Synbiotics are usually associated with promoting the gut health of consumers and thus they have seen an increase in demand. Kefir is a milk-based fermented beverage containing beneficial microorganisms, whereas, basil seed is a natural polysaccharide that acts as a prebiotic and fat-replacing agent. In the present study, kefir grains (3

Introduction: Cardiac fibrosis is a key pathological driver of cardiovascular diseases (CVDs). N6-methyladenosine (m6A) regulates mRNA metabolism. While our previous findings established m6A's role in endothelial cell angiogenesis following ischemic injury, its impact on cardiac fibrosis remains unknown. This study explores the role of m 6 A mRNA methylation in fibroblast activation, focusing on METTL3-mediated β-catenin signaling. We hypothesize that ischemia-induced METTL3 activation stabilizes CTNNB1 mRNA, promoting fibrosis, while METTL3 inhibition reduces myofibroblast activation and adverse remodeling. Method: We first reanalyzed available datasets to identify fibrosis-regulating targets in Sham and myocardial infarction (MI) hearts. Methylation patterns were then examined in vivo using myoFB METTL 3 knockout (KO) mice and through pharmacological inhibition of METTL3 with STM 2457 in MI mice. Cardiac function was assessed via echocardiography. In vitro methylation dynamics, fibrotic gene expression, and β-catenin regulation were studied in ischemic cardiac fibroblasts for mechanistic analysis. An Actinomycin D assay was performed to evaluate the role of methylation in β-catenin mRNA stability. Result: The reanalysis of the available dataset revealed an upregulation of β-catenin expression with multiple methylation sites. Myocardial infarction increased global m 6 A methylation in the mouse heart. In contrast, METTL 3 inhibition via STM 2457 significantly improved cardiac function post-MI by reducing β-catenin mRNA methylation, β-catenin expression, and fibrosis. MyoFBMETTL3KO mice exhibited decreased METTL3 expression, enhanced cardiac function (percentage ejection fraction and fractional shortening), and reduced fibrosis. Interestingly, ischemic stress induces mRNA methylation that stabilizes β-catenin mRNA in cardiac fibroblasts. Conclusion: Increased β-Catenin m6A mRNA methylation exacerbated cardiac fibrosis post- MI. Inhibition of mRNA methylation reduced MI-induced cardiac fibrosis, highlighting STM 2457 as a potential therapeutic candidate for treating heart failure.

Fibrosis is an irreversible process that occurs in all major organs in the body; however, excessive fibrosis results in organ dysfunction and possible death. This process involves robust proliferation of fibroblasts and, in turn, excessive accumulation of extracellular matrix (ECM) within and around damaged tissues, leading to abnormal architectural remodeling (Cannito S, Novo E, Parola M, Adv Drug Deliv Rev 121:57-84, 2017; Kendall RT, Feghali-Bostwick CA, Front Pharmacol 5:123, 2014). The fibrotic process can be triggered and initiated by various fibrotic factors such as transforming growth factor-β (TGF-β) (Travis MA, Sheppard D, Annu Rev Immunol 32:51-82, 2014), members of the connective tissue growth factor (CTGF) family as well as the platelet-derived growth factor (PDGF) family (Wynn TA, J Pathol 214(2):199-210, 2008). Despite advanced modern medicine, there has been no satisfactory progress in inhibiting or reversing the progression of fibrosis. Therefore, research continues to uncover advanced antifibrotic therapeutics. Among the many known factors involved in the development and progression of organ fibrosis, circular RNAs (circRNAs) have recently drawn attention as having an essential role in organ fibrosis. Their critical involvement in fibrosis opens a new avenue of promising drug therapy. This chapter summarizes the current research progress on the biological function and underlying molecular mechanism(s) of circRNAs in regulating organ fibrosis, including heart, lung, and kidney. Furthermore, we discuss the contribution of circRNAs to antifibrotic therapy and what its future holds.

