Biology subjects

Verma, C.

Publications and source records attributed to Verma, C..

10 recordsLinked to original sources

bioRxiv2025

Beta2* Nicotinic Receptors Tune Prefrontal Behavior Through Distinct Neuronal Populations

Beta2 subunit-containing nicotinic acetylcholine receptors (beta2*nAChRs) are highly expressed in the prefrontal cortex (PFC) and critically regulate behavioral and cognitive domains that are disrupted in neuropsychiatric disorders. Despite their therapeutic potential, the cell-type- and circuit-specific functions of beta2* nAChRs remain poorly defined, partially due to the lack of selective pharmacological tools. Here, we delineate the cellular expression patterns and behavioral roles of beta2* nAChRs expressed in a general vs. highly specific neuronal populations of the mouse PFC. Using fluorescence in situ hybridization (FISH), we mapped beta2 nicotinic subunit expression across cortical layers and major neuronal classes. We then employed CRISPR-mediated knockdown (KD) to reduce beta2* nAChRs expression selectively in two distinct PFC neuronal populations and, for comparison, in a striatal population. The KD of beta2* nAChRs in a mixed population of excitatory and inhibitory neurons in deep PFC layers resulted in a mild impairment of social and exploratory behaviors. In contrast, KD in a highly selective population of superficial-layer interneurons expressing the serotonin receptor 5HT3a induced a robust hypersocial phenotype, accompanied by changes in exploratory behavior. Notably, KD of beta2* nAChRs in a rare NPY-expressing striatal population slightly affected similar behavioral domains, but in the opposite direction to PFC manipulations. Together, these findings reveal opposing, cell-type- and region-specific contributions of beta2* nAChRs to behavioral regulations, highlighting the necessity of circuit-resolved targeting strategies to improve the precision and efficacy of therapeutic intervention for neuropsychiatric disorders.

neuroscience↗

bioRxiv2025

In silico discovery of pathogenic PD-L1 nsSNVs with altered glycosylation and immunotherapy binding

Immune checkpoint inhibitors (ICIs), particularly anti-PD-L1 monoclonal antibodies, block extracellular interactions between programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) to enhance antitumour immunity. Here, we present a streamlined workflow integrating public databases, bioinformatics tools, and in silico molecular dynamics (MD) simulations and in vitro experimental assays to identify consequential pathogenic nonsynonymous PD-L1 variants (PD-L1SNVs). Four variants predicted as pathogenic across six bioinformatic tools (SIFT, PolyPhen-2, PANTHER, SNP-PhD, SNP&GO, and Pmut) destabilized the anti-PD-L1 ICI atezolizumab-drug binding epitope in MD studies, correlating with reduced drug affinity. Live-cell assays directly linked PD-L1SNVs to intracellular trafficking defects and aberrant glycosylation, even distal from N-glycosylation sites. Allostery modeling (AlloSigMA) linked these disruptions to long-range structural perturbations. Our work establishes bioinformatics-driven predictions of functionally impactful pathogenic PD-L1SNVs and highlight how glycosylation/trafficking defects may serve as key mechanisms compromising therapeutic efficacy. These insights in predicting and validating pathogenic PD-L1SNVs has significant implications for guiding the personalized selection of effective PD-L1-targeted therapies in clinical settings. One Sentence Summary: Uncovering the implication of non-synonymous single nucleotide variants (nsSNVs) on the structure and function of Programmed Death Ligand-1.

bioinformatics↗

bioRxiv2024

Modulation of Ideonella sakaiensis PETase active site flexibility and activity on morphologically distinct substrates by surface charge engineering

