Anuradha Chakraborti

The significant genetic diversity among Group A Streptococcal (GAS) strains isolated from various geographical regions presents serious challenges for treatment and vaccine development strategies

The focus of this study is on Rheumatic heart disease (RHD), an autoimmune consequence of rheumatic fever leading to mitral valve damage. Aim is to explore the role of exosomes derived from RHD patients in promoting angiogenesis during RHD pathogenesis. Exosomes were isolated via column chromatography from the pericardial fluid (PF) and serum of RHD patients, pericardial fluid from non-RHD patients undergoing coronary artery bypass grafting (CABG) surgery, and serum of healthy individuals. These exosomes were characterized by transmission electron microscopy (TEM) and western blotting. Human umbilical vein endothelial cells (HUVECs) cultured from umbilical cord(s), were treated with purified exosomes from RHD, CABG patients or healthy individuals. Exosome treatment induced morphological changes in HUVECs, including elongation and tube-like structure formation. The qRT-PCR gene expression analysis revealed significant upregulation of angiogenic markers (VEGF-A 2.7-fold, MMP2 1.5-fold) and inflammatory cytokine (IL-4 15.17-fold), in HUVECs stimulated with RHD serum exosomes. Conversely, there was downregulation of anti- angiogenic genes (RASA-1 0.65-fold and endostatin 0.4-fold). Notably, elevated levels of miR- 150-5p (34.29 + 9.524-fold mean difference; p= 0.0007) were detected in serum-derived exosomes from RHD patients compared to healthy controls. Stimulation of HUVECs with RHD serum exosomes resulted in reduction in the expression of TP53 (0.227-fold; p= 0.006), a known target of miR-150-5p. Furthermore, transfection of HUVECs with miR-150-5p mimics induced the formation of angiogenic endothelial tubes, underscoring the role of miR-150-5p in promoting angiogenesis. Graphical

In most cancers, tumor hypoxia downregulates the expression of C‐C motif chemokine 2 (CCL2), and this downregulation has been implicated in monocyte infiltration and tumor progression; however, the molecular mechanism is not yet clear. We compared noncancerous and lung‐adenocarcinoma human samples for hypoxia‐inducible factor 1‐alpha (HIF‐1A), microRNA‐210‐3p (mir‐210‐3p), and CCL2 levels. Mechanistic studies were performed on lung adenocarcinoma cell lines and 3D tumor spheroids to understand the role of hypoxia‐induced miR‐210‐3p in the regulation of CCL2 expression and macrophage polarization. HIF‐1Α stabilization increases miR‐210‐3p levels in lung adenocarcinoma and impairs monocyte infiltration by inhibiting CCL2 expression. Mechanistically, miR‐210‐3p directly binds to the 3′untranslated region (UTR) of CCL2 mRNA and silences it. Suppressing miR‐210‐3p substantially downregulates the effect of hypoxia on CCL2 expression. Monocyte migration is significantly hampered in miR‐210‐3p mimic‐transfected HIF‐1A silenced cancer cells. In contrast, inhibition of miR‐210‐3p in HIF‐1A‐overexpressed cells markedly restored monocyte migration, highlighting a direct link between the miR‐210‐3p level and tumor monocyte burden. Moreover, miR‐210‐3p inhibition in 3D tumor spheroids promotes monocyte recruitment and skewing towards an antitumor M1 phenotype. Anti‐hsa‐miR‐210‐3p‐locked nucleic acid (LNA) delivery in a lung tumor xenograft zebrafish model caused tumor regression, suggesting that miR‐210‐3p could be a promising target for immunomodulatory therapeutic strategies against lung adenocarcinoma.

Group A streptococci (GAS) are gram-positive, cocci-shaped bacteria that cause a wide variety of infections and are a cause of significant health burden, particularly in lower- and middle-income nations. The GAS genome contains a number of virulence factors such as the M-protein, hyaluronic acid, C5a peptidase, etc. Despite its significant health burden across the globe, a proper vaccine against GAS infections is not yet available. Various candidates for an effective GAS vaccine are currently being researched. These are based on various parts of the streptococcal genome. These include candidates based on the N-terminal region of the M protein, the conserved C-terminal region of the M protein, and other parts of the streptococcal genome. The development of a vaccine against GAS infections is hampered by certain challenges, such as extensive genetic heterogeneity and high protein sequence variation. This review paper sheds light on the various virulence factors of GAS, their epidemiology, the different vaccine candidates currently being researched, and the challenges associated with M-protein and non-M-protein-based vaccines. This review also sheds light on the current scenario regarding the status of vaccine development against GAS-related infections.