Heart failure (HF) is a leading cause of death worldwide. We have shown that pressure overload (PO)-induced inflammatory cell recruitment leads to heart failure in IL-10 knockout (KO) mice. However, it's unclear if PO-induced inflammatory cells also target the gut mucosa, causing gut dysbiosis and leakage. We hypothesized that TAC (transverse aortic constriction) exacerbates immune cell homing to the gut (small intestine and colon), promoting dysbiosis and gut leakage in IL-10 KO mice. HF was induced in 8-10 weeks old C57BL/6J wild-type (WT) and B6.129P2-Il10tm1Cgn/J mutant (IL-10 KO) male and female mice by TAC and cardiac function was measured using visual sonics VEVO 3100. Fourteen days post-TAC, levels of monocytes, macrophages, neutrophils, and proinflammatory cytokines were measured in blood and gut. Gut dysbiosis was assessed via 16S rRNA sequencing in feces at 56 days post-TAC. IL-10 KO mice showed worsened cardiac dysfunction post-TAC. TAC worsened monocytes, and neutrophils infiltration in systemic circulation and facilitated their homing to the gut in IL-10 KO mice. Intriguingly, proinflammatory cytokines level was increased in blood, and gut of IL-10 KO mice following TAC. Furthermore, IL-10 expression was reduced in the colon of WT mice post-TAC. Moreover, TAC exacerbated gut dysbiosis in IL-10 KO mice. Finally, an impaired intestinal permeability was noted in IL-10 KO mice post-TAC. In conclusion, TAC-induced systemic inflammation leads to gut dysbiosis and impaired gut permeability in IL-10 KO mice, indicating IL-10's potential role in regulating intestinal integrity and microbiota balance during heart failure.

Background: Inflammatory bowel disease (IBD) has resulted in various comorbidities in the United States. Recently, an increase in cardiovascular-related deaths among IBD patients has been noted. However, the fundamental molecular mechanisms underlying this trend are still not fully understood. Therefore, we proposed that bacterial metabolites, such as peptidoglycan, released from the gut may trigger cardiac inflammation and, eventually, heart failure in mice after colitis. Methods: We treated C57 black mice with DSS (dextran sodium sulfate) to induce colitis. Further, we collected feaces for 16s rRNA and evaluated gut microbiome. Blood was drawn to quantify the circulating peptidoglycan (PGN) level. In addition, heart and intestine tissues were collected to determine inflammatory genes expression (qPCR), immune cell infiltration (FACS), and intestinal permeability (WB and IHC). Results: DSS mice showed cardiac dysfunction (echo-data). Masson's trichrome staining and WB data showed impaired intestinal villi and reduced tight junction proteins, respectively, suggesting leaky gut in DSS treated mice. Comparative analysis of 16s rRNA sequencing data revealed significant changes in the Firmicutes/Bacteroidetes ratio in DSS treated mice compared to control mice. Moreover, plasma PGN level was significantly increased 3 weeks following DSS treatment. Gene expression data showed increased pro-inflammatory cytokines (TNFα) and fibrosis-associated genes (col1α1, fibronectin) in DSS mice hearts. Expression of CD68 (immunostaining data) was increased in DSS mice hearts suggest the recruitment of inflammatory macrophages. At the molecular level, cyclin-dependent kinase 9 (CDK9) expression was significantly increased in macrophages following PGN treatment, which ultimately led to an increase in NFkB signaling. Finally, CDK9 inhibition attenuated PGN-induced NFkB signaling. Conclusion(s): Our study suggests that colitis-induced leaky gut leads to CDK9-mediated NFκB upregulation, contributing to cardiac inflammation. Ongoing research will shed light on the underlying molecular mechanisms of cardiovascular dysfunction.

Background: Inflammatory Bowel Disease (IBD) affects millions of people all over the world. This has been known to contribute to several other comorbidities. Recently, impaired gut-barrier permeability has been known to contribute to cardiovascular diseases in IBD patients. However, the precise mechanisms remain unclear. In this study, we propose that bacterial metabolites, like peptidoglycan, may play a role in promoting cardiac inflammation and ultimately leading to heart failure in mice following colitis. Methods: We induced colitis in C57 black mice with DSS (dextran sodium sulfate) treatment. Further, we collected fecal sample for 16s rRNA analysis to evaluate microbial flora. Additionally, heart and intestine tissues were collected to determine inflammatory genes expression (qPCR), immune cell infiltration (FACS), and intestinal permeability (WB and immunohistochemistry). Blood was collected to quantify the circulating peptidoglycan (PGN) level. The effect of PGN on NFkB inflammatory pathways was evaluated in macrophages. Results: Echo-data showed cardiac dysfunction in DSS mice. Masson’s trichrome staining showed deteriorated intestinal villi, while western blot data indicated decreased Occludin and increased PV-1 level, suggesting a leaky gut in DSS treated mice. Comparative analysis of 16s rRNA sequencing data showed significantly altered Firmicutes/Bacteroidetes ratio in DSS treated mice as compared to control mice. Moreover, plasma PGN level was significantly increased 3 weeks following DSS treatment. Real-time gene expression data of pro-inflammatory cytokines (TNFα) and fibrosis associated genes (col1α1, fibronectin) were increased in DSS mice heart. Increased CD68 expression in the immunostaining data of DSS mice heart suggests the recruitment of inflammatory macrophages. At the molecular level, CDK9 expression was significantly increased in macrophage following PGN treatment, which ultimately leads to an increase in NFkB signaling. Finally, CDK9 inhibition attenuated PGN-induced NFkB signaling. Conclusion: Together, our study suggests that colitis-induced gut leakage leads to CDK9-mediated NFκB upregulation, contributing to cardiac inflammation. Ongoing research will shed light on the underlying molecular mechanisms of cardiovascular dysfunction.