Enzymatic hydrolysis of polyethylene terephthalate (PET) waste is a compelling strategy for environmentally friendly recycling of a major pollutant. Here, we investigate the effects of surface charge point mutations both proximal and distal to the active site of the mesophilic PET-degrading enzyme from Ideonella sakaienses (IsPETase) and an engineered thermostable variant with superior activity, STAR PETase. The vicinal K95A mutation significantly inhibited IsPETase activity on mechanically processed PET powder. Conversely, this mutation significantly increased hydrolysis of PET powder in the STAR PETase background. Activity of both enzymes on PET film was inhibited by the K95A mutation, highlighting complex interplay between mutation context and substrate morphology. Further installing the distal R132N and R280A surface charge mutations potentiated activity of STAR on all substrates tested. This variant afforded 100% degradation of bottle-grade PET powder in 3 days at 40{degrees}C reaction temperature, a 3-fold improvement over IsPETase. Molecular dynamics simulations reveal modulation of active site flexibility in mutants, which differentially impacts both hydrolysis of morphologically distinct PET substrates and the concentration-dependent inhibition phenomenon observed for PETase.

bioengineering↗

bioRxiv2023

Reproductive traits of the European catfish (Silurus glanis) during the early stages of invasion in the Lower Tagus River

Freshwater ecosystems face severe challenges from biological invasions, leading to biodiversity loss, disruption of ecosystem services, and economic impacts. Human-mediated activities, such as aquarium trade and sport angling, contribute to species introductions, with potential negative consequences for native ecosystems. The European catfish (Silurus glanis) is one of the worlds largest freshwater fish and has been intentionally introduced into diverse regions, impacting native ecosystems. However, limited research exists on its reproductive traits outside its native range. This study addresses this gap by examining the reproductive characteristics of non-native European catfish populations in the Lower Tagus River in Portugal, focusing on size at maturity, spawning period, and fecundity. The observed balanced sex ratio aligns with studies of native populations. Variations in size at first maturity (TL50) among populations highlight the influence of habitat conditions, temperature, food availability, growth rate, and geographical location on this trait. The extended spawning season (March to June) in the Tagus River is consistent with native populations, but variations may occur based on environmental conditions and water temperature. Absolute fecundity ranged from 8,961 to 335,500 oocytes, showing positive relationships with body size and emphasizing the reproductive potential of European catfish in Portugal. Egg size variations, along with asynchronous egg development, contribute to the species reproductive strategy, favoring its invasive success. Management efforts should include monitoring, regulations on introductions, removal programs, and public awareness to mitigate their impact. Future research should focus on understanding how non-native European catfish populations adapt in various regions and continue to impact ecosystems.

ecology↗

bioRxiv2023

Expanding the DNA editing toolbox: novel lambda integrase variants targeting microalgal and human genome sequences.

Recombinase enzymes are extremely efficient at integrating very large DNA fragments into target genomes. However, intrinsic sequence specificities curtail their use to DNA sequences with sufficient homology to endogenous target motifs. Extensive engineering is therefore required to broaden applicability and robustness. Here, we describe the directed evolution of novel lambda integrase variants capable of editing exogenous target sequences identified in the diatom Phaeodactylum tricornutum and the algae Nannochloropsis oceanica. These microorganisms hold great promise as conduits for green biomanufacturing and carbon sequestration. The evolved enzyme variants show >1000-fold switch in specificity towards the non-natural target sites when assayed in vitro. A single-copy target motif in the human genome with homology to the Nannochloropsis oceanica site can also be efficiently targeted using an engineered integrase, both in vitro and in human cells. The developed integrase variants represent useful additions to the DNA editing toolbox, with particular application for targeted genomic insertion of large DNA cargos.

bioengineering↗

bioRxiv2022

Transcriptional profiles of functionally distinct HLADR+CD38+ CD8 T cells subsets from acute febrile dengue patients

