Rajiv Gandhi Centre for Biotechnology

Extracellular vesicles (EVs) are nanoscale membranous structures that play pivotal roles in intercellular communication across various biological contexts, encompassing both health and disease. These lipid bilayer-delimited nanovesicles facilitate the horizontal transfer of biomolecular cargo, including nucleic acids, proteins, lipids, and metabolites, from donor to recipient cells, thereby establishing critical cell-to-cell communication and influencing pathological processes (Figure 1). In the context of physiological health, exosomes are indispensable for maintaining homeostasis and regulating cellular functions. They mediate the exchange of information and biomolecules between cells, contributing to processes such as immune response modulation and tissue regeneration. In cancer, exosomes assume a complex role, often promoting disease progression by transporting oncogenic cargo and modulating the tumor microenvironment. In a distinct context, plant-derived exosomes, also known as plant extracellular vesicles, are emerging as intriguing entities with potential applications in agriculture, nutrition, and biomedicine. These plant exosomes harbor bioactive compounds and genetic material, serving as mediators of communication between plants and other organisms, thereby influencing crop health and defense mechanisms. Understanding the intricate functions and diverse roles of exosomes in these contexts holds promising prospects for advancing disease diagnostics and therapeutics and revolutionizing agricultural practices.

Background The process of transdifferentiating epithelial cells to mesenchymal-like cells (EMT) involves cells gradually taking on an invasive and migratory phenotype. Many cell adhesion molecules are crucial for the management of EMT, integrin β4 (ITGB4) being one among them. Although signaling downstream of ITGB4 has been reported to cause changes in the expression of several miRNAs, little is known about the role of such miRNAs in the process of EMT. Methods and results The cytoplasmic domain of ITGB4 (ITGB4CD) was ectopically expressed in HeLa cells to induce ITGB4 signaling, and expression analysis of mesenchymal markers indicated the induction of EMT. β-catenin and AKT signaling pathways were found to be activated downstream of ITGB4 signaling, as evidenced by the TOPFlash assay and the levels of phosphorylated AKT, respectively. Based on in silico and qRT-PCR analysis, miR-383 was selected for functional validation studies. miR-383 and Sponge were ectopically expressed in HeLa, thereafter, western blot and qRT-PCR analysis revealed that miR-383 regulates GATA binding protein 6 (GATA6) post-transcriptionally. The ectopic expression of shRNA targeting GATA6 caused the reversal of EMT and β catenin activation downstream of ITGB4 signaling. Cell migration assays revealed significantly high cell migration upon ectopic expression ITGB4CD, which was reversed upon ectopic co-expression of miR-383 or GATA6 shRNA. Besides, ITGB4CD promoted EMT in in ovo xenograft model, which was reversed by ectopic expression of miR-383 or GATA6 shRNA. Conclusion The induction of EMT downstream of ITGB4 involves a signaling axis encompassing AKT/miR-383/GATA6/β-catenin.

Mycobacterium abscessus subsp. abscessus is a rapidly growing facultative intracellular pathogen that usually infects human lung and skin epithelium. Recently, we and another group have shown that it also has the potential to colonize human gastric epithelium, but its significance with respect to gastric diseases remains unclear. Although Helicobacter pylori still remains the only definite gastric pathogen, recent studies have shown that M. abscessus subsp. abscessus also has the potential to colonize human gastric epithelium. M. abscessus subsp. abscessus is known to exhibit multidrug resistance and clarithromycin has been used as the drug of choice. We aimed to determine the clarithromycin resistance profile of 117 (74 rough and 43 smooth) gastric M. abscessus subsp. abscessus strains and to detect the point mutations in rrl and erm (41) genes conferring the resistance. Our data showed 79.48% (19 smooth and 74 rough) of M. abscessus subsp. abscessus strains were resistant to clarithromycin (MIC 90 ≤ 512 μg/mL), while 20.51% (24 smooth) were susceptible (MIC 90 ≤ 8 μg/ mL). Nucleotide sequence analysis of the rrl gene with reference strains of M. abscessus subsp. abscessus did not show any mutation that is relevant to the clarithromycin resistance. However, analysis of erm (41) gene showed that M. abscessus subsp. abscessus strains, which were susceptible to clarithromycin had C, C, G, and C at their nucleotide positions 28, 159, 238, and 330, respectively, while the resistant strains showed T, T, A, and A at the same positions. Based on antibiogram and sequence analysis data we recommend further studies involving genomic analysis to identify the other genes involved in high clarithromycin resistance in gastric M. abscessus subsp. abscessus along with the mechanisms involved. MR (2023) Unusually high clarithromycin resistance in Mycobacterium abscessus subsp. abscessus isolated from human gastric epithelium.

