Biology subjects

Mukhopadhyay, S.

Publications and source records attributed to Mukhopadhyay, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Activity of the manganese efflux transporter SLC30A10 in dopaminergic but not GABAergic neurons protects against neurotoxicity

Metals such as copper, iron, and manganese (Mn) are essential for life, but induce neurotoxicity at elevated levels. Yet, the neuronal mechanisms of metal-induced neurological disease are largely unclear. A primary limitation has been an inability to selectively alter metal levels in specific neurons, so that the role of the targeted neurons in disease biology can be isolated. Here, we show that neuron-specific depletion of metal efflux transporters provides the feasibility to overcome this limitation by focusing on Mn, which accumulates in the basal ganglia and induces motor disease, and the Mn-specific efflux transporter SLC30A10. Pan-neuronal/glial Slc30a10 knockout mice exhibited increased basal ganglia Mn levels and hypolocomotor deficits in early-life (pre-adulthood), which were exacerbated by Mn exposure. The locomotor deficits of the pan-neuronal/glial strain was associated with a reduction in evoked striatal dopamine release without dopaminergic (DAergic) neurodegeneration or changes in striatal tissue dopamine levels. Furthermore, DAergic-specific, but not GABAergic-specific, Slc30a10 knockout mice recapitulated the hypolocomotor phenotype of the pan-neuronal/glial knockouts in early-life although Mn levels were elevated in the targeted basal ganglia regions of both the neuron-specific strains. Put together, our results imply that (1) activity of SLC30A10 in DAergic neurons is necessary to protect against early-life Mn neurotoxicity; (2) increasing Mn levels in DAergic neurons is sufficient to induce early-life motor disease, suggesting that Mn targets DAergic neurons in the early-life period to induce motor deficits; and (3) neuron-specific knockout of metal efflux transporters may be a widely applicable strategy to elucidate mechanisms of metal-induced neurotoxicity.

neuroscience↗

bioRxiv2022

A model of persistent post SARS-CoV-2 induced lung disease for target identification and testing of therapeutic strategies

COVID-19 survivors develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal samples. Mouse-adapted SARS-CoV-2 MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute disease through clinical recovery. At 15-120 days post-virus clearance, histologic evaluation identified subpleural lesions containing collagen, proliferative fibroblasts, and chronic inflammation with tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal upregulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

microbiology↗

bioRxiv2022

Single-Droplet Surface-Enhanced Raman Scattering Decodes the Molecular Language of Liquid-Liquid Phase Separation

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) are involved in a myriad of critical cellular functions and debilitating neurodegenerative diseases. Elucidating the role of intrinsic disorder and conformational heterogeneity of intrinsically disordered proteins/regions (IDPs/IDRs) in these phase-separated membrane-less organelles is crucial to understanding the mechanism of formation and regulation of biomolecular condensates. Here we introduce a unique single-droplet surface-enhanced Raman scattering (SERS) methodology that utilizes surface-engineered, plasmonic, metal nanoparticles to unveil the inner workings of mesoscopic liquid droplets of Fused in Sarcoma (FUS) in the absence and presence of RNA. These highly sensitive measurements offer unprecedented sensitivity to capture the crucial interactions, conformational heterogeneity, and structural distributions within the condensed phase in a droplet-by-droplet manner. Such an ultra-sensitive single-droplet vibrational methodology can serve as a potent tool to decipher the key molecular drivers of biological phase transitions of a wide range of biomolecular condensates involved in physiology and disease.

biophysics↗

bioRxiv2022

Presence of sodium taurocholate co-transporting polypeptide and Hepatitis B replication marker on placenta: Another home for the virus

