Alpha-1-antitrypsin in serum exosomes and pericardial fluid exosomes is associated with severity of rheumatic heart disease.

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.

Maackia amurensis agglutinin induces apoptosis in cultured drug resistant human non-small cell lung cancer cells.

The emergence of multi drug resistance in non-small cell lung cancer (NSCLC) patients is a major challenge towards the efficacy of chemotherapy. Thus, there is an urgent need for the newer, better clinically targeted strategies to treat this disease. Earlier studies from our laboratory revealed the apoptotic activity of Maackia amurensis agglutinin (MAA) in human NSCLC cells. In this study, the effect of MAA on drug resistant NSCLC cells was investigated. Two Paclitaxel-resistant NSCLC sub-lines (A549/PTX100 and NCI-H460/PTX100) were developed from A549 & NCI-H460 cell lines respectively. The generation of drug resistance phenotype was confirmed by the expression of cell surface MDR-1. Both the drug resistant sub-lines showed distinct morphological alterations. MAA interacted with the cell-surface protein(s) of apparent Mr ~66 kDa and induced apoptosis in both the sub-lines through intrinsic/mitochondrial pathway, involving reduction in mitochondrial trans-membrane potential, up-regulation of Bax, unaltered/decreased expression of Bcl-XL, release of mitochondrial cytochrome c into the cytosol and activation of pro-caspases (-9&-3). Our findings highlighted the potential of this plant agglutinin to serve as an apoptosis inducing agent in drug resistant NSCLC cells.

Sorafenib response in hepatocellular carcinoma: MicroRNAs as tuning forks.

Hepatocellular carcinoma (HCC) is the primary liver malignancy that contributes towards the second most common cause of cancer-related mortality. The targeted chemotherapeutic agent, sorafenib, is known to show a statistically significant but limited overall survival advantage in advanced HCC. However, the individual patient response towards sorafenib varies drastically, with most experiencing stable disease and few with partial response; complete response is very rare. Progressive disease despite the treatment is also evident in many patients, indicating drug resistance. These varied responses have been linked with the modulation of several intracellular signaling pathways. Notably, the regulation of these pathways through diverse operating biomolecules, including microRNAs (miRNAs), is the focus of recent studies. MicroRNAs are tiny, non-coding RNA molecules that regulate the expression of several target genes. In addition, miRNAs are known to play a role in the progression of HCC carcinogenesis. Interestingly, miRNAs have also been identified to play differential roles in terms of sorafenib response in HCC such as biomarkers and functional modulation of cellular response to sorafenib, hence, they are also being therapeutically evaluated. This review outlines the role of reported miRNAs in different aspects of sorafenib response in HCC.

A cytosol derived factor of Group B streptococcus prevent its invasion into human epithelial cells.

Group B streptococcus (GBS) or Streptococcus agalactiae, is an opportunistic pathogen causing a wide range of infections like pneumonia, sepsis, and meningitis in newborn, pregnant women and adults. While this bacterium has adapted well to asymptomatic colonization of adult humans, it still remains a potentially devastating pathogen to susceptible infants. Advances in molecular techniques and refinement of in vitro and in vivo model systems have elucidated key elements of the pathogenic process, from initial attachment to the maternal vaginal epithelium to penetration of the newborn blood-brain barrier. Still, the formidable array of GBS virulence factors makes this bacterium at the forefront of neonatal pathogens. The involvement of bacterial components in the host-pathogen interaction of GBS pathogenesis and its related diseases is not clearly understood. In this study we demonstrated the role of a 39 kDa factor from GBS which plays an important role in the process of its invasion. We found a homogeneous 39 kDa factor from the cytosol of GBS after following a combination of sequential purification steps involving molecular sieving and ion exchange chromatography using ACTA-FPLC system. Its N-terminal sequence showed a homology with xenobiotic response element type transcriptional regulator protein, a 40 kDa protein of Streptococcus. This factor leads to inhibition of GBS invasion in HeLa and A549 cells. This protein also showed sensitivity and specific cross reactivity with the antibodies raised against it in New Zealand white rabbits by western immunoblotting. This inhibitory factor was further confirmed tolerant for its cytotoxicity. These results add a novel aspect to bacterial pathogenesis where bacteria's own intracellular protein component can act as a potential therapeutic candidate by decreasing the severity of disease thus promoting its invasion inhibition.