27-hydroxycholesterol (27-HC) is a cholesterol metabolite and the first discovered endogenous selective estrogen receptor modulator (SERM) that has been shown to have proliferative and metastatic activity in breast cancer. However, whether 27-HC metabolite modulates the epigenetic signatures in breast cancer and its progression remains unclear. The current study, reports that 27-HC represses the expression of euchromatic histone lysine methyltransferase G9a, further reducing di-methylation at H3K9 in a subset of genes. We also observed reduced occupancy of ERα at the G9a promoter, indicating that 27-HC negatively regulates the ERα occupancy on the G9a promoter and functions as a transcriptional repressor. Further, ChIP-sequencing for the H3K9me2 mark has demonstrated that 27-HC treatment reduces the H3K9me2 mark on subset of genes linked to cancer progression, proliferation, and metastasis. We observed upregulation of these genes following 27-HC treatment which further confirms the loss of methylation at these genes. Immunohistochemical analysis with breast cancer patient tissues indicated a positive correlation between G9a expression and CYP7B1, a key enzyme of 27-HC catabolism. Overall, this study reports that 27-HC represses G9a expression via ERα and reduces the levels of H3K9me2 on a subset of genes, including the genes that aid in breast tumorigenesis and invasion further, increasing its expression in the breast cancer cells.

Background: Lack of druggable targets and complex expression heterogeneity of known targets is common among TNBC subtypes. An enhanced expression of galectin-3 in TNBCs has already been documented. We have observed a tumor progression-dependent galectin-3 expression in TNBCs compared to adjacent epithelium and non TNBCs. Objective: To unravel the association of galectin- 3 in tumor progression, aggressiveness and drug resistance in TNBC patients. Methods: Galectin-3 expression in 489 breast cancer tissues was correlated with clinicopathological features and the results were validated in cell lines and mouse model by silencing galectin-3 using shRNA and the proteins were profiled by western blot and qRT-PCR. Protein interaction was analyzed by GFP Trap and Mass spectrometry. Results: Galectin-3 expression correlated with tumor stage in TNBC and a lower galectin-3 expression was associated with poor patient survival. The positive correlation between galectin-3, vimentin and CD44 expression, pinpoints galectin-3 contribution to epithelial to mesenchymal transition, drug resistance and stemness. Vimentin was found as an interacting partner of galectin-3. Duplexing of galecin-3 and vimentin in patient samples revealed the presence of tumor cells co-expressing both galectin-3 and vimentin. In vitro studies also showed its role in tumor cell survival and metastatic potential, elementary for tumor progression. In vivo studies further confirmed its metastatic potential. Conclusions: Tumor progression dependent expression pattern of galectin 3 was found to indicate prognosis. Co-expression of galectin-3 and vimentin in tumor cells promotes tumor dissemination, survival and its metastatic capability in TNBCs.