BackgroundThe role of sodium taurocholate co-transporting polypeptide (NTCP), in facilitating the binding of Hepatitis B virus (HBV) on surface of hepatocytes is well documented. Expression of NTCP in extra hepatic cells may make these cells susceptible to HBV infection and support cellular proliferation akin to hepatocytes. Placental replication of HBV is not well explored. In this study we have assessed the expression of NTCP and HBV replication markers (HBeAg, HBcAg, and HBV DNA) in placental cells, to investigate if these cells act as host for HBV. MethodsFourty one HBsAg+ve pregnant women along with 10 healthy controls were enrolled after obtaining informed consent. The HBV DNA in placenta was detected by qPCR using primers for X and core ORF. Expression of NTCP in placenta was analyzed by qRT-PCR and further investigated by immunohistochemistry (IHC) along with HBV replication biomarkers, HBeAg, and HBcAg. ResultsHBsAg positive subjects were divided in two groups on the basis of viral load [High Viral Load (HVL) Group; viral load [≥] 2000IU/ml, Low Viral Load (LVL) Group; viral load <2000IU/ml] according to INASL guidelines 2018. HBV infected females showed increased expression of NTCP in trophoblasts of placenta compared to control group (HVL 3.69{+/-}0.13 Vs Control 1.74{+/-}0.15, p=0.0117). Furthermore, significant difference in NTCP expression was also observed between HVL and LVL group (HVL 3.69{+/-}0.13 Vs LVL 1.98{+/-}0.17, p=0.022) and positively correlated with the maternal HBV DNA load. Membranous and/or cytoplasmic immunostaining of NTCP, and cytoplasmic staining of HBeAg and HBcAg in trophoblasts along with presence of HBV DNA indicated that trophoblasts are not only susceptible to HBV infection but may also be a site for viral replication. ConclusionsThis is the pioneer study, which demonstrates expression of NTCP on placenta which may facilitate the entry of HBV. Furthermore, the study establishes the presence of HBeAg in placenta of patients without circulating HBeAg, indicating these cells may act as replication host/reservoir. This pioneering finding hints at the possibility of exploring the potential of NTCP blocking strategies in preventing vertical transmission of HBV.

microbiology↗

bioRxiv2021

Spatiotemporal Modulations in Heterotypic Condensates of Prion and α-Synuclein Control Phase Transitions and Amyloid Conversion

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) of proteins and nucleic acids are thought to govern critical cellular functions. These multicomponent assemblies provide dynamic hubs for competitive homotypic and heterotypic interactions. Here, we demonstrate that the complex coacervation between the prion protein (PrP) and -synuclein (-Syn) within a narrow stoichiometry regime results in the formation of highly dynamic liquid droplets. Domain-specific electrostatic interactions between the positively charged intrinsically disordered N-terminal segment of PrP and the negatively charged C-terminal domain of -Syn drive the formation of these highly tunable, reversible, thermo-responsive condensates. Picosecond time-resolved measurements revealed the existence of relatively ordered electrostatic nanoclusters that are stable on the nanosecond timescale and can undergo breaking-and-making on a much slower timescale giving rise to the liquid-like behavior on the second timescale and mesoscopic length-scale. The addition of RNA to these preformed coacervates yields multiphasic, anisotropic, vesicle-like, hollow condensates. LLPS promotes liquid-to-solid maturation of -Syn-PrP condensates resulting in the rapid conversion into heterotypic amyloids. Our results suggest that synergistic interactions between PrP and -Syn in liquid condensates can offer mechanistic underpinnings of their physiological role and overlapping neuropathological features.

biophysics↗

bioRxiv2021

Mutations at the Alphavirus E2/E1 inter-dimer interface have host-specific phenotypes

Alphaviruses are enveloped viruses transmitted by arthropod vectors to vertebrate hosts. The surface of the virion contains 80 glycoprotein spikes embedded in the membrane and these spikes mediate attachment to the host cell and initiate viral fusion. Each spike consists of a trimer of E2-E1 heterodimers. These heterodimers interact at two interfaces: (1) the intra-dimer interactions between E2 and E1 of the same heterodimer, and (2) the inter-dimer interactions between E2 of one heterodimer and E1 of the adjacent heterodimer (E1). We hypothesized that the inter-dimer interactions are essential for trimerization of the E2-E1 heterodimers into a functional spike. In this work, we made a mutant virus (CPB) where we replaced six inter-dimeric residues in the E2 protein of Sindbis virus (WT SINV) with those from the E2 protein from chikungunya virus, and studied its effect in both mammalian and mosquito cell lines. CPB produced fewer infectious particles in mammalian cells than in mosquito cells, relative to WT SINV. When CPB virus was purified from mammalian cells, particles showed reduced amounts of glycoproteins relative to capsid protein, and contained defects in particle morphology compared to virus derived from mosquito cells. Using cryo-EM, we determined that the spikes of CPB had a different conformation than WT SINV. Last, we identified two revertants, E2-H333N and E1-S247L, that restored particle growth and assembly to different degrees. We conclude the inter-dimer interface is critical for spike trimerization and is a novel target for potential antiviral drug design. IMPORTANCEAlphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as an emerging pathogen, with infections occurring worldwide. The spikes on the surface of the alphavirus particle are absolutely required for the virus to enter a new host cell and initiate an infection. Using a structure-guided approach, we made a mutant virus that alters spike assembly in mammalian cells but not mosquito cells. This is important because it identifies a region in the spike that could be a target for antiviral drug design.