Association of rheumatic fever & rheumatic heart disease with plausible early & late-stage disease markers.

BACKGROUND & OBJECTIVES: Rheumatic fever (RF) and rheumatic heart disease (RHD) are the autoimmune sequelae caused by Group A Streptococcus. RHD still remains a major concern in the developing countries due to its poor diagnosis, lack of vaccines and social awareness among population. This study was aimed to identify the plausible early- and late-stage disease markers associated with RF/RHD. METHODS: A total of 84 patients with confirmed pharyngitis (n=18), RF (n=23) and RHD (n=43) were included in the comparative analysis of different factors involved in host-pathogen interaction during RF/RHD pathogenesis. RESULTS: This study revealed high titre of serum antistreptolysin O (ASO) antibody in pharyngitis compared to RF and RHD patients, whereas procollagen type 1 C-peptide (PICP) level was elevated in RHD which showed an inverse correlation with serum ASO titre. The significant elevation of serum anti-peptide associated with RF (PARF) antibody in RF patients was correlated as a probable stage-specific determinant. In addition, pro-inflammatory cytokine profile revealed high levels of interleukin-12 (IL-12)/IL-23p40, IL-17A in RF, whereas IL-6 concentration was higher in RHD compared to healthy controls. INTERPRETATION & CONCLUSIONS: The overall assessment of the factors/ disease markers involved in host-pathogen interaction in RF/RHD may be suggestive of plausible disease marker in different groups of patients. Further studies with larger sample need to be done to better understand RF/RHD pathogenesis.

Inhibition of enteroaggregative Escherichia coli cell adhesion in-vitro by designed peptides.

Enteroaggregative Escherichia coli (EAEC) bears remarkable capacity to adhere the host intestinal mucosal surface and results in acute or persistent childhood diarrhea worldwide. In this study, an attempt has been made to inhibit EAEC cell adherence in-vitro using synthetic peptides. E. coli isolates (n = 54) were isolated from the stool samples of clinically diagnosed pediatric diarrheal patients. 92.8% isolates showed different types of aggregative adherence patterns with HEp-2 cells. AAF-II (Aggregative Adherence Fimbriae-II) EAEC exhibited the maximum ability to form biofilm and intracellular survival. Peptides were designed against the high antigenic epitopic regions of AAF-II adhesin of EAEC O42 using prediction algorithms like BcePred and ProPred software to block the EAEC cell adhesion in-vitro. Peptides P2 (DITITPATNRDVNV) and P3 (MRIKAWGEANHGQL) demonstrated higher inhibition of EAEC cell adhesion than P1 (GMQGSITPAIPLRPG). Interestingly, increasing the pre-incubation time of the peptides with HEp-2 cells from 1 h to 2 h showed the maximum inhibition. The data suggested the potential role of P2 and P3 peptides in successfully blocking the binding of AAF-II EAEC with HEp-2 cell receptors. Hence, the peptides may be efficacious in designing new chemotherapeutic for the management of EAEC mediated diarrhea.

Small intestinal bacterial overgrowth and toll-like receptor signaling in patients with non-alcoholic fatty liver disease.

BACKGROUND AND AIM: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is multifactorial. There is sparse literature on the role of small intestinal bacterial overgrowth (SIBO) and toll-like receptor (TLR) signaling in NAFLD. The present study evaluated the relationship of SIBO with expression of TLR signaling genes in patients with NAFLD. METHODS: A total of 142 subjects composed of NAFLD (n = 60, mean age 38.7 ± 10.4 years), chronic viral hepatitis (CVH) (n = 32, mean age 39.5 ± 10.6 years), and healthy volunteers (n = 50, mean age 36.56 ± 4.2 years) were enrolled in the study. Duodenal fluid was taken endoscopically in 32 prospective patients with NAFLD for evaluation of SIBO. Hepatic mRNA expression of TLR4, CD14, TLR2, NF-κß, and MD2 and protein expression of TLR4 and TLR2 were studied in 64 patients (NAFLD = 32, CVH = 32) by RT-PCR and immunohistochemistry, respectively. Serum levels of TNF-α, adiponectin, insulin, and endotoxins were also evaluated. RESULTS: Small intestinal bacterial overgrowth was present in 12 (37.5%) out of 32 patients with NAFLD with Escherichia coli as the predominant bacterium. In comparison with those without SIBO, patients with SIBO had significantly higher endotoxin levels and higher CD14 mRNA, nuclear factor kappa beta mRNA, and TLR4 protein expression. Patients with NASH had significantly higher endotoxin levels and higher intensity of TLR4 protein expression in comparison with patients without NASH. Serum levels of TNF-α, endotoxins, and insulin were significantly higher and of adiponectin lower in NAFLD in comparison with CVH and healthy volunteers. CONCLUSIONS: Our study provides the first direct evidence of role of SIBO and endotoxemia and its relation with TLR signaling genes and liver histology in patients with NAFLD.

