Biology subjects

Pandey, K.

Publications and source records attributed to Pandey, K..

29 records · Page 2Linked to original sources

bioRxiv2021

Inference of cell state transitions and cell fate plasticity from single-cell with MARGARET

Despite recent advances in inferring cellular dynamics using single-cell RNA-seq data, existing trajectory inference (TI) methods face difficulty in accurately reconstructing cell-state manifold and inferring trajectory and cell fate plasticity for complex topologies. We present MARGARET, a novel TI method that utilizes a deep unsupervised metric learning-based approach for inferring the cellular embeddings and employs a novel measure of connectivity between cell clusters and a graph-partitioning approach to reconstruct complex trajectory topologies. MARGARET utilizes the inferred trajectory for determining terminal states and inferring cell-fate plasticity using a scalable absorbing Markov Chain model. On a diverse simulated benchmark, MARGARET out-performed state-of-the-art methods in recovering global topology and cell pseudotime ordering. When applied to experimental datasets from hematopoiesis, embryogenesis, and colon differentiation, MARGARET reconstructed major lineages and associated gene expression trends, better characterized key branching events and transitional cell types, and identified novel cell types, and branching events that were previously uncharacterized.

genomics↗

bioRxiv2021

Defining the Immune Responses for SARS-CoV-2-Human Macrophage Interactions

Host innate immune response follows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and it is the driver of the acute respiratory distress syndrome (ARDS) amongst other inflammatory end-organ morbidities. Such life-threatening coronavirus disease 2019 (COVID-19) is heralded by virus-induced activation of mononuclear phagocytes (MPs; monocytes, macrophages, and dendritic cells). MPs play substantial roles in aberrant immune secretory activities affecting profound systemic inflammation and end organ malfunctions. All follow an abortive viral infection. To elucidate SARS-CoV-2-MP interactions we investigated transcriptomic and proteomic profiles of human monocyte-derived macrophages. While expression of the SARS-CoV-2 receptor, the angiotensin-converting enzyme 2, paralleled monocyte-macrophage differentiation it failed to affect productive viral infection. In contrast, simple macrophage viral exposure led to robust pro-inflammatory cytokine and chemokine expression but attenuated type I interferon (IFN) activity. Both paralleled dysregulation of innate immune signaling pathways specifically those linked to IFN. We conclude that the SARS-CoV-2-infected host mounts a robust innate immune response characterized by a pro-inflammatory storm heralding consequent end-organ tissue damage.

immunology↗

bioRxiv2021

Discovery and in-vitro evaluation of potent SARS-CoV-2 entry inhibitors

SARS-CoV-2 infection initiates with the attachment of spike protein to the ACE2 receptor. While vaccines have been developed, no SARS-CoV-2 specific small molecule inhibitors have been approved. Herein, utilizing the crystal structure of the ACE2/Spike receptor binding domain (S-RBD) complex in computer-aided drug design (CADD) approach, we docked [~]8 million compounds within the pockets residing at S-RBD/ACE2 interface. Five best hits depending on the docking score, were selected and tested for their in vitro efficacy to block SARS-CoV-2 replication. Of these, two compounds (MU-UNMC-1 and MU-UNMC-2) blocked SARS-CoV-2 replication at sub-micromolar IC50 in human bronchial epithelial cells (UNCN1T) and Vero cells. Furthermore, MU-UNMC-2 was highly potent in blocking the virus entry by using pseudoviral particles expressing SARS-CoV-2 spike. Finally, we found that MU-UNMC-2 is highly synergistic with remdesivir (RDV), suggesting that minimal amounts are needed when used in combination with RDV, and has the potential to develop as a potential entry inhibitor for COVID-19.

molecular biology↗

bioRxiv2021

Blockade of SARS-CoV-2 infection in-vitro by highly potent PI3K-α/mTOR/BRD4 inhibitor