microbiology↗

bioRxiv2021

Two step aggregation kinetics of Amyloid-β proteins from fractal analysis

Self-aggregation in proteins has long been studied and modeled due to its ubiquity and importance in many biological contexts. Several models propose a two step aggregation mechanism, consisting of linear growth of fibrils and branch formation. Single molecule imaging techniques such as total internal reflection fluorescence (TIRF) microscopy can provide direct evidence of such mechanisms, however, analyzing such large datasets is challenging. In this paper, we analyze for the first time, images of growing amyloid fibrils obtained from TIRF microscopy using the techniques of fractal geometry, which provides a natural framework to disentangle the two types of growth mechanisms at play. We find that after an initial linear growth phase, identified by a plateau in the average fractal dimension with time, the occurrence of branching events leads to a further increase in the fractal dimension with a final saturation value {approx} 2. We also simulate the aggregation process using the identified linear growth and secondary nucleation mechanisms, using an event driven algorithm. We theoretically model this system using a set of coupled nonlinear differential equations describing a mean field model for branching and linear growth, which we use to characterize the growth process observed in simulations as well as experiments. Finally, we provide estimates for the parameter regimes that govern the two step aggregation process observed in experiments.

biophysics↗

bioRxiv2021

Capsid-E2 interactions rescue core assembly in viruses that cannot form cytoplasmic nucleocapsid cores

Alphavirus capsid proteins (CPs) have two domains: the N-terminal domain (NTD) that interacts with the viral RNA, and the C-terminal domain (CTD) that forms CP-CP inter-actions and interacts with the cytoplasmic domain of the E2 spike protein (cdE2). In this study, we examine how mutations in the CP NTD affect CP CTD interactions with cdE2. We changed the length and/or charge of the NTD of Ross River virus CP and found that changing the charge of the NTD has a greater impact on core and virion assembly than changing the length of the NTD. The NTD CP insertion mutants are unable to form cyto-plasmic cores during infection but they do form cores or core-like structures in virions. Our results are consistent with cdE2 having a role in core maturation during virion assembly and rescuing core formation when cytoplasmic cores are not assembled. We go on to find that the isolated cores from some mutant virions are now assembly competent in that they can be disassembled and reassembled back into cores. These results show how the two domains of CP may have distinct yet coordinated roles. IMPORTANCEStructural viral proteins have multiple roles during entry and assembly. The capsid protein (CP) of alphaviruses has one domain that interacts with the viral genome and another domain that interacts with the E2 spike protein. In this work we determine that the length and/or charge of the CP affects cytoplasmic core formation. However, defects in cytoplasmic core formation can be overcome by E2-CP interactions, thus assembling a core or core-like complex in the virion. In the absence of both cytoplasmic cores and CP-E2 interactions, CP is not even packaged in the released virions, but some infectious particles are still released presumably as RNA packaged in a glycoprotein containing membrane shell. This suggests that the virus has multiple mechanisms in place to ensure the viral genome is surrounded by a capsid core during its lifecycle.

microbiology↗

bioRxiv2021

Interactions between TULP3 tubby domain cargo site and ARL13B amphipathic helix promote lipidated protein transport to cilia

The tubby family protein-TULP3 coordinates with the intraflagellar transport complex-A (IFT-A) in trafficking certain transmembrane proteins to cilia. These transmembrane cargoes have short motifs that are necessary and sufficient for TULP3-mediated trafficking. However, whether TULP3 regulates trafficking of membrane-associated proteins is not well understood. Here we show that TULP3 is required for transport of the atypical GTPase ARL13B into cilia, and for ciliary enrichment of ARL13B-dependent farnesylated and myristoylated proteins. ARL13B transport requires TULP3 binding to IFT-A core but not to phosphoinositides, unlike transmembrane cargo transport that requires binding to both by TULP3. A conserved lysine in TULP3s tubby domain mediates direct ARL13B binding and trafficking of lipidated and transmembrane cargoes. An N-terminal amphipathic helix in ARL13B flanking the palmitoylation site mediates binding to TULP3 and directs trafficking to cilia even in absence of palmitoylation and RVxP sorting motif. Therefore, TULP3 transports transmembrane proteins and ARL13B into cilia by capture of short sequences through a shared tubby domain site.

