Dynamics and spatial organization of Kv1.3 at the immunological synapse of human CD4+ T cells.

Formation of the immunological synapse (IS) is a key event during initiation of an adaptive immune response to a specific antigen. During this process, a T cell and an antigen presenting cell form a stable contact that allows the T cell to integrate both internal and external stimuli in order to decide whether to activate. The threshold for T cell activation depends on the strength and frequency of the calcium (Ca2+) signaling induced by antigen recognition, and it must be tightly regulated to avoid undesired harm to healthy cells. Potassium (K+) channels are recruited to the IS to maintain the negative membrane potential required to sustain Ca2+ entry. However, the precise localization of K+ channels within the IS remains unknown. Here, we visualized the dynamic subsynaptic distribution of Kv1.3, the main voltage-gated potassium channel in human T cells. Upon T cell receptor engagement, Kv1.3 polarized toward the synaptic cleft and diffused throughout the F-actin rich distal compartment of the synaptic interface-an effect enhanced by CD2-CD58 corolla formation. As the synapse matured, Kv1.3 clusters were internalized at the center of the IS and released in extracellular vesicles. We propose a model in which specific distribution of Kv1.3 within the synapse indirectly regulates the channel function and that this process is limited through Kv1.3 internalization and release in extracellular vesicles.

Pseudotime dynamics of T cells in pancreatic ductal adenocarcinoma inform distinct functional states within the regulatory and cytotoxic T cells.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest types of cancer and has a 5-year survival of less than 8% owing to its complex biology. As PDAC is refractory to immunotherapy, we need to understand the functional dynamics of T cells in the PDAC microenvironment to develop alternative therapeutic strategies. In this study, we performed RNA velocity-based pseudotime analysis on a scRNA-seq dataset from surgically resected human PDAC specimens to gain insight into temporal gene expression patterns that best characterize the cell fates. The tumor microenvironment was seen to encompass a range of terminal states for the T cell trajectories with suppressive and non-tumor-responsive T cells dominating them. However, the results also reveal the existence of a functional branch of the T cell population that was not transitioning to exhausted and senescent states. These findings reveal various microenvironmental signals driving T cell patterns which can be useful in identifying new therapeutic avenues.

Drug repurposing and sequence analysis in S-glycoprotein variants reveals critical signature patterns and destabilization of receptor-binding domain in omicron variant.

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus since its emergence in 2019 has yielded several new viral variants with varied infectivity, disease severity, and antigenicity. Although most mutations are expected to be relatively neutral, mutations at the Spike region of the genome have shown to have a major impact on the viral transmission and infection in humans. Therefore, it is crucial to survey the structures of spike protein across the global virus population to contextualize the rate of therapeutic success against these variants. In this study, high-frequency mutational variants from different geographic regions were pooled in order to study the structural evolution of the spike protein through drug docking and MD simulations. We investigated the mutational burden in the spike subregions and have observed that the different variants harbour unique signature patterns in the spike subregions, with certain domains being highly prone to mutations. Further, the MD simulations and docking study revealed that different variants show differential stability when docked for the same set of drug targets. This work sheds light on the mutational burden and the stability landscape of the spike protein across the variants from different geographical regions.Communicated by Ramaswamy H. Sarma.

T-cell trans-synaptic vesicles are distinct and carry greater effector content than constitutive extracellular vesicles.

The immunological synapse is a molecular hub that facilitates the delivery of three activation signals, namely antigen, costimulation/corepression and cytokines, from antigen-presenting cells (APC) to T cells. T cells release a fourth class of signaling entities, trans-synaptic vesicles (tSV), to mediate bidirectional communication. Here we present bead-supported lipid bilayers (BSLB) as versatile synthetic APCs to capture, characterize and advance the understanding of tSV biogenesis. Specifically, the integration of juxtacrine signals, such as CD40 and antigen, results in the adaptive tailoring and release of tSV, which differ in size, yields and immune receptor cargo compared with steadily released extracellular vesicles (EVs). Focusing on CD40L+ tSV as model effectors, we show that PD-L1 trans-presentation together with TSG101, ADAM10 and CD81 are key in determining CD40L vesicular release. Lastly, we find greater RNA-binding protein and microRNA content in tSV compared with EVs, supporting the specialized role of tSV as intercellular messengers.

The interplay between membrane topology and mechanical forces in regulating T cell receptor activity.

T cells are critically important for host defense against infections. T cell activation is specific because signal initiation requires T cell receptor (TCR) recognition of foreign antigen peptides presented by major histocompatibility complexes (pMHC) on antigen presenting cells (APCs). Recent advances reveal that the TCR acts as a mechanoreceptor, but it remains unclear how pMHC/TCR engagement generates mechanical forces that are converted to intracellular signals. Here we propose a TCR Bending Mechanosignal (TBM) model, in which local bending of the T cell membrane on the nanometer scale allows sustained contact of relatively small pMHC/TCR complexes interspersed among large surface receptors and adhesion molecules on the opposing surfaces of T cells and APCs. Localized T cell membrane bending is suggested to increase accessibility of TCR signaling domains to phosphorylation, facilitate selective recognition of agonists that form catch bonds, and reduce noise signals associated with slip bonds.