Pathogenic viruses like SARS-CoV-2 and HIV hijack the host molecular machinery to establish infection and survival in infected cells. This has led the scientific community to explore the molecular mechanisms by which SARS-CoV-2 infects host cells, establishes productive infection, and causes life-threatening pathophysiology. Very few targeted therapeutics for COVID-19 currently exist, such as remdesivir. Recently, a proteomic approach explored the interactions of 26 of 29 SARS-CoV-2 proteins with cellular targets in human cells and identified 67 interactions as potential targets for drug development. Two of the critical targets, the bromodomain and extra-terminal domain proteins (BETs): BRD2/BRD4 and mTOR, are inhibited by the dual inhibitory small molecule SF2523 at nanomolar potency. SF2523 is the only known mTOR PI3K-/(BRD2/BRD4) inhibitor with potential to block two orthogonal pathways necessary for SARS-CoV-2 pathogenesis in human cells. Our results demonstrate that SF2523 effectively blocks SARS-CoV-2 replication in lung bronchial epithelial cells in vitro, showing an IC50 value of 1.5 {micro}M, comparable to IC50 value of remdesivir (1.1 {micro}M). Further, we demonstrated that the combination of doses of SF2523 and remdesivir is highly synergistic: it allows for the reduction of doses of SF2523 and remdesivir by 25-fold and 4-fold, respectively, to achieve the same potency observed for a single inhibitor. Because SF2523 inhibits two SARS-CoV-2 driven pathogenesis mechanisms involving BRD2/BRD4 and mTOR signaling, our data suggest that SF2523 alone or in combination with remdesivir could be a novel and efficient therapeutic strategy to block SARS-CoV-2 infection and hence be beneficial in preventing severe COVID-19 disease evolution. One Sentence SummaryEvidence of in silico designed chemotype (SF2523) targeting PI3K-/mTOR/BRD4 inhibits SARS-CoV-2 infection and is highly synergistic with remdesivir.

pharmacology and toxicology↗

bioRxiv2020

Unusual mode of dimerization of retinitis pigmentosa-associated F220C rhodopsin

Mutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wildtype and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.

biophysics↗

bioRxiv2020

Preclinical validation of a second generation leishmanization vaccine against vector transmitted fatal visceral leishmaniasis

Visceral Leishmaniasis (VL) is fatal if untreated. There is no licensed vaccine available against human leishmaniasis. We recently demonstrated protection in mice against L. major infection using a CRISPR genome edited attenuated Leishmania major strain (LmCen-/-). Here, as a pre-clinical step, we evaluated the protective efficacy of LmCen-/- against VL induced by sand fly transmitted Leishmania donovani in hamsters. Intradermal immunization of hamsters with LmCen-/- did not develop any lesion; while still priming a pro-inflammatory immune response. When challenged with L. donovani either by intradermal needle injection or by infected sand flies, LmCen-/--immunized hamsters were protected, not showing spleen or liver pathology averting VL fatality compared to control animals. Spleen cells from LmCen-/- immunized and infected sand fly challenged hamsters produced significantly higher Th1-associated cytokines and chemokines including IFN-{gamma} and TNF-, and significantly reduced expression of the anti-inflammatory cytokines IL-10 and IL-21, compared to non-immunized challenged animals. We further developed a GLP-grade LmCen-/- which showed equal protection as laboratory-grade LmCen-/- parasites in hamsters. Importantly, GLP-grade LmCen-/- parasites also induced a proinflammatory immune response in the PBMCs isolated from healthy people living in non-endemic and endemic for VL as well as cured VL people living in endemic region. Together, this study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and it is a strong candidate vaccine to be tested in a human clinical trial.

immunology↗

bioRxiv2020

Chronic morphine administration differentially modulates viral reservoirs in SIVmac251 infected rhesus macaque model

HIV persists in cellular reservoirs despite effective anti-retroviral therapy with rebound of viremia upon therapy interruption. Opioids modulate the immune system and suppress antiviral gene responses, which significantly impact people living with HIV (PLWH). However, the effects of opioids on viral reservoir remains elusive. Herein, we describe a morphine dependent SIVmac251 infected Rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. RMs were ramped up with morphine (n=10) or saline (n=9) for two weeks to a final dosage of 5mg/kg administered twice daily, which was maintained for seven weeks, and then infected with SIVmac251. Combined anti-retroviral therapy (cART) was initiated in approximately half the animals in each group five weeks post-infection and morphine/saline administration continued for 10 months. Among drug naive macaques, there were no differences in plasma/CSF viral load nor in cell-associated DNA/RNA loads. However, within the cART-suppressed macaques, there was a reduction in cell-associated DNA load, intact proviral DNA copy numbers, and inducible SIV reservoir in both peripheral blood and lymph nodes (LNs) of morphine-administered RMs compared to saline controls. Further, PBMCs of morphine administered RMs, a reduction in Th1 polarized CD4+ T cells and in LNs there was a reduction in the total Tfh and Th1 like Tfh cells were observed, indicating probably have impact on reduction of viral reservoirs. In distinction to PBMC and LNs, within the CNS size of latent SIV reservoirs was higher in the CD11b+ microglia/macrophages of morphine-dependent RMs. These data suggest that morphine plays a role in modulating SIV reservoirs, reduces the CD4+ T-cell reservoir in both peripheral blood and LNs, and increases microglia/macrophage reservoirs in CNS. These findings will aid in understanding of molecular mechanism(s) of opioid-mediated differential modulation of viral reservoirs and evaluation of therapeutic strategies to reduce/eliminate HIV reservoirs in opioid-dependent PLWH. Author summaryOpioids are commonly used as well as abused by HIV infected individuals, are known to suppress immune responses. However, their effects on modulating viral reservoir dynamics is not known. Here we developed a morphine dependent SIVmac251 infected rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs and immune cells. We found that there was no difference in viral loads or cell-associated DNA/RNA loads among morphine dependent vs. saline treated control macaques. On the other hand, when macaques were treated with cART, there was a reduction in SIV reservoirs both in periphery and lymphoid tissues in morphine administered RMs, and the size of latent SIV reservoir was higher in the CNS CD11b+ microglia/macrophages as compared to control macaques. Therefore, these new data form macaque models suggest that PLWH who suffering from opioid use disorders have higher reservoirs in CNS as compared to lymphoid system. Thus, through understanding these reservoirs among PLWH who uses opioids are critical for better designing HIV cure strategies.