cell biology↗

bioRxiv2021

Optimized structure of monoubiquitinated FANCD2 (human) at Lys 561: a theoretical approach

Fanconi anaemia pathway repairs inter-strand cross linking damage (ICL) of the DNA. Monoubiquitination of FANCD2 and FANCI is very crucial for ICL repairing. In this work we have tried to understand the monoubiquitinated FANCD2 structure, which facilitates the FANCD2 for binding the damage part of the chromatin. Crystal structure of the monoubiquitinated FANCD2 alone is not available, therefore we have developed the optimized structure of the human monoubiquitinated (Lys 561) FANCD2. As there is no suitable software or web server we have developed a method for building up monoubiquitinated product and validated on simplest monoubiquitinated protein, diubiquitin. We have predicted the structure of human monoubiquitinated FANCD2 by using our method and studied the interaction with DNA by docking studies. Molecular Dynamics (MD) simulation was used to understand the stability of the structure. Large structural differences have been observed between FANCD2 and monoubiquitinated FANCD2. DNA docking studies suggest that the binding site varies for the FANCD2 and monoubiquitinated FANCD2.

bioinformatics↗

bioRxiv2021

Sub-stoichiometric Hsp104 regulates the genesis and persistence of self-replicable amyloid seeds of a yeast prion protein

The prion-like self-perpetuating conformational conversion is involved in both transmissible neurodegenerative diseases and non-Mendelian inheritance traits. The transmissibility of amyloid-like aggregates is dependent on the stoichiometry of chaperones such as heat shock proteins. To provide the mechanistic underpinning of the generation and persistence of prefibrillar amyloid seeds that are critical for the prion-like propagation, we studied the effect of Hsp104 disaggregase on the assembly mechanism of a yeast prion determinant of Saccharomyces cerevisiae Sup35. At low sub-stoichiometric concentrations, Hsp104 exhibits a dual role and considerably accelerates the formation of seeding-competent prefibrillar amyloids by shortening the lag phase but also prolongs their persistence by introducing unusual kinetic halts and delaying their conversion into matured fibers. Hsp104-mediated amyloid species comprise a more ordered packing and display an enhanced autocatalytic self-templating ability compare to amyloids formed without Hsp104. Our findings underscore the key functional and pathological roles of sub-stoichiometric chaperones in prion-like propagation.

biophysics↗

bioRxiv2021

Selection and structural characterisation of anti-TREM2 scFvs that reduce levels of shed ectodomain

Single point mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration including Alzheimers disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signalling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterised. Here, we present phage display generated scFv antibody binders to human TREM2 ectodomain. Cocrystal structures revealed the binding of two scFvs to an epitope on the globular TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterisation of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.

molecular biology↗

bioRxiv2021

An Intrinsically Disordered Pathological Variant of the Prion Protein Y145Stop Transforms into Self-Templating Amyloids via Liquid-Liquid Phase Separation

Biomolecular condensation via liquid-liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is thought to govern critical cellular functions, whereas, aberrant phase transitions are associated with a range of deadly neurodegenerative diseases. Here we show, a naturally occurring pathological truncation variant of the prion protein (PrP) by a mutation of a tyrosine residue at 145 to a stop codon (Y145Stop) yielding a highly disordered N-terminal IDR that spontaneously phase-separates into liquid-like droplets. Phase separation of this N-terminal segment that is rich in positively charged and aromatic residues is promoted by the electrostatic screening and a multitude of other transient, intermolecular, noncovalent interactions. Single-droplet Raman measurements in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies revealed that the intrinsic disorder and dynamics are retained in the liquid-like condensates. Lower concentrations of RNA promote the phase transition of Y145Stop at low micromolar protein concentrations under physiological condition. Whereas, higher RNA to protein ratios inhibit condensation indicating the role of RNA in modulating the phase behavior of Y145Stop. Highly dynamic liquid-like droplets eventually transform into dynamically-arrested, ordered, {beta}-rich, amyloid-like aggregates via liquid-to-solid transition upon aging. These amyloid-like aggregates formed via phase separation display the self-templating characteristic and are capable of recruiting and autocatalytically converting monomeric Y145Stop into amyloid fibrils. In contrast to this disease-associated intrinsically disordered Y145 truncated variant, the wild-type full-length PrP exhibited a much lower propensity for phase separation and liquid-to-solid maturation into amyloid-like aggregates hinting at a potentially crucial, chaperone-like, protecting role of the globular C-terminal domain that remains largely conserved in vertebrate evolution. Such an intriguing interplay in the modulation of the protein phase behavior will have much broader implications in cell physiology and disease.