Aza-BODIPY biotin-based nano-self-assembly has been designed with a biotin receptor for selective NIR turn-on fluorescence activity against cancer cells. The nanostructured aza-BODIPY assemblies exhibit distinctive fluorescence characteristics via an avidin-induced partial disintegration process. The in vitro studies for nanoprobes NSA-DPR1a and NSA-DPR1b showed effective recognition and can discriminate biotin receptor-positive cancer cells (MDA-MB-231 and MCF7) compared to non-cancerous cells (MCF10A).

Background. Spermatogonial stem cells (SSCs) were considered to be stem cells with limited potencies due to their existence in adult organisms. However, the production of spermatogonial stem cell colonies with broader differentiation capabilities in primary germ cell cultures from mice of select genetic backgrounds (C57BL6/Tg14, ddY, FVB and 129/Ola) indicated that SSCs from these strains were pluripotent. Methods. We established primary cultures of SSCs from neonatal and adult Swiss 3T3 Albino mice. Stemness of SSC colonies were evaluated by performing real-time PCR and immunofluorescence analysis for a panel of chosen stemness markers. Differentiation potentials of SSCs were examined by attempting the generation of embryoid bodies and evaluating the expression of ectodermal, mesodermal and endodermal markers using immunofluorescence and real-time PCR analysis. Results. Spermatogonial stem cells from neonatal and mature mice testis colonised in vitroand formed compact spermatogonial stem cell colonies in culture. Alkaline phosphatase positivity and the presence of stem cell marker Oct-4 indicated stemness in these colonies. The differentiation potential of these SSC colonies was demonstrated by their transformation into embryoid bodies upon withdrawal of growth factors from the culture medium. SSC colonies and embryoid bodies formed were evaluated using immunofluorescence and real-time PCR analysis. Embryoid body like structures derived from both neonatal and adult mouse testis were quite similar in terms of the expression of germ layer markers. Conclusion. These results strongly suggest that SSC-derived EB-like structures could be used for further differentiation into cells of interest in cell-based therapeutics.

Fibrosis that occurs after nonfatal myocardial infarction (MI) is an irreversible reparative cardiac tissue remodeling process characterized by progressive deposition of highly cross-linked type I collagen. No currently available therapeutic strategy prevents or reverses MI-associated fibrotic scarring of myocardium. In this study, we used an epicardial graft prepared of porcine cholecystic extracellular matrix to treat experimental nonfatal MI in rats. Graft-assisted healing was characterized by reduced fibrosis, with scanty deposition of type I collagen. Histologically, the tissue response was associated with a favorable regenerative reaction predominated by CD4-positive helper T lymphocytes, enhanced angiogenesis, and infiltration of proliferating cells. These observations indicate that porcine cholecystic extracellular matrix delayed the fibrotic reactionand support its use as a potential biomaterial for mitigating fibrosis after MI. Delaying the progression of cardiac tissueremodeling may widen the therapeutic window for management of scarring after MI.

Sphingolipids are essential components of all eukaryotic membranes. The bioactive sphingolipid molecule, Sphingosine 1-Phosphate (S1P), regulates various important biological functions. This review aims to provide a comprehensive overview of the role of S1P signaling pathway in various immune cell functions under different pathophysiological conditions including bacterial and viral infections, autoimmune disorders, inflammation, and cancer. We covered the aspects of S1P pathways in NOD/TLR pathways, bacterial and viral infections, autoimmune disorders, and tumor immunology. This implies that targeting S1P signaling can be used as a strategy to block these pathologies. Our current understanding of targeting various components of S1P signaling for therapeutic purposes and the present status of S1P pathway inhibitors or modulators in disease conditions where the host immune system plays a pivotal role is the primary focus of this review.

