Biology subjects

Ramesh, S.

Publications and source records attributed to Ramesh, S..

24 records · Page 2Linked to original sources

bioRxiv2022

CNTNAP4 signaling regulates osteosarcoma disease progression

Improved treatment strategies for sarcoma rely on clarification of the molecular mediators of disease progression. Recently, we reported that the secreted glycoprotein NELL-1 modulates osteosarcoma (OS) disease progression in part via altering the sarcomatous extracellular matrix (ECM) and cell-ECM interactions. Of known NELL-1 interactor proteins, Contactin-associated protein-like 4 (Cntnap4) encodes a member of the neurexin superfamily of transmembrane molecules best known for its presynaptic functions in the central nervous system. Here, CRISPR/Cas9 gene deletion of CNTNAP4 reduced OS tumor growth, sarcoma-associated angiogenesis, and pulmonary metastases. CNTNAP4 knockout (KO) in OS tumor cells largely phenocopied the effects of NELL-1 KO, including reductions in sarcoma cell attachment, migration, and invasion. Further, CNTNAP4 KO cells were found to be unresponsive to the effects of NELL-1 treatment. Transcriptomic analysis combined with protein phospho-array demonstrated notable reductions in the MAPK/ERK signaling cascade with CNTNAP4 deletion, and the ERK1/2 agonist isoproterenol restored cell functions among CNTNAP4 KO tumor cells. Finally, human primary cells and tissues in combination with sequencing datasets confirmed the significance of CNTNAP4 signaling in human sarcomas. In summary, our findings demonstrate the biological importance of NELL-1/CNTNAP4 signaling axis in disease progression of human sarcomas and suggest that targeting the NELL-1/CNTNAP4 signaling pathway represents a strategy with potential therapeutic benefit in sarcoma patients.

cancer biology↗

bioRxiv2021

Altered interactions between circulating and tissue-resident CD8 T cells with the colonic mucosa define colitis associated with immune checkpoint inhibitors

Therapeutic blockade of co-inhibitory immune receptors PD-1 and CTLA-4 has revolutionized oncology, but treatments are limited by immune-related adverse events (IRAEs). IRAE Colitis (irColitis) is the most common, severe IRAE affecting up to 25% of patients on dual PD-1 and CTLA-4 inhibition. Here, we present a systems biology approach to define the cell populations and transcriptional programs driving irColitis. We collected paired colon mucosal biopsy and blood specimens from 13 patients with irColitis, 8 healthy individuals, and 8 controls on immune checkpoint inhibitors (ICIs), and analyzed them with single-cell/nuclei RNA sequencing with paired TCR and BCR sequencing, multispectral fluorescence microscopy, and secreted factor analysis (Luminex). We profiled 299,407 cells from tissue and blood and identified 105 cell subsets that revealed significant tissue remodeling in active disease. Colon mucosal immune populations were dominated by tissue-resident memory (TRM) ITGAE-expressing CD8 T cells representing a phenotypic spectrum defined by gene programs associated with T cell activation, cytotoxicity, cycling, and exhaustion. CD8 TRM and effector CD4 T cells upregulated type 17 immune programs (IL17A, IL26) and Tfh-like programs (CXCL13, PDCD1). We also identified for the first time an increased abundance of two KLRG1 and ITGB2-expressing CD8 T cell populations with circulatory cell markers, including a GZMK TRM-like population and a CX3CR1 population that is predicted to be intravascular. These two populations were more abundant in irColitis patients treated with dual PD-1/CTLA-4 inhibition than those receiving anti-PD-1 monotherapy. They also had significant TCR sharing with PBMCs, suggesting a circulatory origin. In irColitis we observed significant epithelial turnover marked by fewer LGR5-expressing stem cells, more transit amplifying cells, and upregulation of apoptotic and DNA-sensing programs such as the cGAS-STING pathway. Mature epithelial cells with top crypt genes upregulated interferon-stimulated pathways, CD274 (PD-L1), anti-microbial genes, and MHC-class II genes, and downregulated aquaporin and solute-carrier gene families, likely contributing to epithelial cell damage and absorptive dysfunction. Mesenchymal remodeling was defined by increased endothelial cells, both in irColitis patients and specifically in patients on dual PD-1/CTLA-4 blockade. Cell-cell communication analysis identified putative receptor-ligand pairs that recruit CD8 T cells from blood to inflamed endothelium and positive feedback loops such as the CXCR3 chemokine system that retain cells in tissue. This study highlights the cellular and molecular drivers underlying irColitis and provides new insights into the role of CTLA-4 and PD-1 signaling in maintaining CD8 TRM homeostasis, regulating CD8 T recruitment from blood, and promoting epithelial-immune crosstalk critical to gastrointestinal immune tolerance and intestinal barrier function.

immunology↗

bioRxiv2021

Engineering site-selective incorporation of fluorine into natural product analogs

While bioactive compounds are commonly derived both by human design as well as from living organisms, man-made and natural products typically display very different structural characteristics. As such, a longstanding goal in the discovery of new molecular function is to develop approaches to incorporate the advantageous elements of both groups of molecules, thereby expanding the molecular space accessible for this purpose. In this work, we report the engineering a fluorine-selective enzyme that can complement mutated acyltransferase (AT) domains of a modular polyketide synthase, which are the main determinants of the identity and location of substituents on polyketides, to produce different fluorinated regioisomers of the erythromycin precursor in vitro. We further show that by engineering cell uptake of fluorinated building blocks, we can control fluorine selectivity in vivo to produce selectively fluorinated polyketides using engineered E. coli. These results demonstrate that it is possible to introduce fluorine, a key synthetic design element for drug development, selectively into the scaffold of a complex natural product and produce these analogs by microbial fermentation.