biophysics↗

bioRxiv2021

Gpr161 ciliary pools prevent hedgehog pathway hyperactivation phenotypes specifically from lack of Gli transcriptional repression

The role of compartmentalized cAMP signaling in primary cilia is not well understood. The cilia-localized G-protein-coupled receptor--Gpr161 represses hedgehog pathway via cAMP signaling. Here, by generating knock-in mutant mouse at endogenous Gpr161 locus (mut1), we show that ciliary and extraciliary receptor pools repress hedgehog signaling in a tissue-specific manner. Gpr161mut1 was competent in generating cAMP but did not transit through cilia. Compared to knockout, Gpr161mut1 had delayed embryonic lethality, less upregulation of hedgehog targets and partially down-regulated Gli3-repressor. Ventral-most progenitor expansion in neural tube of Gpr161 knockout occurred in a Gli2-activator-dependent manner but was not seen in Gpr161mut1. Intermediate-level ventralization occurred from Gpr161 lack specifically in cilia but was restored by increased extraciliary mutant generating Gli3-repressor. Morphogenesis in limb buds and midface that require Gli-repressor manifested hedgehog hyperactivation phenotypes-- polydactyly and midfacial widening--in Gpr161mut1. Thus, ciliary and extraciliary cAMP signaling establishes tissue-specific Gli-repressor thresholds in dictating morpho-phenotypic outcomes. Impact statementO_ST_ABSSignaling by CiliaC_ST_ABSSignaling by cAMP specifically in cilia directs tissue-specific morphogenesis

developmental biology↗

bioRxiv2020

Impact of clade specific mutations on structural fidelity of SARS-CoV-2 proteins

The SARS-CoV-2 is a positive stranded RNA virus with a genome size of ~29.9 kilobase pairs which spans 29 open reading frames. Studies have revealed that the genome encodes about 16 non-structural proteins (nsp), four structural proteins, and six or seven accessory proteins. Based on prevalent knowledge on SARS-CoV and other coronaviruses, functions have been assigned for majority of the proteins. While, researchers across the globe are engrossed in identifying a potential pharmacological intervention to control the viral outbreak, none of the work has come up with new antiviral drugs or vaccines yet. One possible approach that has shown some positive results is by treating infected patients with the plasma collected from convalescent COVID-19 patients. Several vaccines around the world have entered their final trial phase in humans and we expect that these will in time be available for application to worldwide population to combat the disease. In this work we analyse the effect of prevalent mutations in the major pathogenesis related proteins of SARS-COV2 and attempt to pinpoint the effects of those mutations on the structural stability of the proteins. Our observations and analysis direct us to identify that all the major mutations have a negative impact in context of stability of the viral proteins under study and the mutant proteins suffer both structural and functional alterations as a result of the mutations. Our binary scoring scheme identifies L84S mutation in ORF8 as the most disruptive of the mutations under study. We believe that, the virus is under the influence of an evolutionary phenomenon similar to Mullers ratchet where the continuous accumulation of these mutations is making the virus less virulent which may also explain the reduction in fatality rates worldwide.

bioinformatics↗

bioRxiv2020

Postnatal fluoxetine treatment alters perineuronal net formation and maintenance in the hippocampus