molecular biology↗

bioRxiv2020

Structural basis to design multi-epitope vaccines against Novel Coronavirus 19 (COVID19) infection, the ongoing pandemic emergency: an in silico approach

The 2019 novel coronavirus (COVID19 / Wuhan coronavirus), officially named as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is a positive-sense single-stranded RNA coronavirus. SARS-CoV-2 causes the contagious COVID19 disease also known as 2019-nCoV acute respiratory disease and has led to the ongoing 2019-20 pandemic COVID19 outbreak. The effective counter measures against SARS-CoV-2 infection require the design and development of specific and effective vaccine candidate. In the present study, we have screened and shortlisted 38 CTL, 33 HTL and 12 B cell epitopes from the eleven Protein sequences of SARS-CoV-2 by utilizing different in silico tools. The screened epitopes were further validated for their binding with their respective HLA allele binders and TAP (Transporter associated with antigen processing) molecule by molecular docking. The shortlisted screened epitopes were further utilized to design novel two multi-epitope vaccines (MEVs) composed of CTL, HTL and B cell epitopes overlaps with potential to elicit humoral as well as cellular immune response against SARS-CoV-2. To enhance the immune response for our vaccine design, truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1 (Ov-ASP-1) has been utilized as an adjuvant at N terminal of both the MEVs. Further molecular models for both the MEVs were prepared and validated for their stable molecular interactions with Toll-Like Receptor 3 (TLR 3). The codon-optimized cDNA of both the MEVs were further analyzed for their potential of high level of expression in a human cell line. The present study is very significant in terms of molecular designing of prospective CTL and HTL vaccine against SARS-CoV-2 infection with the potential to elicit cellular as well as humoral immune response. (SARS-CoV-2), Coronavirus, Human Transporter associated with antigen processing (TAP), Toll-Like Receptor (TLR), Epitope, Immunoinformatics, Molecular Docking, Molecular dynamics simulation, Multi-epitope Vaccine Graphical abstractThe designed CTL (Cytotoxic T lymphocyte) and HTL (Helper T lymphocyte) multi-epitope vaccines (MEV) against COVID19 infection. Both the CTL and HTL MEV models show a very stable and well fit conformational complex formation tendency with the Toll like receptor 3. CTL and HTL MEVs: ribbon; Toll like receptor 3: gray cartoon; Adjuvant [truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1]: orange ribbon regions; Epitopes: cyan ribbons regions; 6xHis Tag: magenta ribbon regions. O_FIG O_LINKSMALLFIG WIDTH=87 HEIGHT=200 SRC="FIGDIR/small/019299v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@6cb749org.highwire.dtl.DTLVardef@1752d54org.highwire.dtl.DTLVardef@1f2fc16org.highwire.dtl.DTLVardef@18415a9_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2019

Genetic changes of P. vivax tempers host tissue-specific responses in Anopheles stephensi