Background and Aim The host dietary fibre is fermented into short-chain fatty acids (SCFA) by intestinal microbiota as bacterial metabolites like propionate, acetate and butyrate. Among these metabolites, the role of butyrate is well documented to provide energy to intestinal epithelial cells. Also, butyrate has anti-inflammatory and anti-tumour properties and decrease in its level by unbalanced diet can develops cancer. Lately, some research has suggested that sodium butyrate as an inhibitor of histone deacetylase (HDAC) may have anticancer potential for hepatocellular carcinoma (HCC), the most common type of liver cancer. Since, HCC is asymptomatic it is usually diagnosed at its advanced stage. Sorafenib with antiproliferative and antiangiogenic effects is the first line of treatment in advanced HCC. However, prolonged drug treatment to HCC patients develops adaptive resistance towards the sorafenib. Sorafenib resistance can also be enhanced by differentially expressed microRNAs. However, the significance of butyrate in HCC sorafenib resistance and its association with sorafenib-targeted microRNAs is yet to be unfurled. Here, an attempt has been made to explore the role of bacterial metabolite butyrate on sorafenib resistant HCC as well as on sorafenib-targeted microRNAs (miR-7641 and miR-199) to curtail sorafenib resistance in HCC. Methods Initially, in-silico analysis was performed using Human Metabolome Database (HMDB) so to identify specific butyrate producing faecal bacteria. Then, their specific 16s rRNA expression was compared between HCC patients and healthy individuals using qRT-PCR. Additionally, the cell viability (MTT) and apoptosis assays were performed in both parental and sorafenib resistant HepG2 cells to evaluate the role of sodium butyrate in sorafenib resistant HCC. Moreover, the association of sodium butyrate with sorafenib-targeted miR-7641 and miR-199 was also assessed using real time PCR, cell viability, cell apoptosis and transfection assays. Results In silico analysis demonstrated Roseburia cecical, Roseburia intestinalis, Eubacterium rectal, Faecalibacterium prausnitzii as specific butyrate producing faecal bacteria and their 16s rRNA expression was downregulated in HCC patients. In vitro study revealed the presence of sodium butyrate also decreased the cell viability as well as enhanced cell apoptosis of both parental and resistant HepG2 cells. Interestingly, sodium butyrate also decreased the expression of both sorafenib-targeted miR-7641 and miR-199. Further, combination of both sodium butyrate and antimiR-7641 or antimiR-199 also increased apoptosis and decreased viability of resistant cells. Conclusion This is first study to unravel the association of butyrate producing bacteria with HCC patients and the significance of bacterial metabolite butyrate as anti-tumour in sorafenib resistant hepatocellular carcinoma. The study also demonstrated the plausible new aspects of bacterial metabolite butyrate association with sorafenib-targeted miRNAs (miR-7641 and miR-199). Hence, the study highlighted the therapeutic potential of bacterial metabolite butyrate that might improve the clinical management of hepatocellular carcinoma.

Rheumatic heart disease (RHD) is an autoimmune sequel of pharyngitis and rheumatic fever that leads to permanent heart valve damage, especially the mitral valves. The mitral valves, which are responsible for the binding of auto-antibodies during immune response generation, lead to valve scarring and eventually valves dysfunction. Recently, exosomes (EXOs), the nano-sized vesicles, which range in size from 30 to 150 nm, are reported in various cardiovascular physiological and pathological processes. These vesicles are found in several body fluids such as plasma, serum, and also in cell culture media. Exosomal cargo contains proteins, which are taken up by the recipient cells and modulate the cellular characteristics. The role of exosomal proteins in RHD is still obscure. Hence, the present study has been designed to unveil the exosomal proteins in disease severity during RHD. In this study, the exosomes were isolated from biological fluids (serum and pericardial fluid) of RHD patients as well as from their respective controls. Protein profiling of these isolated exosomes revealed that alpha-1 antitrypsin is up-regulated in the biological fluids of RHD patients. The enhanced levels of exosomal alpha-1 antitrypsin, were further, validated in biological samples and mitral valve tissues of RHD patients, to correlate with the disease severity. These findings suggest an association of increased levels of exosomal alpha-1 antitrypsin with the RHD pathogenesis.

