Mechanisms of chronic alcohol exposure-induced aggressiveness in cellular model of HCC and recovery after alcohol withdrawal.

Alcohol-related liver disease is the most prevalent chronic liver disease worldwide, accounting for 30% of hepatocellular carcinoma (HCC) cases and HCC-specific deaths. However, the knowledge on mechanisms by which alcohol consumption leads to cancer progression and its aggressiveness is limited. Better understanding of the clinical features and the mechanisms of alcohol-induced HCC are of critical importance for prevention and the development of novel treatments. Early stage Huh-7 and advanced SNU449 liver cancer cell lines were subjected to chronic alcohol exposure (CAE), at different doses for 6 months followed by 1-month alcohol withdrawal period. ADH activity and ALDH expression were much lower in SNU449 compared with Huh-7 cells and at the 270 mM dose, CAE decreased cell viability by about 50% and 80%, respectively, in Huh-7 and SNU449 cells but induced mortality only in Huh-7 cells. Thus, Huh-7 may be more vulnerable to ethanol toxicity because of the higher levels of acetaldehyde. CAE induced a dose-dependent increase in cell migration and invasion and also in the expression of cancer stem cells markers (CD133, CD44, CD90). CAE in Huh-7 cells selectively activated ERK1/2 and inhibited GSK3ß signaling pathways. Most of the changes induced by CAE were reversed after alcohol withdrawal. Interestingly, we confirmed the increase in CD133 mRNA levels in the tumoral tissue of patients with ethanol-related HCC compared to other HCC etiologies. Our results may explain the benefits observed in epidemiological studies showing a significant increase of overall survival in abstinent compared with non-abstinent patients.

Novel genetic polymorphisms identified in the clinical isolates of Mycobacterium tuberculosis PE_PGRS33 gene modulate cytokines expression and promotes survival in macrophages.

OBJECTIVE: PE_PGRS33 is a member of multi-gene family restricted to pathogenic Mycobacteria and their functions remain elusive. PE_PGRS33 is highly polymorphic in nature to evade host's immune response. Therefore, we investigated PE_PGRS33 gene polymorphisms in clinical isolates and functional characterization using in vitro experiments. METHODS: A total of 103 clinical isolates were recruited. Genomic DNA was extracted, PE_PGRS33 gene amplification, sequencing. Afterward, we have cloned, expressed PE_PGRS33 wild type and three polymorphic alleles in M. smegmatis. Further, performed in vitro stresses assays, THP-1 differentiated macrophage infection assays followed by quantification of cytokine expression. RESULTS: We have identified nine novel polymorphisms and also demonstrated that in comparison to M. smegmatis expressing wild type/Mut_alleles displayed altered in growth kinetics and colony morphology. M. smegmatis harbouring Mut_allele3 survived better under oxidative, acidic stress and were resistant to lysozyme treatment. qRT-PCR of cytokines TNF-α, IL-12 and IL-10 after infection with recombinant M. smegmatis showed two Mut_allele (Mut_allele1 and Mut_allele3) induced higher expression of TNF-α, IL-12, while IL-10 expression was decreased in both mutant alleles as compared to wild type PE_PGRS33 at each experimental time point. CONCLUSION: Results of our functional study suggest that PE_PGRS33 gene polymorphisms aid in the survival or persistence of M. tuberculosis and differentially modulate the expression of various cytokines. Overall this study suggests that Mtb clinical strains harbouring different PE_PGRS33 alleles could act as a virulence determinant by differentially regulating pathways essential for the pathogen's ability to adapt inside the host.

VEGFA Promoter Polymorphisms rs699947 and rs35569394 Are Associated With the Risk of Anterior Cruciate Ligament Ruptures Among Indian Athletes: A Cross-sectional Study.

BACKGROUND: Associations of genetic variants within certain fibril-forming genes have previously been observed with anterior cruciate ligament (ACL) injuries. Evidence suggests a significant role of angiogenesis-associated cytokines in remodeling the ligament fibril matrix after mechanical loading and maintaining structural and functional integrity of the ligament. Functional polymorphisms within the vascular endothelial growth factor A (VEGFA) gene have emerged as plausible candidates owing to their role in the regulation of angiogenic responses. HYPOTHESIS: VEGFA promoter polymorphisms rs699947 and rs35569394 are associated with ACL injury risk among athletes. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: A total of 90 Indian athletes with radiologically confirmed or surgically proven isolated ACL tears and 76 matched-control athletes were selected for the present cross-sectional genetic association study. Oral mouthwash samples were collected from all the case and control athletes and genotyped for VEGFA rs699947 and rs35569394 using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: The A allele (rs699947) was significantly overrepresented in the ACL group (C vs A allele: odds ratio [OR], 1.68 [95% CI, 1.08-2.60]; P = .021) (CC vs CA + AA: OR, 2.69 [95% CI, 1.37-5.26]; P = .004). There was a greater frequency of the AA genotype in the ACL group in comparison with the control group (OR, 3.38 [95% CI, 1.23-9.28]; P = .016) when only male athletes were compared. Likewise, there was a greater frequency of the I allele (rs35569394) in the ACL group (D vs I allele: OR, 1.64 [95% CI, 1.06-2.55]; P = .025) (DD vs ID + II: OR, 2.61 [95% CI, 1.31-5.21]; P = .006). The A-I haplotype was overrepresented in the ACL group compared with the control group (OR, 1.68 [95% CI, 1.08-2.60]; χ2 = 5.320; P = .021), and both the polymorphisms were found to be in complete linkage disequilibrium (r 2 = 0.929; logarithm of the odds score = 63.74; D' = 1.0). Female athletes did not show any difference in genotype or allele frequency. CONCLUSION: This is the first study to investigate the association of VEGFA promoter polymorphisms in ACL tears among Indian athletes. Increased frequencies of the A allele (rs699947) and I allele (rs35569394) were observed in the ACL group. These results suggest that sequence variants in the VEGF gene are associated with ACL injury risk among athletes. Further research with long-term follow-ups measuring VEGF expression levels during recovery is warranted to establish its role in ACL injuries and healing.