Expression of astrocytic genes coding for proteins implicated in neural excitation and brain edema is altered after acute liver failure.

In vitro and in vivo studies have suggested that reduced astrocytic uptake of neuronally released glutamate, alterations in expression of glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQP-4) contribute to brain edema in acute liver failure (ALF). However, there is no evidence to date to suggest that these alterations occur in patients with ALF. We analyzed the mRNA expression of excitatory amino acid transporters (EAAT-1, EAAT-2), GFAP, and AQP-4 in the cerebral cortex obtained at autopsy from eight patients with ALF and from seven patients with no evidence of hepatic or neurological disorders by real-time PCR, and protein expression was assessed using immunoblotting and immunohistochemistry. We demonstrated a significant decrease in GFAP mRNA and protein levels in ALF patients compared to controls. While the loss of EAAT-2 protein in ALF samples was post-translational in nature, EAAT-1 protein remained within normal limits. Immunohistochemistry confirmed that, in all cases, the losses of EAAT-2 and GFAP were uniquely astrocytic in their localization. AQP-4 mRNA expression was significantly increased and its immunohistochemistry demonstrated increased AQP-4 immunoreactivity in the glial end-feet process surrounding the microvessels. These findings provide evidence of selective alterations in the expression of genes coding for key astrocytic proteins implicated in central nervous system (CNS) excitability and brain edema in human ALF. We investigated the gene expression of astrocytic proteins involved in astrocyte swelling causing brain edema in autopsied brain tissues of patients with acute liver failure. This study demonstrated loss of GFAP expression and up-regulation of AQP-4 protein expression leading to cerebral edema, and loss of EAAT-2 expression implicated in excitatory neurotransmission. These findings may provide new drug targets against CNS complications of acute liver failure.

Differences in virulence repertoire and cell invasive potential of group A Streptococcus emm1-2 in comparison to emm1 genotype.

Group A streptococcus (GAS, Streptococcus pyogenes) type emm1 is widely associated with streptococcal invasive disease. This type is prevalent worldwide but is rare in India. Instead, emm1-2 type which is closely related to emm1 but is a distinct type is more prevalent. Although emm1 has been well characterized, information available on emm1-2 is rare. In this study we present a comparative study of both types. DNA microarray analysis showed segregation of emm1 and emm1-2 isolates into two distinct clusters. Out of 229 arrayed genes, 83-87% were present, 6-9% absent and 4-8% genes were ambiguous in emm1 isolates. emm1-2 strains harboured only 68-77%, 11-13% were absent and 10-20% ambiguous genes. Fourteen genes, present in all emm1, were completely absent in the emm1-2 isolates. sfb1 is a gene which encodes for Streptococcal fibronectin binding adhesin and invasin which has restricted distribution among different emm types of GAS. A variant of sfb1 (sfb1-2) was the only gene which was present in all emm1-2 isolates, but absent from all emm1 strains. Sfb1 and Sfb1-2 differ in sequences in the aromatic domain and the proline rich repeat region, whereas the fibronectin binding region was conserved and exhibited similar fibronectin binding activity. The presence of Sfb1-2 in emm1-2 strains was concomitant with significantly higher fibronectin-binding and invasion efficiency of HEp-2 cells when compared to emm1 isolates. The role of Sfb1-2 in invasion was confirmed by latex bead assay. emm1-2 isolates follow membrane ruffling mechanism during invasion and intracellularly follow classical endocytic pathway. Further studies are required to understand the correlation between the presence of emm1-2 isolates and the disease pattern in North India.