Previous studies showed that a discrete population of the CD8 T cells with HLADR+CD38+ phenotype expand massively during the acute febrile phase of dengue natural infection. Although about a third of these massively expanding HLADR+CD38+ CD8 T cells were of CD69high phenotype, only a small fraction of them produced IFN{gamma} upon in vitro peptide stimulation. What other cytokines/ chemokines do these peptides stimulated HLADR+CD38+ CD8 T cells express, what transcriptional profiles distinguish the CD69+IFN{gamma}+, CD69+IFN{gamma}-, and CD69-IFN{gamma}- subsets, and whether the expansion of the total HLADR+CD38+ CD8 T cells or the IFN{gamma} producing CD8 T cells differ depending on disease severity remained unclear. This study addresses these knowledge gaps. We find that the CD69+IFN{gamma}+ subset uniquely expressed key genes involved in protein translation, cellular metabolism, proliferation and dendritic cell cross talk. Both the CD69+IFN{gamma}+ and CD69+IFN{gamma}- subsets had an antigen responsive gene signature with genes involved in cytotoxic effector functions, regulation of T cell receptor signaling, signaling by MAPK, chemotaxis and T cell trafficking to inflamed tissues with the expression being more robust in the IFN{gamma}+ CD69+ subset. On the other hand, the CD69- IFN{gamma}- subset was biased towards expression of genes that both augment and dampen T cell responses. Lastly, the expansion of total HLADR+ CD38+ CD8 T cells and also the IFN{gamma} producing HLADR+ CD38+ CD8 T cells was similar in patients with different grades of disease. Taken together, this study provides valuable insights into the inherent diversity of the effector CD8 T cell response during dengue.

immunology↗

bioRxiv2022

Dual scRNA-Seq analysis reveals rare and uncommon parasitized cell populations in chronic L. donovani infection

Although phagocytic cells are documented targets of Leishmania parasites, it is unclear whether these parasites can infect other cell types. In this study, we describe a computational approach that exploits scRNA-seq to simultaneously analyze the transcriptomic signatures of the host cell and to identify rare and uncommon cells that harbor Leishmania donovani in the spleen and bone marrow. Individual cells were annotated as parasitized based on the presence of L. donovani transcripts that were detected with high accuracy. This unbiased approach allowed identification of heterogenous parasitized cell populations that cannot be detected by conventional methods. Consistent with previous studies, analysis of spleen cells isolated from L. donovani infected mice revealed inflammatory monocytes as the dominant parasitized cells. In addition, megakaryocytes, basophils, and NK cells were found to be the rare cells infected in the spleen. Unexpectedly, hematopoietic stem cells (HSCs), not known to be phagocytic, were the dominant cells parasitized cell in the bone marrow. In addition, eosinophils, megakaryocytes, and basal cells were the rare bone marrow cells found to be infected. scRNA-seq analysis revealed known phagocytic receptors Fc{gamma}R and CD93 are expressed on HSCs. In vitro studies using purified HSCs showed that these cells can phagocytize L. donovani. Parasitized HSCs were also detectable in the bone marrow of mouse infected with L donovani.. This unbiased dual scRNA-seq approach enables identification of rare and uncommon parasitized cells that could be involved in pathogenesis, persistence, and protective immunity. Further, such approach could be used to study pathogenesis of other infectious agents.