Several studies over the last decade demonstrate the recruitment of immune cells, increased inflammatory cytokines, and chemokine in patients with metabolic diseases, including heart failure, parenchymal inflammation, obesity, tuberculosis, and diabetes mellitus. Metabolic rewiring of immune cells is associated with the severity and prevalence of these diseases. The risk of developing COVID-19/SARS-CoV-2 infection increases in patients with metabolic dysfunction (heart failure, diabetes mellitus, and obesity). Several etiologies, including fatigue, dyspnea, and dizziness, persist even months after COVID-19 infection, commonly known as Post-Acute Sequelae of CoV-2 (PASC) or long COVID. A chronic inflammatory state and metabolic dysfunction are the factors that contribute to long COVID. Here, this study explores the potential link between pathogenic metabolic and immune alterations across different organ systems that could underlie COVID-19 and PASC. These interactions could be utilized for targeted future therapeutic approaches.

Background The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cell is mediated through the binding of the SARS-CoV-2 Spike protein via the receptor binding domain (RBD) to human angiotensin-converting enzyme 2 (hACE2). Identifying compounds that inhibit Spike-ACE2 binding would be a promising and safe antiviral approach against COVID-19. Methods In this study, we used a BSL-2 compatible replication-competent vesicular stomatitis virus (VSV) expressing Spike protein of SARS-CoV-2 with eGFP reporter system (VSV-eGFP-SARS-CoV-2) in a recombinant permissive cell system for high-throughput screening of viral entry blockers. The SARS-CoV-2 permissive reporter system encompasses cells that stably express hACE2-tagged cerulean and H2B tagged with mCherry, as a marker of nuclear condensation, which also enables imaging of fused cells among infected EGFP positive cells and could provide real-time information on syncytia formation. Results A limited high-throughput screening identified six natural products that markedly inhibited VSV-eGFP-SARS-CoV-2 with minimum toxicity. Further studies of Spike-S1 binding using the permissive cells showed Scillaren A and 17-Aminodemethoxygeldanamycin could inhibit S1 binding to ACE2 among the six leads. A real-time imaging revealed delayed inhibition of syncytia by Scillaren A, Proscillaridin, Acetoxycycloheximide and complete inhibition by Didemnin B indicating that the assay is a reliable platform for any image-based drug screening. Conclusion A BSL-2 compatible assay system that is equivalent to the infectious SARS-CoV-2 is a promising tool for high-throughput screening of large compound libraries for viral entry inhibitors against SARS-CoV-2 along with toxicity and effects on syncytia. Studies using clinical isolates of SARS-CoV-2 are warranted to confirm the antiviral potency of the leads and the utility of the screening system.

Visualizing and quantifying the numerous factors that regulate murine pre-implantation embryonic development is technically challenging. Here, we present a protocol for the isolation of pre-implantation embryos at multiple stages of embryonic development to study gene expression. We describe steps for isolating RNA and cDNA synthesis from a small number of embryos. We then detail an immunofluorescence assay for the detection and localization of protein of interest by confocal microscopy in the pre-implantation embryos. For complete details on the use and execution of this protocol, please refer to Varghese et al.1.