Levels of hepatitis B virus replicative intermediate in serum samples of chronic hepatitis B patients.

Hepatitis B virus (HBV) cccDNA levels is an absolute marker of HBV replication in the liver of HBV infected patients. This study aimed to quantify the HBV cccDNA levels in sera and liver tissue samples of treatment naïve patients with chronic hepatitis B. Eighty one chronic hepatitis B (CHB) treatment naïve patients were enrolled from January 2009 to June 2011. Total HBV DNA and HBV cccDNA levels were quantified using sensitive real time PCR assay. The mean age of recruited patients was 34 ± 11.5 years. Fifty four (66.7%) patients were HBeAg negative. Liver tissue samples were available from 2 HBeAg positive and 21 HBeAg negative CHB patients. The amount of total intrahepatic HBV DNA ranged from 0.09 to 1508.92 copies/cell. The median intrahepatic HBV cccDNA was 0.31 and 0.20 copies/cell in HBeAg positive and HBeAg negative cases, respectively. Serum HBV cccDNA was detectable in 85.2 % HBeAg positive and 48.1% HBeAg negative CHB patients. Median serum HBV cccDNA was 46,000 and 26,350 copies/mL in HBeAg positive and HBeAg negative subjects, respectively. There was a significant positive correlation between the levels of intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009). A positive correlation was also seen between serum HBV cccDNA levels and serum HBV DNA levels (r = 0.871, p < 0.001). It was concluded that serum HBV cccDNA could be detectable in higher proportion of HBeAg positive patients compared to HBeAg negative patients. Moreover, the median level of serum HBV cccDNA was significantly higher in HBeAg positive patients in contrast to HBeAg negative subjects.

Streptococcal pharyngitis, rheumatic fever and rheumatic heart disease: Eight-year prospective surveillance in Rupnagar district of Punjab, India.

BACKGROUND: Rheumatic fever (RF)/rheumatic heart disease (RHD) continue to be a neglected public health priority. We carried out a registry-based control project, prospective surveillance and sample surveys to estimate the burden of disease. METHODS: We trained healthcare providers and established a surveillance system for the 1.1 million population of Rupnagar district in Punjab. In sample surveys conducted among schools, physicians examined the sampled children. Children with a cardiac murmur were investigated by echocardiography. Throat swabs were obtained from a sub-sample, and group A streptococci (GAS) were identified and emm typed by standard laboratory methods. We estimated the morbidity rates for RF/RHD from surveillance data and school surveys using a correction factor to account for under-registration of cases in the registry. RESULTS: A total of 813 RF/RHD cases were registered from 2002 to 2009. Of the 203 RF and 610 RHD cases, respectively, 51.2% and 36.7% were males. In the age group of 5-14 years, RF was more common (80%) than RHD (27%). The prevalence of RF/RHD in 5-14-year-old students was 1.0/1000 (95% CI 0.8-1.3). The school survey indicated that about two-thirds of the RF/RHD cases were enrolled in the hospital-based registries. Based on the school survey, the prevalence of RF/RHD was estimated to be 143/100,000 population. In the registry, the annual incidence of acute RF was estimated to be at least 8.7/100 000 children in the age group of 5-14 years. The prevalence of GAS was 2% (13/656) in children with sore throat and 0.5% (14/2920) among those not having sore throat. Typing of 27 GAS revealed 16 emm types. We estimate that about 1000 episodes of GAS pharyngitis lead to one episode of acute RF. CONCLUSION: RF/RHD continue to be a public health problem in Punjab, India.

Overexpression of microRNA-122 enhances in vitro hepatic differentiation of fetal liver-derived stem/progenitor cells.

MicroRNAs (miRNAs) are a versatile class of tiny non-coding RNAs involved in regulation of various biological processes. miRNA-122 (miR-122) is specifically and abundantly expressed in human liver. However, the role of miR-122 in differentiation of fetal liver stem/progenitor cells into hepatocytes remains unclear. In this study, dual positive CD34+/CD117+ expressing human fetal liver stem/progenitor cells was enriched by magnetic cell sorting and cultured in vitro. The level of miR-122 was found to be increased at specific time intervals. Interestingly, during the differentiation process of hepatocyte-like cells, the increase in expression of miR-122 was positively correlated with expression of hepatocyte-specific genes. The status of differentiation process was improved by transfection of miR-122 into enriched stem/progenitor cells. The expression level of hepatic-specific genes as well as liver-enriched transcription factors (LETFs) was significantly increased by overexpression of miR-122 in fetal liver stem/progenitor cells. Thus, the study delineated the role of hepato-specific miR-122 in differentiation of fetal liver stem/progenitor cells into hepatocyte-like cells which could be used as a therapeutic target molecule to generate abundant hepatocytes.