COLIA1 + 1245 G > T Sp1 Binding Site Polymorphism is Not Associated with ACL Injury Risks Among Indian Athletes.

OBJECTIVE: Type I collagen polypeptides contribute significantly to the structural composition of ligament tissue matrix. Since anterior cruciate ligament (ACL) tears account for roughly 50% of all knee injuries in sports, the objective of the study was to investigate association of Sp1-transcription factor binding site polymorphism COLIA1 Sp1 + 1245 G > T with ACL injury risk among Indian athletes. METHODS: A total of 166 athletes (90 with ACL tears and 76 as control) were recruited and were genotyped for COLIA1 Sp1 + 1245 G > T polymorphism using allele-specific PCR (AS-PCR) method. RESULT: Both the groups were matched for nature of sports, training regimen, and other demographic characteristics. We observed no significant difference between ACL cases and control group in GT or TT genotype frequency distribution (p = 0.967) and T-allele frequency distribution (p = 0.861) for COLIA1 Sp1 + 1245 G > T polymorphism. Also, the three models of inheritance of minor allele failed to show any statistical significance in the present study. CONCLUSION: COLIA1 Sp1 + 1245 G > T polymorphism has been studied in relation to many connective tissue pathologies. This is probably the first study to investigate the association of collagen protein genes with ACL injury risk on Indian athletes. Further studies with more SNPs in genes encoding fibril-forming collagen and large sample sizes are necessary to fully understand the genetic link to ACL injuries among athletes.

Local Activation of the Alternative Pathway of Complement System in Mycotic Keratitis Patient Tear.

Aspergillus flavus and Fusarium solani are the predominant causative agents of mycotic keratitis in the tropical part of the world. Tear proteins play a major role in the innate immune response against these fungal infections as has been shown by the presence of complement proteins and neutrophil extracellular trap proteins in keratitis patients tear. In this study, we established the presence of the components of the alternate pathway of complement system and their functional state in the tear film of mycotic keratitis patients. The complement proteins namely, C3 and CFH were found only in the open-eye tear of patients but not in control individuals. In vitro analysis showed binding of purified C3b and CFH to fungal spores, which confirmed that the spores can provide a foreign surface for forming the complement complex. Analysis of spore bound tear proteins by mass spectrometry exhibited the presence of known proteins of the alternate pathway complement cascade in keratitis patient tear. Hemolytic assay using rabbit RBC confirmed the presence of a functional alternate pathway of complement cascade in the tear proteome of the patients. The presence of negative regulators, CFH and CFI, in the patient tear indicate that the complement activity is tightly regulated during fungal infection. Mass spectrometry data show vitronectin and clusterin, two known inhibitors of the membrane attack complex only in the patient tear. These data demonstrate the activation of the alternate pathway of complement cascade during the early stages of infection. Interestingly, the production of multiple negative regulators of complement cascade implies the pathogen can effectively evade the host complement system during infection.

Dataset for the spore surface proteome and hydrophobin A/RodA proteoforms of A.flavus.

Fungal keratitis is a major sight-threatening corneal infection: and mycotic keratitis is more common in tropical parts of the world including India. Aspergillus flavus and Fusarium are the predominant causative agents of corneal infection. We extracted conidial surface proteins of A. flavus from saprophyte and clinical isolates and analyzed the proteins using high resolution mass spectrometry. The data revealed ecotype specific alteration in surface proteome since the proteome profile of the clinical isolates and saprophyte showed significant differences. Detailed examination of the mass spec data of RodA proteins extracted from polyacrylamide gels revealed the presence of two proteoforms of this protein. We also identified the mechanism of formation of these two isoforms. Detailed analysis of this data and the conclusions derived are described in the article, "Identification of the proteoforms of surface localized Rod A of A. flavus and determination of the mechanism of proteoform generation" [1].

Spoligotyping, phenotypic and genotypic characterization of katG, rpoB gene of M. tuberculosis isolates from Sahariya tribe of Madhya Pradesh India.