In our preceding study (Sharma et al., 2019; BioRxiv) we showed that in the gut lumen Plasmodium vivax follows a unique strategy of immuno-suppression by disabling gut flora proliferation. Here, we further demonstrate that post gut invasion, a shrewd molecular relationship with individual tissues such as midgut, hemocyte, salivary glands, and strategic changes in the genetic makeup of P. vivax favors its survival in the mosquito host. A transient suppression of metabolic machinery by early oocysts, and increased immunity against late oocysts suggested a unique mechanism of gut homeostasis restoration and Plasmodium population regulation. Though a hyper immune response of hemocyte was a key to remove free circulating sporozoites, but a strong suppression of salivary metabolic activities, may favor successful survival of invaded sporozoites. Finally, genetic alteration of P. vivax ensures evasion of mosquito responses. Conclusively, our system-wide RNAseq analysis provides first genetic evidences of direct mosquito-Plasmodium interaction and establishes a functional correlation.\n\nAuthor SummaryMalaria transmission dynamics is heavily influenced by mosquito -parasite interaction. When passing through tissue specific barriers, Plasmodium have to compromise by losing its own population, but genetic relation is unknown. To win the developmental race Plasmodium need to overcome two important immuno-physiological barriers. First one accounts an indirect 24-30hr long pre-invasive gut-microbe-parasite interaction in the gut lumen. And second one follows a direct post gut invasive 14-18 days interaction with midgut, hemocyte and salivary glands. During pre-invasive phase of interaction, we showed Plasmodium vivax follows immuno-suppression strategy by restricting microbial growth in the gut lumen. Here, we demonstrate that switch of parasite from one stage to another stage within mosquito vector is accompanied by genetic changes of parasite. Our data suggests genetic makeup change enables the parasite to manipulate the metabolism of mosquito tissues. This strategy not only clear off multifaceted mosquitos tissue specific immune responses, but also favors Plasmodium own survival and transmission. Comprehending this tissue specific interaction between host and parasite at molecular level could provide new tool to intervene the plasmodium life cycle within vector.

genomics↗

bioRxiv2019

Microbiome-Gut-Brain-Axis communication regulates metabolic switch in the mosquito Anopheles culicifacies

Periodic ingestion of a protein-rich blood meal by adult female mosquitoes causes a drastic metabolic change in their innate physiological status, which is referred to as metabolic switch. Although the down-regulation of olfactory factors is key to restrain host-attraction, how the gut metabolic switch modulates brain functions, and resilience physiological homeostasis remains unexplored. Here, we demonstrate that the protein-rich diet induces mitochondrial function and energy metabolism, possibly shifting the brains engagement to manage organismal homeostasis. A dynamic expression pattern of neuro-signaling and neuro-modulatory genes in both the brain and gut indicates an optimal brain-distant organ communication. Even after decapitation, significant modulation of the neuro-modulator receptor genes as well as quantitative estimation of neurotransmitters (NTs), together confer the guts ability to serve as a second brain. Finally, data on comparative metagenomic analysis and altered NTs dynamics of naive and aseptic mosquitoes provide the initial evidence that gut-endosymbionts are key modulators for the synthesis of major neuroactive molecules. Conclusively, our data establish a new conceptual understanding of microbiome-gut-brain-axis communication in mosquitoes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/774430v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@4fed8corg.highwire.dtl.DTLVardef@17494f3org.highwire.dtl.DTLVardef@df1cd7org.highwire.dtl.DTLVardef@1de7923_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG HighlightsO_LIHighly proteinaceous blood meal uptake causes gut metabolic switch activity in mosquitoes. C_LIO_LIGuts calling shifts the brains administrative function from external communication to inter-organ management. C_LIO_LI Gut, as a Second brain plays a crucial role in the maintenance of physiological homeostasis. C_LIO_LIMetabolic switch and proliferation of symbiotic bacteria establish microbiome-gut-brain axis communication in mosquitoes. C_LI

animal behavior and cognition↗

bioRxiv2019

Water for sterol: an unusual mechanism of sterol egress from a StARkin domain

Previously we identified a new family of endoplasmic reticulum membrane proteins that possess sterol-binding StARkin domains (Gatta et al. eLife 2015). These Lam/GramD1 proteins are implicated in intracellular sterol homeostasis, a function that requires them to be able to bind sterols. Here we show how these proteins exchange sterol molecules with membranes. An aperture at one end of the StARkin domain enables sterol to enter/exit the binding pocket. Strikingly, the wall of the pocket is fractured along its length, exposing bound sterol to solvent. We considered whether hydration of the pocket could mediate sterol entry/exit. Large-scale atomistic molecular dynamics simulations reveal that sterol egress involves widening of the fracture, penetration of water into the cavity and consequent destabilization of the bound sterol. The simulations also identify polar residues along the fracture that are important for sterol release. Their replacement with alanine affects the ability of the StARkin domain to bind sterol, catalyze inter-vesicular sterol exchange and alleviate the nystatin-sensitivity of lam2{Delta} yeast cells. These data suggest an unprecedented, water-controlled mechanism of sterol acquisition and discharge from a StARkin domain.

biochemistry↗