Hepatitis B virus reverse transcriptase mutations in treatment Naïve chronic hepatitis B patients.

Mutations in the reverse transcriptase (RT) region of the hepatitis B virus (HBV) genome lead to decreased susceptibility to nucleos(t)ide analogs approved for treatment of HBV infection. The aim of this study was to detect and analyze pre-existing HBV RT mutations in treatment naïve patients with chronic hepatitis B. Seventy one chronic HBV treatment naïve patients were enrolled from January 2009 to June 2011. HBV RT sequence analysis was done by using direct bidirectional sequencing of semi-nested PCR products. HBV genotypes were determined by multiplex PCR. Genotype D was found in 64 patients (90.1%) followed by genotype C and A which were present in 5 (7.0%) and 2 (2.8%) patients respectively. The results of the RT sequence analysis showed mutations in 34 (47.9%) patients. The rtH248N mutation was the most common mutation, accounting for 47.1% patients. Other common mutations included rtD263E/S, rtM129L, rtF122L/V/I, rtS135Y/H, rtQ149K, rtL91I, rtH126R, rtC256S/G, rtY257W, rtS259T and rtE271D, which were present in 26.5% (9/34), 29.4% (10/34), 20.6% (7/34), 20.6% (7/34), 20.6% (7/34), 17.6% (6/34), 14.7% (5/34), 14.7% (5/34), 11.8% (4/34), 11.8% (4/34) and 11.8% (4/34) patients respectively. The known primary drug resistance mutations were found in 3 (8.8%) patients. The present study shows the presence of RT amino acid substitutions in treatment-naïve patients with chronic hepatitis B, which may decrease susceptibility to available oral antiviral drugs. On the basis of the finding of this study, genotypic testing is recommended before the start of therapy in naïve patients, so that suitable antiviral drugs can be prescribed.

Serum levels of angiogenic and anti-angiogenic factors: their prognostic relevance in locally advanced hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a prototype tumor wherein angiogenesis plays a vital role in its progression. The role of VEGF, a major angiogenic factor in HCC is known; however, the role of anti-angiogenic factors simultaneously with the angiogenic factors has not been studied before. Hence, in this study, the serum levels of major angiogenic [Vascular Endothelial Growth Factor (VEGF), angiopoietin-2 (Ang-2)] and anti-angiogenic (endostatin, angiostatin) factors were analyzed and correlated with clinico-radiological features and with outcome. A total of 150 patients (50 HCC, 50 cirrhosis and 50 chronic hepatitis) and 50 healthy controls were enrolled in this study. Serum levels of VEGF, Ang-2, endostatin, and angiostatin were estimated by enzyme-linked immunosorbent assay. HCC shows significantly elevated serum levels of angiogenic factors VEGF and Ang-2 and of anti-angiogenic factors endostatin and angiostatin. ROC curve analysis for serum VEGF yielded an optimal cut-off value of 225.14 pg/ml, with a sensitivity of 78 % and specificity of 84.7 % for a diagnosis of HCC and its distinction from other group. Using this value, the univariate and multivariate analysis revealed significantly poor outcome in patients with higher levels of serum VEGF (p = 0.009). Combinatorial analysis revealed that patients with higher levels of both angiogenic and anti-angiogenic factors showed poor outcome. Serum VEGF correlates with poor survival of HCC patients and, therefore, serves as a non-invasive biomarker of poor prognosis. Moreover, elevated levels of anti-angiogenic factors occur endogenously in HCC patients.

Angiogenic and anti-angiogenic factor gene transcript level quantitation by quantitative real time PCR in patients with hepatocellular carcinoma.