BACKGROUND: Sahariya, a primitive tribal group, residing in Gwalior and Sheopur districts of Madhya Pradesh, India, show high incidence and prevalence of pulmonary tuberculosis (PTB). The study was designed to understand the genetic diversity and phenotype - genotype association of drug resistant M. tuberculosis strains, infecting Sahariya tribe. MATERIALS AND METHODS: A total of 103 pulmonary tuberculosis patients from Sahariya tribe were recruited from Gwalior and Sheopur districts. Sputum samples were collected and cultured on LJ slants and drug sensitivity tests were carried out. Genomic DNA was extracted, followed by spoligotyping and genotyping of drug target genes, katG and rpoB, using MAS-PCR, PCR-RFLP and DNA sequencing. RESULT: Seventeen different spoligotypes were identified, in which, EAI3_IND/ST11 M. tuberculosis strain appeared predominant, followed by CAS1_Delhi/ST26. Results of our phenotypic drug susceptibility test identified high incidence (12.6%) of isoniazid-resistant tuberculosis, while 4.85% isolates were multi drug resistant (MDR). Further genotyping of drug target genes identified 8.7% of isoniazid-R isolates to have a mutation at katG codon 463, while 3.8% isolates showed mutations at two sites, katG codons 315 and 463. In case of MDR-TB isolates, all from CAS lineage, 3.85% had mutations on katG and rpoB genes, at codon 463 and codon 526, respectively, while 0.97% isolates were harbouring mutations at codons 315, 463 and 531. CONCLUSION: Our findings have revealed that EAI3_IND strain is the predominant strain infecting Sahariya. The incidence of isoniazid-R M. tuberculosis strain infection is high, with an increased propensity to evolve into MDR-TB. Therefore, the TB centres should also consider isoniazid-R status of the isolates along with CBNAAT before deciding the drug regimen for the patients.

Identification of the proteoforms of surface localized Rod A of Aspergillus flavus and determination of the mechanism of proteoform generation.

Fungal keratitis is a serious, potentially sight-threatening corneal infection that is more prevalent in the tropical parts of the world including India, and A. flavus and Fusarium solani are the predominant etiological agents. The surface of fungal conidia is covered by hydrophobin family proteins, effectively masking the conidial antigens from immune cells. In this study, we report that the outer cell wall layer of A. flavus conidia contain Rod A as well as other hydrophobins, which could be extracted by formic acid. Analysis of these surface proteins by mass spectrometry showed the presence of rodlet forming hydrophobins and other membrane and antigenic proteins. Our analysis revealed that Rod A existed as two proteoforms on the conidial surface. These proteoforms were separated using polyacrylamide gel electrophoresis and the amino acid sequence of these proteoforms was determined by high resolution mass spectrometry. PCR analysis of the mRNA encoding the Rod A showed the retention of intron one, which results in the formation of a truncated proteoform two. This is the first report in which the presence of RodA and its proteoforms and their mechanism of formation has been demonstrated in the corneal pathogenic fungus A. flavus. SIGNIFICANCE: A. flavus is a common fungal pathogen in tropical countries playing a predominant role in causing mycotic keratitis in humans. Surface of fungal conidia is immunologically inert primarily due to the hydrophobin family proteins forming a rodlet layer and masking the conidia from immune cells. In this study we demonstrated the existence two proteoforms of RodA/hydrophobin A and intron retention is shown to be responsible for the formation of one of the proteoforms. In addition, the spore surface proteins of A.flavus corneal isolates and saprophyte are distinctly different, which indicate the spore surface protein profile is ecotype specific. This is the first report showing the presence of two proteoforms of RodA on A.flavus conidial surface and demonstration of the mechanism of formation of the proteoforms.

Identification of SLAMF3 (CD229) as an inhibitor of hepatocellular carcinoma cell proliferation and tumour progression.

Although hepatocellular carcinoma (HCC) is one of the most common malignancies and constitutes the third leading cause of cancer-related deaths, the underlying molecular mechanisms are not fully understood. In the present study, we demonstrate for the first time that hepatocytes express signalling lymphocytic activation molecule family member 3 (SLAMF3/CD229) but not other SLAMF members. We provide evidence to show that SLAMF3 is involved in the control of hepatocyte proliferation and in hepatocellular carcinogenesis. SLAMF3 expression is significantly lower in primary human HCC samples and HCC cell lines than in human healthy primary hepatocytes. In HCC cell lines, the restoration of high levels of SLAMF3 expression inhibited cell proliferation and migration and enhanced apoptosis. Furthermore, SLAMF3 expression was associated with inhibition of HCC xenograft progression in the nude mouse model. The restoration of SLAMF3 expression levels also decreased the phosphorylation of MAPK ERK1/2, JNK and mTOR. In samples from resected HCC patients, SLAMF3 expression levels were significantly lower in tumorous tissues than in peritumoral tissues. Our results identify SLAMF3 as a specific marker of normal hepatocytes and provide evidence for its potential role in the control of proliferation of HCC cells.