immunology↗

bioRxiv2021

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

RAS is the most commonly mutated oncogene in human cancers and RAS-driven tumors are amongst the most difficult to treat. Historically, discovery of therapeutics targeting this protein has proven challenging due to a lack of deep hydrophobic pockets to which a small molecule might bind. The single such pocket available is normally occupied by GDP or GTP which have millimolar cellular concentrations and picomolar affinities for KRAS and hence is challenging to target. The recent FDA approval of sotorasib, a covalent modifier of the KRASG12C mutant protein, has clinically validated KRAS as an oncology target. However, traditional challenges remain for targeting the more common KRAS mutations such as G12D and G12V. As an alternative approach, we investigated the superior binding capacity of macrocyclic peptides to identify KRAS inhibitory molecules. We focused on the recently reported disulfide-cyclized arginine-rich peptide KRpep-2d, discovered through phage display and previously independently confirmed by us as a bona fide KRAS binder. To mitigate intracellular disulfide reduction and loss of binding, we identified an alternate cyclization motif by inverting the configuration of Cys5 and linking it to Cys15 through a thioacetal bridge. The corresponding peptide bound KRAS through cis isomerization of the peptide bond between D-Cys5 and Pro6 as observed through x-ray crystallography. Prototypic molecules displayed measurable cellular inhibition of RAS signaling without membrane lysis and counter-screen off-target activity. An analogue containing azido-lysine confirmed the cell penetrant nature of this molecule using our recently reported NanoClick assay. To improve cellular activity, we sought to improve proteolytic stability. Structure-activity relationship studies identified key scaffold residues critical for binding and revealed that replacement of N- and C-terminal arginine residues with D-arginines and introduction of an -methyl moiety at Ser10 resulted in a molecule with improved proteolytic stability as indicated by its persistence in whole cell homogenate. The resulting peptide MP-3995 had improved and sustained cell-based efficacy. On-target activity was validated through confirmation of target engagement, lack of signaling in irrelevant pathways, and use of non-binding control peptides. Mechanism of action studies suggested that peptide binding to both GDP- and GTP-states of KRAS may contribute to cellular activity. Although validated as bona fide and on-target inhibitors of cell based KRAS signaling, this series is unlikely to advance to the clinic in its current form due to its arginine-dependent cell entry mechanism. Indeed, we showed a strong correlation between net positive charge and histamine release in an ex vivo assay making this series challenging to study in vivo. Nonetheless, these molecules provide valuable templates for further medicinal chemistry efforts aimed at leveraging this unique inhibitory binding site on KRAS. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/459913v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@17f4764org.highwire.dtl.DTLVardef@12818daorg.highwire.dtl.DTLVardef@177bdf0org.highwire.dtl.DTLVardef@57046c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Src activates retrograde membrane traffic through phosphorylation of GBF1

The Src tyrosine kinase controls cancer-critical protein glycosylation through Golgi to ER relocation of GALNTs enzymes. How Src induces this trafficking event is unknown. Golgi to ER transport depends on the GTP Exchange factor (GEF) GBF1 and small GTPase Arf1. Here we show that Src induces the formation of tubular transport carriers containing GALNTs through promotion of a GBF1-Arf1 complex. The complex is initiated by phosphorylation on GBF1 on 10 tyrosine residues; two of them, Y876 and Y898 are located near the C-terminus of the Sec7 GEF domain. Their phosphorylation promotes partial melting of the Sec7 domain, favoring binding to the GTPase. Perturbation of these rearrangements prevent GALNTs relocation. In sum, Src promotes GALNTs relocation by favoring binding of GBF1 to Arf1. Regulation of a GEF-Arf axis by tyrosine phosphorylation appears to be a highly conserved and wide-spread mechanism.

cell biology↗

bioRxiv2018

Phosphorylation regulates auto-inhibition of kinesin KIF3A

Kinesin are molecular motors that move along the microtubules. They function to transport cargoes, vesicles and organelles to designated locations in the cells. KIF3A belongs to the Kinesin-2 family and forms a heterotrimeric complex with KIF3B and KAP3. We have earlier shown that the cargo trafficking activity of KIF3 motor can be regulated by CaMKII kinase and POPX2 phosphatase. In this study, we elucidated the mechanism of KIF3A regulation. We find that KIF3A adopts an auto-inhibited state through the interaction between the motor and tail domains. The motor-tail interaction also hinders the ATPase activity of the motor domain. We show that the phosphorylation status of serine-689/690 (mouse/human) at the C-terminal region of KIF3A is crucial for the motor-tail interaction. The motor domain does not interact well with the tail domain when serine-689 is phosphorylated by CaMKII or mutated to aspartic acid to mimic phosphorylation. Molecular dynamics simulations suggest that the non-phosphorylated tail domain folds into the hydrophobic pocket formed by the motor dimers. Phosphorylation of serine-689 results in conformational changes that leads to the relieve of auto-inhibition.

biochemistry↗