pathway analysis is a key analytical stage in the interpretation of such transcriptomic data, providing a powerful method for detecting alterations in important biological processes. Current pathway analysis tools are built on models developed for bulk-RNA sequencing, limiting their effectiveness when applied to more complex single cell RNA-sequencing (scRNA-seq) datasets. We recently developed a sensitive and distribution-free statistical framework for multisample distribution testing, which we implement here in the open-source R package Single Cell Pathway Analysis (SCPA). After demonstrating the effectiveness of SCPA over commonly used methods, we generate a scRNA-seq T cell dataset and characterize pathway activity over early cellular activation and between T cell populations. This revealed unexpected regulatory pathways in T cells, such as an intrinsic type I interferon system regulating T cell survival and a reliance on arachidonic acid metabolism throughout T cell activation. A systems level characterization of pathway activity in T cells across multiple human tissues also revealed alpha defensin expression as a hallmark of bone marrow derived T cells. Overall, our work here provides a widely applicable tool for single cell pathway analysis, and highlights unexpected regulatory mechanisms of T cells using a novel T cell dataset.

immunology↗

bioRxiv2022

Merkel cell polyomavirus regulates miR183 cluster and piR62011 in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer, a major subset of which is caused by the clonal integration of Merkel Cell Polyomavirus (MCV). Recent studies by Cheng et al. (2017) reported that virus-derived small T antigen protein-bound EP400 complex drives expression of genes essential for cellular transformation. On close analysis of their ChIP-Seq data, we uncovered that the complex binds to the promoter region of the microRNA-183 cluster. The miRNA183 cluster is a cluster of 3 miRNAs (miR183, 182 & 96) expressed and regulated together. These miRNAs are conserved across species, highly expressed in human embryonic stem cells and necessary for sensory/ mechanosensory organ development. We hypothesized that the MCV oncoproteins regulate host miRNA expression directly; an interaction novel in polyomaviruses. We tested miRNA expression via qPCR in both virus positive and negative MCC cell lines and found the former showed a much higher level. Further, fibroblasts expressing T antigens displayed an increase in miR182 expression in comparison to control. Knock-down of T antigens in MCC cells correspondingly decreased miR182 levels. To investigate its regulation we performed luciferase assays for the miRNA predicted promoter that showed increased activity in the presence of T antigens. Intriguingly, the seed sequence of miR182 completely matches to a piRNA called piR62011. Upon reanalysis of a MCC small RNA library, piR62011 emerged as the highest expressed. We found it expressed in MKL-1, a MCV positive cell line as well. Finally, to translate our findings into therapy for MCC, we screened small molecule (CMBL) library by performing surface plasmon resonance (SPR) assay and identified small molecules that binds to pre-miRNA182 and are testing them for their activity to kill MCC.

molecular biology↗

bioRxiv2022

Liquid condensate is a common state of proteins and polypeptides at the regime of high intermolecular interactions

Liquid-liquid phase separation (LLPS) has emerged as a crucial biological mechanism for sequestering macromolecules (such as proteins and nucleic acids) into membraneless organelles in cells. Unstructured and intrinsically disordered domains are known to facilitate multivalent interactions driving protein LLPS. We hypothesized that LLPS could be an intrinsic property of proteins/polypeptides at their high intermolecular interaction regime. To examine this, we studied many (a total of 23) proteins/polypeptides with different structures and sequences for LLPS study using molecular crowder polyethylene glycol (PEG-8000). We showed that all proteins and even highly charged polypeptides (under study) can undergo liquid condensate formation, however with different phase space and conditions. Using a single component and combinations of protein multicomponent (co-LLPS) systems, we establish that a variety of intermolecular interactions can drive proteins/polypeptides LLPS.

biophysics↗

bioRxiv2021

A combination of potently neutralizing monoclonal antibodies isolated from an Indian convalescent donor protects against the SARS-CoV-2 delta variant

Although efficacious vaccines have significantly reduced the morbidity and mortality due to COVID-19, there remains an unmet medical need for treatment options, which monoclonal antibodies (mAbs) can potentially fill. This unmet need is exacerbated by the emergence and spread of SARS-CoV-2 variants of concern (VOCs) that have shown some resistance to vaccine responses. Here we report the isolation of two highly potently neutralizing mAbs (THSC20.HVTR04 and THSC20.HVTR26) from an Indian convalescent donor, that neutralize SARS-CoV-2 VOCs at picomolar concentrations including the delta variant (B.1.617.2). These two mAbs target non-overlapping epitopes on the receptor-binding domain (RBD) of the spike protein thereby preventing the virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Furthermore, the mAb cocktail demonstrated protection against the Delta variant at low antibody doses when passively administered in the K18 hACE2 transgenic mice model, highlighting their potential as cocktail for prophylactic and therapeutic applications. Developing the capacity to rapidly discover and develop mAbs effective against highly transmissible pathogens like coronaviruses at a local level, especially in a low- and middle-income country (LMIC) such as India, will enable prompt responses to future pandemics as an important component of global pandemic preparedness. HighlightsO_LIIdentification of an Indian convalescent donor prior to emergence of SARS-CoV-2 Delta variant whose plasma demonstrated neutralization breadth across SARS-CoV-2 variants of concern (VOCs). C_LIO_LITwo (THSC20.HVTR04 and THSC20.HVTR26) monoclonal antibodies isolated from peripheral memory B cells potently neutralize SARS-CoV-2 VOCs: Alpha, Beta, Gamma, Delta and VOIs: Kappa and Delta Plus. C_LIO_LITHSC20.HVTR04 and THSC20.HVTR26 target non-competing epitopes on the receptor binding domain (RBD) and represent distinct germline lineages. C_LIO_LIPassive transfer of THSC20.HVTR04 and THSC20.HVTR26 mAbs demonstrated protection against Delta virus challenge in K18-hACE2 mice at low antibody doses. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/474152v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1f1b55corg.highwire.dtl.DTLVardef@1b9b438org.highwire.dtl.DTLVardef@e6d2a6org.highwire.dtl.DTLVardef@f92cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