Activated Regulatory T-Cells, Dysfunctional and Senescent T-Cells Hinder the Immunity in Pancreatic Cancer.

Pancreatic cancer has one of the worst prognoses of any human malignancy and leukocyte infiltration is a major prognostic marker of the disease. As current immunotherapies confer negligible survival benefits, there is a need to better characterise leukocytes in pancreatic cancer to identify better therapeutic strategies. In this study, we analysed 32 human pancreatic cancer patients from two independent cohorts. A multi-parameter mass-cytometry analysis was performed on 32,000 T-cells from eight patients. Single-cell RNA sequencing dataset analysis was performed on a cohort of 24 patients. Multiplex immunohistochemistry imaging and spatial analysis were performed to map immune infiltration into the tumour microenvironment. Regulatory T-cell populations demonstrated highly immunosuppressive states with high TIGIT, ICOS and CD39 expression. CD8+ T-cells were found to be either in senescence or an exhausted state. The exhausted CD8 T-cells had low PD-1 expression but high TIGIT and CD39 expression. These findings were corroborated in an independent pancreatic cancer single-cell RNA dataset. These data suggest that T-cells are major players in the suppressive microenvironment of pancreatic cancer. Our work identifies multiple novel therapeutic targets that should form the basis for rational design of a new generation of clinical trials in pancreatic ductal adenocarcinoma.

A comprehensive SARS-CoV-2 genomic analysis identifies potential targets for drug repurposing.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is a novel human coronavirus strain (HCoV) was initially reported in December 2019 in Wuhan City, China. This acute infection caused pneumonia-like symptoms and other respiratory tract illness. Its higher transmission and infection rate has successfully enabled it to have a global spread over a matter of small time. One of the major concerns involving the SARS-COV-2 is the mutation rate, which enhances the virus evolution and genome variability, thereby making the design of therapeutics difficult. In this study, we identified the most common haplotypes from the haplotype network. The conserved genes and population level variants were analysed. Non-Structural Protein 10 (NSP10), Nucleoprotein, Papain-like protease (Plpro or NSP3) and 3-Chymotrypsin like protease (3CLpro or NSP5), which were conserved at the highest threshold, were used as drug targets for molecular dynamics simulations. Darifenacin, Nebivolol, Bictegravir, Alvimopan and Irbesartan are among the potential drugs, which are suggested for further pre-clinical and clinical trials. This particular study provides a comprehensive targeting of the conserved genes. We also identified the mutation frequencies across the viral genome.

Heart failure and the glutathione cycle: an integrated view.

Heart failure results from the heart's inability to carryout ventricular contraction and relaxation, and has now become a worldwide problem. During the onset of heart failure, several signatures are observed in cardiomyocytes that includes fetal reprogramming of gene expression where adult genes are repressed and fetal genes turned on, endoplasmic reticulum stress and oxidative stress. In this short review and analysis, we examine these different phenomenon from the viewpoint of the glutathione cycle and the role of the recently discovered Chac1 enzyme. Chac1, which belongs to the family of γ-glutamylcyclotransferases, is a recently discovered member of the glutathione cycle, being involved in the cytosolic degradation of glutathione. This enzyme is induced during the Endoplasmic Stress response, but also in the developing heart. Owing to its exclusive action on reduced glutathione, its induction leads to an increase in the oxidative redox potential of the cell that also serves as signaling mechanism for calcium ions channel activation. The end product of Chac1 action is 5-oxoproline, and studies with 5-oxoprolinase (OPLAH), an enzyme of the glutathione cycle has revealed that down-regulation of OPLAH can lead to the accumulation of 5-oxproline which is an important factor in heart failure. With these recent findings, we have re-examined the roles and regulation of the enzymes in the glutathione cycle which are central to these responses. We present an integrated view of the glutathione cycle in the cellular response to heart failure.

Pharmacophore-driven identification of N-methyl-D-receptor antagonists as potent neuroprotective agents validated using in vivo studies.

Alzheimer's disease (AD), apparently the most widespread reason behind dementia, is delineated by a continuous cognitive weakening in the aged. During its progression, N-methyl-D-aspartate receptor (NMDAR) antagonists are known to play a pivotal part in the mechanisms of learning and memory. Since there is an unmet medical need for the treatment of AD, we aim to identify possible chemical compounds targeted toward N-methyl-D-aspartate receptors. Three-dimensional models are developed to unveil some of the essential characteristics of the N-methyl-D-aspartate receptors by using a collection of already discovered N-methyl-D-aspartate receptor inhibitors. This is followed by virtual screening, which results in novel chemical compounds having the potential to inhibit N-methyl-D-aspartate receptors. Molecular docking studies and analysis promulgated two lead compounds with a high LibDock score. The compounds are shortlisted based on high estimated activity, fit values, LibDock score, no violation of Lipinski's, and availability for procuring. Finally, the shortlisted compounds are tested by employing in vivo studies, which we further propose as potential NMDA inhibitors for treating AD.