Elevation of serotonin via postnatal fluoxetine (PNFlx) treatment during critical temporal windows is hypothesized to perturb the development of limbic circuits thus establishing a substratum for persistent disruption of mood-related behavior. We examined the impact of PNFlx treatment on the formation and maintenance of perineuronal nets (PNNs), extracellular matrix (ECM) structures that deposit primarily around inhibitory interneurons, and mark the closure of critical period plasticity. PNFlx treatment evoked a significant decline in PNN number, with a robust reduction in PNNs deposited around parvalbumin (PV) interneurons, within the CA1 and CA3 hippocampal subfields at postnatal day 21 in Sprague-Dawley rat pups. While the reduction in CA1 subfield PNN number was still observed in adulthood, we observed no change in colocalization of PV-positive interneurons with PNNs in the hippocampi of adult PNFlx animals. PNFlx treatment did not alter hippocampal parvalbumin, calretinin, or reelin-positive neuron numbers in PNFlx animals at P21 or in adulthood. We did observe a small, but significant increase in somatostatin (SST)-positive interneurons in the DG subfield of PNFlx-treated animals in adulthood. This was accompanied by altered GABA-A receptor subunit composition, increased dendritic complexity of apical dendrites of CA1 pyramidal neurons, and enhanced neuronal activation revealed by increased c-Fos-positive cell numbers within hippocampi of PNFlx-treated animals in adulthood. These results indicate that PNFlx treatment alters the developmental trajectory of PNNs within the hippocampus, raising the possibility of a disruption of critical period plasticity and the establishment of an altered excitation-inhibition balance within this key limbic brain region. Significance StatementClinical and preclinical studies indicate that developmental exposure to fluoxetine programs persistent dysregulation of mood-related behaviors. This is hypothesized to involve the disruption of the normal development of key brain regions, such as the hippocampus that regulate mood behaviors. We show that postnatal exposure to fluoxetine alters hippocampal perineuronal nets (PNNs), extracellular matrix structures that regulate plasticity and mark the closure of critical periods. The decline in PNNs is noted in early postnatal life, and persists into adulthood in specific hippocampal subfields. Adult animals with a history of postnatal fluoxetine exposure exhibit altered numbers of somatostatin interneurons, GABA receptor subunit expression and neuronal activation within the hippocampus. This indicates that postnatal fluoxetine disrupts the normal developmental trajectory of the hippocampus.

neuroscience↗

bioRxiv2020

Identification, Mapping and Relative Quantitation of SARS-Cov2 Spike Glycopeptides by Mass-Retention Time Fingerprinting

We describe a novel analytical method for rapid and robust identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein. The method may be executed using any LC-TOF mass spectrometer, requires no specialised knowledge of glycan analysis and makes use of the differential resolving power of reversed phase HPLC. While this separation technique resolves peptides with high efficiency, glycans are resolved poorly, if at all. Consequently, glycopeptides consisting of the same peptide bearing different glycan structures will all possess very similar retention times and co-elute. While this has previously been viewed as a disadvantage, we show that shared retention time can be used to map multiple glycan species to the same peptide and location. In combination with MSMS and pseudo MS3, we have constructed a detailed mass-retention time database for Spike. This database allows any ESI-TOF equipped lab to reliably identify and quantify spike glycans from a single overnight elastase protein digest in less than 90 minutes.

biochemistry↗

bioRxiv2020

Comparative severity analysis of colitis in C57BL/6 than BALB/c mice: A novel and rapid model of DSS induced colitis

Colitis is a complex and multifactorial disease with unknown etiology. To understand colitis, a number of models were developed in mouse. Colitis, in these models, was induced using either chemicals or select bacteria. Dextran sulfate sodium (DSS) induced colitis model was used widely, over other models, because of the simpler properties of DSS and similarities of the model with colitis in humans. The available models of DSS induced colitis require either longer time to understand the chronic stages of the disease or a shorter time period to study the acute phase. Currently, no composite models exist that can be used to study the acute, chronic and recovery stages of colitis within a reasonably shorter period of time. In the current study, we have developed a newer model system in two differently immune biased (Th-1 and Th-2) mice strains using DSS. We have developed the DSS model to compare the response in C57BL/6 (Th1 biased) and BALB/c (Th2 biased) mice models. We have standardized the dosage for both C57BL/6 and BALB/c mice with zero mortality rates. Using the model developed we studied the acute, chronic and recovery phases of colitis. The differential responses of C57BL/6 and BALB/c revealed that the disease was more severe in C57Bl/6. BALB/c, on the contrary, recovered from the diseased condition more quickly. The current report will further help to unravel the disease etiology based on immunological background of the host.

immunology↗