Exosomes are double-layered lipid membranous nanovesicles that are endosomal in origin and secreted by almost all cells. They are 30-130 nm in size and contain various molecular signatures such as miRNAs, mRNAs, DNA, lipids, and proteins. Due to their highly heterogeneous content, exosomes have a major role in influencing cellular physiology and pathology. Although exosome research has been in progress for a long time, its biomedical applications have recently been expanding due to its bio-friendly nature. However, the most challenging part is its isolation to obtain quality exosomes with good yield. Therefore, in this chapter, we have described appropriate protocols for exosome isolation and characterization along with alternative purification methods.

AbstractCardiac fibrosis is the hallmark of cardiovascular disease (CVD), which is leading cause of death worldwide. Previously, we have shown that interleukin‐10 (IL10) reduces pressure overload (PO)‐induced cardiac fibrosis by inhibiting the recruitment of bone marrow fibroblast progenitor cells (FPCs) to the heart. However, the precise mechanism of FPC involvement in cardiac fibrosis remains unclear. Recently, exosomes and small extracellular vesicles (sEVs) have been linked to CVD progression. Thus, we hypothesized that pro‐fibrotic miRNAs enriched in sEV‐derived from IL10 KO FPCs promote cardiac fibrosis in pressure‐overloaded myocardium. Small EVs were isolated from FPCs cultured media and characterized as per MISEV‐2018 guidelines. Small EV's miRNA profiling was performed using Qiagen fibrosis‐associated miRNA profiler kit. For functional analysis, sEVs were injected in the heart following TAC surgery. Interestingly, TGFβ‐treated IL10‐KO‐FPCs sEV increased profibrotic genes expression in cardiac fibroblasts. The exosomal miRNA profiling identified miR‐21a‐5p as the key player, and its inhibition with antagomir prevented profibrotic signalling and fibrosis. At mechanistic level, miR‐21a‐5p binds and stabilizes ITGAV (integrin av) mRNA. Finally, miR‐21a‐5p‐silenced in sEV reduced PO‐induced cardiac fibrosis and improved cardiac function. Our study elucidates the mechanism by which inflammatory FPC‐derived sEV exacerbate cardiac fibrosis through the miR‐21a‐5p/ITGAV/Col1α signalling pathway, suggesting miR‐21a‐5p as a potential therapeutic target for treating hypertrophic cardiac remodelling and heart failure.

PURPOSE:Genetically diverse parasites enhances resistance against antimalarials, vaccines and host immune responses. The present study was designed to evaluate the role played by Plasmodium falciparum genetic diversity in predicting the real world malarial population.METHODS:Initially, the incidence pattern of all four northern Indian malarial species was examined using 18S rRNA gene and performed principal component analysis (PCA) based on frequencies of Plasmodium species. Consequently, genetic variance of Plasmodium falciparum histidine-rich protein-2 (Pfhrp2) gene among different malarial populations were compared using phylogenetic analysis. Multi-dimensional scaling was performed to assess genetic similarities and distances among studied populations.RESULTS:Of total 2168 patients screened, 561 patients with fever of unknown origin were included. 18S rRNA and Pfhrp2 genes were amplified in 78 and 45 samples, respectively. Among them 13.9%(78/561) patients had Plasmodium infection. Infections by P. falciparum, P. vivax and mixed infections were diagnosed among 47(60.2%) and 28(35.9%) and 3(3.8%) patients, respectively. We found eight types of Pfhrp2 amino acid sequence repeats among northern Indian population. The PCA findings were in line with genetic diversity and phylogenetic data. Temporal analysis showed the proportion of total diversity present in total subpopulation (ΔS/ΔT) was maximum for P. falciparum.CONCLUSIONS:Higher incidence of Pfhrp2 sequence variation through genetic recombination among multiple strains during sexual reproduction is potentially correlated with high transmission activity. This sequence variation might alter RDT detection sensitivities for different parasites by modulating the structure and frequency of antigenic epitopes.