Yeast glutaredoxin, GRX4, functions as a glutathione S-transferase required for red ade pigment formation in Saccharomyces cerevisiae.

The adenine biosynthetic mutants ade1 and ade2 of Saccharomyces cerevisiae accumulate a characteristic red pigment in their vacuoles under adenine limiting conditions. This red pigmentation phenotype, widely used in a variety of genetic screens and assays, is the end product of a glutathione-mediated detoxification pathway, where the glutathione conjugates are transported into the vacuole. The glutathione conjugation step, however, has still remained unsolved. We show here, following a detailed analysis of all the members of the thioredoxinfold superfamily, the involvement of the monothiol glutaredoxin GRX4 as essential for pigmentation. GRX4 plays multiple roles in the cell, and we show that the role in ade pigmentation does not derive from its regulatory role of the iron transcription factor, Aft1p, but a newly identified GST activity of the protein that we could demonstrate using purified Grx4p. Further, we demonstrate that the GRX domain of GRX4 and its active site cysteine C171 is critical for this activity. The findings thus solve a decades old enigma on a critical step in the formation of this red pigmentation.

Analysing non-synonymous mutations in XDR and MDR tuberculosis drugs.

Tuberculosis is a bacterial disease caused by Mycobacterium tuberculosis. It is known to be the second-largest cause of death and models a severe risk to public health throughout the world. Though it affects people of almost every age, individuals with weakened immune systems, (e.g., HIV infection) are more likely to get infected. The present study deals with analyzing non-synonymous mutations in anti-tuberculosis drugs, which may have a significant role in causing XDR and MDR tuberculosis drug resistance. Continued use of tuberculosis drugs, discontinuation of medicines and various other factors can promote drug resistance in the host's body. To understand the actual cause of resistance, we have identified some patterns of mutations which might be responsible for a change in the structure of the protein, ultimately causing drug resistance. Here, we aim to present some of the unique mutation patterns in the genes associated with the marketed drugs that might have a deleterious effect. In this study, we have used molecular docking approach for understanding the ligand binding affinity of the mutated drugs. The results are further validated by molecular dynamics studies.

Meta-analysis of genomic variants and gene expression data in schizophrenia suggests the potential need for adjunctive therapeutic interventions for neuropsychiatric disorders.

Schizophrenia (SZ) is a debilitating mental illness with a multigenic aetiology and significant heritability. Despite extensive genetic studies, the molecular aetiology has remained enigmatic. A recent systems biology study suggested a protein-protein interaction network for SZ with 504 novel interactions. The onset of psychiatric disorders is predominant during adolescence, often accompanied by subtle structural abnormalities in multiple regions of the brain. The availability of BrainSpan Atlas data allowed us to re-examine the genes present in the SZ interactome as a function of space and time. The availability of genomes of healthy centenarians and nonpsychiatric Exome Aggregation Consortium database allowed us to identify the variants of criticality. The expression of the SZ candidate genes responsible for cognition and disease onset was studied in different brain regions during particular developmental stages. A subset of novel interactors detected in the network was further validated using gene expression data of post-mortem brains of patients with psychiatric illness. We have narrowed down the list of drug targets proposed by theprevious interactome study to 10 proteins. These proteins belonging to 81 biological pathways are targeted by 34 known Food and Drug Administration-approved drugs that have distinct potential for the treatment of neuropsychiatric disorders. We also report the possibility of targeting key genes belonging to celecoxib pharmacodynamics, Gα signalling and cGMP-PKG signalling pathwaysthat are not known to be specific to SZ aetiology.

Intronic miRNA mediated gene expression regulation controls protein crowding inside the cell.

Gene regulatory effects of microRNAs at a posttranscriptional level have been established over the last decade. In this study, we analyze the interaction networks of mRNA translation regulation through intronic miRNA, under various tissue-specific cellular contexts, taking into account the thermodynamic affinity, chemical kinetics, co-localization, concentration levels, network parameters and the presence of competitive interactors. This database, and analysis has been made available through an open-access web-server, miRiam, to promote further exploration. Here we report that expression of genes involved in Apoptosis Processes, Immune System Processes, Translation Regulator Activities, and Molecular Transport Activities within the cell are predominately regulated by miRNA mediation. Our findings further indicate that this regulatory effect has a profound effect in controlling protein crowding inside the cell. A miRNA mediated gene expression regulation serves as a temporal regulator, allowing the cellular machinery to temporarily 'pause' the translation of mRNA, indicating that the miRNA-mRNA interactions may be important for governing the optimal usage of cell volume.