Tumor angiogenesis, a major requirement for tumor growth and metastasis, is regulated by pro- and anti-angiogenic factors. The aim of this study was to quantify the expression of angiogenic (VEGF, HIF-1α, Angiopiotein-2) and anti-angiogenic (endostatin, angiostatin and Thrombospondin-1) factors and to discern their clinical relevance. A total 90 patients (67 HCC, 9 cirrhosis and 14 chronic hepatitis) were enrolled in the study. Tissue transcript levels of angiogenic (VEGF, HIF-1α, Ang-2) and anti-angiogenic (endostatin, angiostatin and TSP-1) factors were analyzed by quantitative real time-polymerase chain reaction (qRT-PCR) in the tissue samples. The tissue transcript levels of VEGF, HIF-1α and endostatin were found to be significantly higher in HCC in comparison to cirrhosis and chronic hepatitis. Although Ang-2, angiostatin and TSP-1 tissue transcript levels were higher in HCC group than the others groups but the difference was not statistically significant. In univariate analysis both VEGF and HIF-1α were found to be associated with poor survival of HCC patients. Multivariate analysis by the cox proportional hazard model revealed only VEGF as an independent factor predicting poor survival of the HCC patients. Angiogenic and anti-angiogenic factors are all highly expressed in HCC patients. Upregulation of tissue anti-angiogenic factors indicates the urgency for the alternative of anti-angiogenic therapies.

Asp299Gly and Thr399Ile polymorphism of TLR-4 gene in patients with prostate cancer from North India.

BACKGROUND: The etiological factors associated with prostate cancer (CaP) have not been completely understood as yet. Genetic predisposition and inflammation is fast emerging as risk factors for CaP is a key player in the innate immune response and plays role in immune- surveillance and inflammation. The present study was conducted to evaluate TLR-4 gene polymorphism in patients with CaP. MATERIAL AND METHODS: DNA was isolated from blood samples of 198 patients with CaP, 200 cases of Benign Prostatic Hyperplasia (BPH) and 119 controls. TLR-4 gene polymorphisms Asp299Gly and Thr399Ile were determined by Restriction Fragment Length Polymorphism (RFLP) technique using Nco1 and Hinf 1 restriction enzymes. All statistical calculations were performed using SPSS for windows, version 13 (SPSS Inc., Chicago, Illinois, USA). RESULTS: A significantly high proportion of patients with CaP had AG genotype (16.6%) as compared to control (4.2%) [OR-4.4, 95% CI (1.57-13.26), P =0.0013] with respect to Asp299Gly single nucleotide polymorphism (SNP). AA genotype showed a protective effect towards CaP development [OR-0.39, 95% CI (0.18-0.83), P=0.007). A trend was observed towards development of BPH with respect to AG genotype (P=0.06). Thr399Ile SNP was not significantly different among the population groups studied. CONCLUSIONS: This finding highlights the genetic predispositions to CaP with respect to TLR-4 gene. Individuals with Asp299Gly polymorphism having AG genotype appear to have four fold higher risk for development of Prostate cancer.

Bacterial metabolite butyrate in modulating sorafenib-targeted microRNAs to curtail its resistance in hepatocellular carcinoma.

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.

Association of HLA-DRB1*14 with rheumatic heart disease patients from Chandigarh, North India.

CONTEXT: Acute rheumatic fever (ARF)/rheumatic heart disease (RHD) is a major cause of morbidity and mortality in India, yet, few studies are available on susceptibility markers. OBJECTIVE: To associate human leukocyte antigen (HLA) class II alleles with north Indian RHD patients as genetic susceptibility markers. MATERIALS AND METHODS: HLA alleles were analysed using sequence specific primer-polymerase chain reaction and nucleotide sequencing, while HLA-B27 expression by flowcytometry. RESULTS: Few HLA-DQB1/DRB1 alleles were associated with RHD and HLA-DRB1*14 gene polymorphism revealed two single nucleotide polymorphisms (SNPs) in patients. Bioinformatic predictions showed influence of SNPs on protein function. HLA-B27 was positive in 42.85% ARF patients. CONCLUSION: The study showed association of different HLA class II alleles with RHD in North Indian population.

Antimicrobial Susceptibility Pattern and Serotype Distribution of Streptococcus pneumoniae Isolates From a Hospital-Based Study in Chandigarh, North India.

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.

miR-23b-3p Modulating Cytoprotective Autophagy and Glutamine Addiction in Sorafenib Resistant HepG2, a Hepatocellular Carcinoma Cell Line.

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.