Background The environmental microbiome has a direct influence on human health and disease. Previous reports suggest that urbanization and anthropogenic activities can alter natural microbial flora and potentially spread infectious disease-causing agents by emergence of pathogenic strains of bacteria. The nature of microbes present in urban settings and the flow of genetic elements between environmental and clinically relevant pathogenic bacteria, however, remains largely unknown. Results To unravel the bacterial diversity and resistome profile of multiple hotspot setups of a tropical urban system such as transport hubs, wet markets, hospital surroundings, waste dumps, and urban coastal area (beaches) metagenomics analyses of sediment samples from around Thiruvananthapuram city were done. Our study revealed the presence of 3,735 species belonging to 46 phyla of bacteria and archaea. The phylum Pseudomonadota was the most abundant bacterial community, followed by Bacteriodota and Actinomycetota. The genus Cloacibacterium had the highest overall relative abundance, while Pseudomonas was the most prevalent bacterial genus in hospital surroundings and coastal area (beaches) settings. We identified 291 antimicrobial resistance genes (ARGs) in the urban resistome, conferring resistance to more than 15 drug classes. The hospital settings had the highest number of ARGs across different drug classes. From the culturomics microcosm setups, we reconstructed 62 high-quality metagenome-assembled genomes (MAGs) which shows high resemblance to pathogenic bacterias such as Klebsiella pneumoniae, Escherichia coli and Acinetobacter baumannii etc. The ARGs detected in these genomes include genes encoding antibiotic-modifying enzymes (ArnT, eptA, eptB), beta-lactamase (ampC, ampC1, ampH), transcription regulating proteins (KpnE, KpnF, KpnG), efflux pumps (oqxA, oqxB). Furthermore, eight MAGS belonging to Acinetobacter kookii, Acinetobacter pitti, Acinetobacter baumannii, Acinetobacter gerneri, Escherichia coli, Klebsiella pneumoniae and Klebsiella quasipneumoniae were found to contain virulence factors such as siderophores (acinetobactin, aerobactin, enterobactin etc.), capsule, secretion systems belonging to type III group) (T3SS, TTSS etc) or type II (T2SS), fimbriae (type 3 and I), efflux pump (AdeFGH), or quorum sensing (abaR) associated with pathogenicity. Conclusions The study provides insights into bacterial composition, antimicrobial resistance, and virulence potential in urban environments, highlighting the importance of monitoring and managing antimicrobial resistance in urban ecosystems.

Owing to its life cycle involving multiple hosts and species-specific biological complexities, a vaccine against Plasmodium, the causative agent of Malaria remains elusive. This makes chemotherapy the only viable means to address the clinical manifestations and spread of this deadly disease. However, rapid surge in antimalarial resistance poses significant challenges to our efforts to eliminate Malaria since the best drug available to-date; Artemisinin and its combinations are also rapidly losing efficacy. Sodium ATPase (PfATP4) of Plasmodium has been recently explored as a suitable target for new antimalarials such as Cipargamin. Prior studies showed that multiple compounds from the Medicines for Malaria Venture (MMV) chemical libraries were efficient PfATP4 inhibitors. In this context, we undertook a structure- based virtual screening approach combined to Molecular Dynamic (MD) simulations to evaluate whether new molecules with binding affinity towards PfATP4 could be identified from the Pandemic Response Box (PRB), a 400-compound library of small molecules launched in 2019 by MMV. Our analysis identified new molecules from the PRB library that showed affinity for distinct binding sites including the previously known G358 site, several of which are clinically used anti-bacterial (MMV1634383, MMV1634402), antiviral (MMV010036, MMV394033) or antifungal (MMV1634494) agents. Therefore, this study highlights the possibility of exploiting PRB molecules against Malaria through abrogation of PfATP4 activity.

Chikungunya virus (CHIKV) hijacks host cell machinery to support its replication. Nucleophosmin 1 (NPM1/B23), a nucleolar phosphoprotein, is one of the host proteins known to restrict CHIKV infection; however, the mechanistic details of the antiviral role of NPM1 are not elucidated. It was seen in our experiments that the level of NPM1 expression affected the expression levels of interferon-stimulated genes (ISGs) that play antiviral roles in CHIKV infection, such as IRF1, IRF7, OAS3, and IFIT1, indicating that one of the antiviral mechanisms could be through modulation of interferon-mediated pathways. Our experiments also identified that for CHIKV restriction, NPM1 must move from the nucleus to the cytoplasm. A deletion of the nuclear export signal (NES), which confines NPM1 within the nucleus, abolishes its anti-CHIKV action. We observed that NPM1 binds CHIKV nonstructural protein 3 (nsP3) strongly via its macrodomain, thereby exerting a direct interaction with viral proteins to limit infection. Based on site-directed mutagenesis and coimmunoprecipitation studies, it was also observed that amino acid residues N24 and Y114 of the CHIKV nsP3 macrodomain, known to be involved in virus virulence, bind ADP-ribosylated NPM1 to inhibit infection. Overall, the results show a key role of NPM1 in CHIKV restriction and indicate it as a promising host target for developing antiviral strategies against CHIKV. IMPORTANCE Chikungunya, a recently reemerged mosquito-borne infection caused by a positive-sense, single-stranded RNA virus, has caused explosive epidemics in tropical regions. Unlike the classical symptoms of acute fever and debilitating arthralgia, incidences of neurological complications and mortality were reported. Currently there are no antivirals or commercial vaccines available against chikungunya. Like all viruses, CHIKV uses host cellular machinery for establishment of infection and successful replication. To counter this, the host cell activates several restriction factors and innate immune response mediators. Understanding these host-virus interactions helps to develop host-targeted antivirals against the disease. Here, we report the antiviral role of the multifunctional host protein NPM1 against CHIKV. The significant inhibitory effect of this protein against CHIKV involves its increased expression and movement from its natural location within the nucleus to the cytoplasm. There, it interacts with functional domains of key viral proteins. Our results support ongoing efforts toward development of host-directed antivirals against CHIKV and other alphaviruses.