synthetic biology↗

bioRxiv2020

Structural and Functional Implications of Non-synonymous Mutations in the Spike protein of 2,954 SARS-CoV-2 Genomes

Protein-protein interactions between virus and host are crucial for infection. SARS-CoV-2, the causative agent of COVID-19 pandemic is an RNA virus prone to mutations. Formation of a stable binding interface between the Spike (S) protein Receptor Binding Domain (RBD) of SARS-CoV-2 and Angiotensin-Converting Enzyme 2 (ACE2) of host actuates viral entry. Yet, how this binding interface evolves as virus acquires mutations during pandemic remains elusive. Here, using a high fidelity bioinformatics pipeline, we analysed 31,403 SARS-CoV-2 genomes across the globe, and identified 444 non-synonymous mutations that cause 49 distinct amino acid substitutions in the RBD. Molecular phylogenetic analysis suggested independent emergence of these RBD mutants during pandemic. In silico structure modelling of interfaces induced by mutations on residues which directly engage ACE2 or lie in the near vicinity revealed molecular rearrangements and binding energies unique to each RBD mutant. Comparative structure analysis using binding interface from mouse that prevents SARS-CoV-2 entry uncovered minimal molecular determinants in RBD necessary for the formation of stable interface. We identified that interfacial interaction involving amino acid residues N487 and G496 on either ends of the binding scaffold are indispensable to anchor RBD and are well conserved in all SARS-like corona viruses. All other interactions appear to be required to locally remodel binding interface with varying affinities and thus may decide extent of viral transmission and disease outcome. Together, our findings propose the modalities and variations in RBD-ACE2 interface formation exploited by SARS-CoV-2 for endurance. ImportanceCOVID-19, so far the worst hit pandemic to mankind, started in January 2020 and is still prevailing globally. Our study identified key molecular arrangements in RBD-ACE2 interface that help virus to tolerate mutations and prevail. In addition, RBD mutations identified in this study can serve as a molecular directory for experimental biologists to perform functional validation experiments. The minimal molecular requirements for the formation of RBD-ACE2 interface predicted using in silico structure models may help precisely design neutralizing antibodies, vaccines and therapeutics. Our study also proposes the significance of understanding evolution of protein interfaces during pandemic.

evolutionary biology↗

bioRxiv2019

Genetic diversity and haplotypic structure of a Saudi population sample using Investigator Argus X-12 amplification kit

X-chromosome short tandem repeat (X-STR) markers have shown a great capability in forensic identity investigations and paternity testing involving kinship analysis.\n\nIn the current study, the distribution of 12 X-STR loci located in four linkage groups was evaluated using Investigator(R) Argus X-12 Amplification Kit in 200 unrelated healthy individuals (105 males and 95 females) from the central region of the Saudi Arabia in order to create a DNA database.\n\nOur results indicated that DXS10146 locus was the most informative with 21 alleles while DXS8378 locus was the least with five alleles. Forensic parameters showed that all X-STRs loci either as individual markers or as linkage groups provide genetic information with high discrimination that is appropriate for forensic purposes with Paternity Informed Consent (PIC), Power of exclusion (PE), and Paternity index (PI) varied from 0.61211 to 0.917979, 0.38722 to 0.842949, and 0.038416 to 0.16367, respectively. A significant Linkage disequilibrium (LD) with p-value after Bonferroni correction p [≤] 0.05/66= 0.0008 was observed for 17 pairs of loci in male samples and 4 pairs of loci in female. In the male group, LG3 showed relatively high values of Haplotype diversity. The pairwise genetic distance fixation index (Fst) results showed that the Saudi population is genetically close to the Egyptian and Emirati populations and distant to the Turkish population.\n\nThe current study revealed that Investigator(R) Argus 12 X-STR kit would support forensic application, kinship testing involving female offspring, and human identification in Saudi populations.

genetics↗

bioRxiv2017

A comparison of root and shoot hydraulics, aquaporin expression and leaf gas exchange between two grapevine cultivars reveals differences in hydraulic control mediated by aquaporins

Hydraulics of plants that take different strategies of stomatal control under water stress are still relatively poorly understood. Here we explore how root and shoot hydraulics, gas exchange, aquaporin expression and abscisic acid (ABA) concentration in leaf xylem sap ([ABA]xylem) may be involved and coordinated. A comparison in responses to mild water stress and ABA application was made between two cultivars of Vitis vinifera L. previously classified as isohydric (Grenache) and anisohydric (Syrah). Grenache showed stronger adjustments of leaf, plant, and root hydraulic conductances to decreased soil moisture and a steeper correlation of stomatal conductance (gs) to [ABA]xylem than Syrah resulting in greater conservation of soil moisture, but not necessarily more isohydric behaviour. Under well-watered conditions, changes in vapour pressure deficit (VPD) had a strong influence on gs in both cultivars with adjustments of leaf hydraulic conductance. Grenache was more sensitive to decreases in soil water availability compared to Syrah that rather responded to VPD. There were stronger correlations between plant hydraulic parameters and changes in aquaporin gene expression in leaves and roots of Grenache. Overall, the results reinforce the hypothesis that both hydraulic and chemical signals significantly contribute to the differences in water conservation behaviours of the two cultivars.

plant biology↗