Background: Cardiac fibrosis is pivotal in heart failure progression, where excessive extracellular matrix (ECM) secretion by activated fibroblasts leads to adverse remodeling and dysfunction. While Wnt/β-catenin signaling influences fibroblast activation and cardiac fibrosis post-MI, its precise regulation remains unclear. Emerging evidence suggests N6-methyladenosine (m 6 A) mRNA methylation's role in disease pathology, yet its specific contribution to post-MI cardiac fibrosis is not well understood. Thus, we hypothesized that “ MI-induced METTL3 (a Key m6A mRNA methyltransferase) activation stabilizes δ-catenin mRNA, facilitating cardiac fibrosis and adverse remodeling ”. Methods: The mice underwent sham/MI surgeries for 4 weeks, after which heart tissues were collected for biochemical and histological analysis following heart function measurements. Additionally, methyl-immunoprecipitation followed by RNA sequencing (MeRIP-sequencing) was performed on the heart tissue post-surgery, and the data were analyzed to identify fibrosis-associated targets. Results: Ischemic injury significant increase in m6A mRNA methylation in heart tissues. This increase in m 6 A RNA was also observed in adult cardiac fibroblasts (AMFs) following TGFb treatment. Interestingly, METTL3 inhibition (METTL3 siRNA) resulted in a significant reduction in TGFb-induced periostin and fibronectin gene expression, while METTL3 overexpression enhanced the expression of fibrotic genes in AMFs. To identify differentially regulated m6A target genes, MeRIP-seq was performed on RNA isolated from mice hearts post-MI. The sequencing data suggested hypermethylation of fibrosis-associated genes, including δ-catenin, post-MI. Notably, TGFb-induced increased δ-catenin mRNA methylation led to δ-catenin mRNA stabilization. In contrast, METTL3 inhibition using siMETTL3 in AMFs and in METTL3 KO mice significantly reduced fibroblast activation and cardiac fibrosis. Conclusion: Our data suggests that hypermethylation of δ-catenin mRNA plays a significant role in the progression of cardiac fibrosis following AMI. Therefore, regulating METTL3 could be a potential therapeutic target for attenuating cardiac fibrosis.

Background: Diabetic patients are more susceptible to heart failure (HF). High blood glucose level in diabetic patients eventually triggers the body's inflammatory response and causes cardiac fibroblast activation, endothelial cell dysfunction (ECD), and, ultimately, HF. Previously, we have shown that activated fibroblast-mediated ECD leads to HF. However, the molecular mechanism of fibroblast-induced ECD in diabetics is not yet well defined. Therefore, we hypothesized that “myofibroblast in the diabetic heart secretes exosomes loaded with antiangiogenic/profibrotic factors, which impair EC function.” Methods: Exosomes were isolated from diabetic mice plasma and fibroblast conditioned media by ultracentrifugation and characterized by nanosight & electron microscopy. We cultured mouse primary heart endothelial cells in EC growth media. ECs were treated with exosomes derived from fibroblasts (treated with either 25mM glucose, 500nM Angiotensin II (AngII) or both) for 48 hr. Mannitol (25mM) served as control. Pathway-based miRNA array was screened with exosome-derived miR, and targets were predicted bioinformatically for selected miR. Results: Glu-AngII cotreatment significantly activated fibroblasts as shown by qPCR (Col1α, and FN expression) and western blot (pSmad2/Smad2, p-p38/p38). Interestingly, exosomes derived from activated fibroblasts significantly induced ECD (eNOS, VEGF, CD31 genes, and proteins expression). We further checked the effect of diabetic mice plasma exosomes on ECs function. We found significantly impaired endothelial function, as shown by Matrigel tube formation and Boyden chamber migration assays. Micro RNA array and qPCR data showed that miR-216a-5p was highly upregulated in exosomes derived from FBs cotreated with Glu-AngII. Pathway-based analysis revealed that PGM5 could be a potential target in miR-216-induced ECD. Interestingly, miR-216a-5p inhibition significantly rescued diabetes-induced PGM5 inhibition and improved endothelial function Conclusions: Taken together, this study demonstrates that fibroblast in the diabetic heart releases miR-216a-5p through exosomes, which promote endothelial dysfunction via the miR-216a-5p/PGM5 axis.