Background: Hepatocellular carcinoma (HCC) is the second most common malignancy with increasing cancer deaths worldwide. HCC is mainly diagnosed at its advanced stage, and treatment with FDA-approved sorafenib, the multikinase inhibitor drug, is advised. Acquired resistance against sorafenib develops through several pathways involving hypoxia, autophagy, high glycolysis, or glutaminolysis. Small non-coding RNAs, similar to microRNAs (miRNAs), are also known to affect sorafenib resistance in HCC. However, there is a lack of information regarding the significance of differentially expressed miRNA (if any) on autophagy and glutamine regulation in sorafenib-resistant HCC. Methods: The expression of autophagy and glutaminolysis genes was checked in both parental and sorafenib resistant HepG2 cell lines by real-time PCR. MTT and Annexin/PI assays were also performed in the presence of inhibitors such as chloroquine (autophagy inhibitor) and BPTES (glutaminolysis inhibitor). Next generation sequencing and in silico analysis were performed to select autophagy and glutamine addiction-specific microRNA. Selected miRNA were transfected into both HepG2 cells to examine its effect on autophagy and glutamine addiction in regulating sorafenib-resistant HCC. Results: Our in vitro study depicted a higher expression of genes encoding autophagy and glutaminolysis in sorafenib-resistant HepG2 cells. Moreover, inhibitors for autophagy (chloroquine) and glutaminolysis (BPTES) showed a diminished level of cell viability and augmentation in cell apoptosis of sorafenib-resistant HepG2 cells. NGS and real-time PCR demonstrated the downregulated expression of miR-23b-3p in sorafenib-resistant cells compared to parental cells. In silico analysis showed that miR-23b-3p specifically targeted autophagy through ATG12 and glutaminolysis through GLS1. In transfection assays, mimics of miR-23b-3p demonstrated reduced gene expression for both ATG12 and GLS1, decreased cell viability, and increased cell apoptosis of sorafenib-resistant HepG2 cells, whereas the antimiRs of miR-23b-3p demonstrated contrasting results. Conclusion: Our study highlights the cytoprotective role of autophagy and glutamine addiction modulated by miR-23b-3p (tumor suppressor), suggesting new approaches to curb sorafenib resistance in HCC.

Rheumatic heart is an autoimmune sequel of pharyngitis and rheumatic fever that leads to permanent heart valves damage, especially the mitral valves. The mitral valves, which are responsible for the binding of auto-antibodies during immune response generation, leads to valves scarring and eventually mitral valve dysfunction. Recently, exosomes (EXOs), the nano-sized-vesicles, which ranges in size from 30-150 nm, are involved in various cardiovascular physiological and pathological processes. These vesicles are found in several body fluids such as plasma, serum and also in cell culture media. Exosomal cargo contains proteins, which are taken up by the recipient cells and modulates the cellular characteristics. The role of exosomal proteins in RHD is still obscure. Hence, the present study is designed to unveil the role of exosomal proteins in increasing disease severity during RHD. In this study, the exosomes were isolated from biological fluids (plasma and pericardial fluid) of RHD patients as well as from their respective controls. Protein profiling of these isolated exosomes revealed that alpha-1 antitrypsin is up-regulated in the biological fluids of RHD patients. The augmented levels of exosomal alpha-1 antitrypsin, was further, validated in biological samples and mitral valve tissues of RHD patients, which was further correlated with the disease severity. These findings suggest that increased levels of exosomal alpha-1 antitrypsin is responsible for increased severity during RHD.

Streptococcus pneumoniae (pneumococcus) causes significant infection-related morbidity and mortality worldwide. The genome plasticity of pneumococcus is an essential factor in antibiotic resistance, serotype switching, and the emergence of nonvaccine serotypes. Information regarding the serotype distribution as well as antimicrobial susceptibility in pneumococcus clinical isolates responsible for various infections in Northern India is limited. Here, we have explored the antibiotic resistance and serotype pattern associated with S. pneumoniae infections from both invasive and noninvasive sites of patients of all ages, visiting out-patient department of a tertiary care hospital (PGIMER, Chandigarh, India). This study was carried out on 68 S. pneumoniae isolates and the isolates exhibited the highest resistance (76.5%) to cotrimaxozole followed by resistance toward tetracycline (36.8%) and erythromycin (23.5%). All isolates showed vancomycin susceptibility and 86.8% of isolates showed sensitivity to chloramphenicol. Multidrug resistance was found in 32% (n=22) of the S. pneumoniae isolates showing resistance toward three different antibiotics. Serotype 19F was found to be the most prevalent serotype (39%) followed by serotypes 6A/B/C (19%) and 1 (12%). These data shed light on the latest trends in antibiotic susceptibility and prevalent serotype patterns of hospital-based S. pneumoniae isolates. This information can be helpful in designing future disease-preventive strategies.