The importance of regulatory features in health and disease is increasing, making it crucial to identify the hallmarks of these features. Self-attention networks (SAN) have given rise to numerous models for the prediction of complex phenomena. But the potential of SANs in biological models was limited because of high memory requirement proportional to input token length and lack of interpretability of self-attention scores. To overcome these constraints, we propose a deep learning model named Interpretable Self-Attention Network for REGulatory interactions (ISANREG) that combines both block self-attention and attention-attribution mechanisms. This model predicts transcription factor-bound motif instances and DNA-mediated TF-TF interactions using self-attention attribution scores derived from the network, overcoming the limitations of previous deep learning models. ISANREG will serve as a framework for other biological models in interpreting the contribution of the input with single-nucleotide resolution.

Mammalian lipoxygenases (LOXs) are involved in the biosynthesis of mediators of anaphylactic reactions and have been implicated in cell maturation, the pathogenesis of bronchial asthma, atherosclerosis, rheumatoid arthritis, cardiovascular diseases, Alzheimer’s disease and osteoporosis. Hence LOX inhibition in chronic conditions can lead to reducing the disease progression, which can be a good target for treating these diseases. The present study deals with designing methyl gallate derivatives and their anti-inflammatory effect by in silico, in vitro and in vivo methods. Designed derivatives were docked against LOX enzyme, and molecular dynamic simulations were carried out. Following the synthesis of derivatives, in vitro LOX inhibition assay, enzyme kinetics and fluorescence quenching studies were performed. One of the derivatives of methyl gallate (MGSD 1) was demonstrated as an anti-inflammatory agent for the treatment of rheumatoid arthritis in the animal model. Amelioration of Freund’s complete adjuvant (FCA)-induced arthritis by methyl gallate and its derivative with a concentration of 10–40 mg.kg⁻¹ has been assessed in vivo in a 28-day-long study. TNF-α and COX-2 gene expression were also studied. Methyl gallate synthetic derivatives (MGSDs) inhibited LOX with an IC50 of 100 nM, 304 nM, and 226 nM for MGSD 1, MGSD 2, and MGSD 3, respectively. Fluorescence quenching methods also prove their binding characteristics, and 200 ns simulations studies showed that the RMSDs for the entire complex were less than 2.8 Å. The in vivo results showed that methyl gallate was required approximately five times diclofenac for the same level of effect, and the synthesised (MGSD 1) compound required only approximately 1/12 of diclofenac for the same level of effect in in-vivo studies. The preeminent expression of COX-2 and TNF-α genes was significantly decreased after the treatment of the methyl gallate derivative. Hence, the in vivo results showed that the referenced synthetic derivative might have more arthritis-reducing properties than the parent compound methyl gallate and is more potent than the standard drug diclofenac, with no apparent induced toxicity.