Objective: Cardiac fibrosis is one of the most warranted cardiac impairments which ultimately leads to heart failure. N6-methyladenosine (m6A) is an abundant and conserved chemical modification in eukaryotic mRNA and is associated with mRNA metabolism. Recently, we showed that m6A mRNA methylation regulates the angiogenic potential of the endothelial cells following ischemic injury. However, the role of m6A mRNA methylation in cardiac fibrosis following ischemic injury is still elusive and needs improvement in treatment strategy. Methods: First we analyzed the available data set for the fibrosis-regulating targets in Sham and MI hearts. Further, we studied methylation patterns in vivo by inhibiting methylation in MI mice using STM2457. Then we assessed cardiac function using echocardiography and harvested tissue samples for further biochemical analysis. We also performed in vitro experiment to investigate the expression pattern of the screened gene (Fibronectin) in hypoxic cardiac fibroblast as an ischemic model. We also performed an Actinomycin D assay to check the role of methylation on FN mRNA stability. Fibrosis and other phenotypic statuses were studied using several molecular techniques including ICC/IHC. Finally, siMETTL3 was used to study rescue experiments. Results: We have shown upregulated FN expression post-MI, and also several methylation sites on FN mRNA in-silico. Histological and biochemical analysis showed increased fibrosis and m6A mRNA methylation in mice post-MI. Hypoxic stress in fibroblast induces mRNA methylation, which leads to FN mRNA stabilization and deposition of FN at the scar site by activated fibroblast. In contrast, METTL3 inhibition using STM 2457 significantly improved cardiac function. METTL3 inhibition significantly reduced FN mRNA methylation and FN expression, and ultimately reduced cardiac fibrosis. Conclusion: We observed that increased FN mRNA (m6A) methylation enhanced cardiac fibrosis following MI. Inhibition of mRNA methylation using STM2457 in in-vivo, and siRNA in-vitro reduced hypoxia-induced FN deposition and fibrosis, which suggests STM2457 could be a potential therapeutic molecule either alone or in combination with current regimens to curb methylation-induced cardiac fibrosis.

Background: Millions of people in the United States are affected by Inflammatory Bowel Disease (IBD). Recently, gut dysbiosis and impaired gut-barrier permeability are known to contribute to cardiovascular diseases in IBD patients. However, its mechanisms are poorly understood. Here, we hypothesized that gut derived bacterial metabolites (such a peptidoglycan in this case) in a mice colitis model, promotes cardiac inflammation and heart failure. Methods: To induced colitis WT mice were treated with control/DSS (dextran sodium sulfate). Heart and intestine were collected to evaluate inflammatory genes expression(qPCR), immune cell infiltration (FACS), and intestinal permeability (WB and immunohistochemistry). Blood was collected to quantify peptidoglycan (PGN) level. The effect of PGN on NFκB inflammatory pathways was evaluated in macrophages. Results: Echo-data showed cardiac dysfunction in DSS mice. Deteriorated intestinal villi in Masson’s trichrome staining and decreased Occludin and increased PV-1 in western data revealed leaky gut in DSS treated mice. Remarkably, PGN level was significantly increased in blood plasma 3 weeks following DSS treatment. Real-time gene expression data of pro-inflammatory cytokines (IL-6, IL-1 and TNFα) and fibrosis associated genes (col1α1, fibronectin) were increased in DSS mice heart. Increased CD68 expression in the immunostaining data of DSS mice heart indicated the recruitment of inflammatory macrophages. Intriguingly, we observed that CDK9 expression significantly increased upon PGN treatment leading to an increase in NFκB. Next, we inhibited CDK9 before PGN treatment and surprisingly we found that CDK9 inhibition reduces the effect of PGN. Conclusion: Taken together, data suggest that in gut dysbiosis state, PGN leaks into the blood and causes CDK9 mediated NFκB upregulation and cardiac inflammation.