Intracellular ROS scavenging and antioxidant regulation of WL15 from cysteine and glycine-rich protein 2 demonstrated in zebrafish in vivo model.

Antioxidant peptides are naturally present in food, especially in fishes, and are considered to contain rich source of various bioactive compounds that are structurally heterogeneous. This study aims to identify and characterize the antioxidant property of the WL15 peptide, derived from Cysteine and glycine-rich protein 2 (CSRP2) identified from the transcriptome of a freshwater food fish, Channa striatus. C. striatus is already studied to contain high levels of amino acids and fatty acids, besides traditionally known for its pharmacological benefits in the Southeast Asian region. In our study, in vitro analysis of WL15 peptide exhibited strong free radical scavenging activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), superoxide anion radical and hydrogen peroxide (H2O2) scavenging assay. Further, to evaluate the cytotoxicity and dose-response, the Human dermal fibroblast (HDF) cells were used. Results showed that the treatment of HDF cells with varying concentrations (10, 20, 30, 40 and 50 µM) of WL15 peptide was not cytotoxic. However, the treatment concentrations showed enhanced antioxidant properties by significantly inhibiting the levels of free radicals. For in vivo assessment, we have used zebrafish larvae for evaluating the developmental toxicity and for determining the antioxidant property of the WL15 peptide. Zebrafish embryos were treated with the WL15 peptide from 4 h of post-fertilization (hpf) to 96 hpf covering the embryo-larval developmental period. At the end of the exposure period, the larvae were exposed to H2O2 (1 mM) for inducing generic oxidative stress. The exposure of WL15 peptide during the embryo-larval period showed no developmental toxicity even in higher concentrations of the peptide. Besides, the WL15 peptide considerably decreased the intracellular reactive oxygen species (ROS) levels induced by H2O2 exposure. WL15 peptide also inhibited the H2O2-induced caspase 3-dependent apoptotic response in zebrafish larvae was observed using the whole-mount immunofluorescence staining. Overall results from our study showed that the pre-treatment of WL15 (50 µM) in the H2O2-exposed zebrafish larvae, attenuated the expression of activated caspase 3 expressions, reduced Malondialdehyde (MDA) levels, and enhanced antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT). The gene expression of antioxidant enzymes such as glutathione S-transferase (GST), glutathione peroxide (GPx) and γ-glutamyl cysteine synthetase (GCS) was found to be upregulated. In conclusion, it can be conceived that pre-treatment with WL15 could mitigate H2O2-induced oxidative injury by elevating the activity and expression of antioxidant enzymes, thereby decreasing MDA levels and cellular apoptosis by enhancing the antioxidant response, demonstrated by the in vitro and in vivo experiments.

Context-Dependent Regulation of Nrf2/ARE Axis on Vascular Cell Function during Hyperglycemic Condition.

Diabetes mellitus is associated with an increased risk of micro and macrovascular complications. During hyperglycemic conditions, endothelial cells and vascular smooth muscle cells are exquisitely sensitive to high glucose. This high glucose-induced sustained reactive oxygen species production leads to redox imbalance, which is associated with endothelial dysfunction and vascular wall remodeling. Nrf2, a redox-regulated transcription factor plays a key role in the antioxidant response element (ARE)-mediated expression of antioxidant genes. Although accumulating data indicate the molecular mechanisms underpinning the Nrf2 regulated redox balance, understanding the influence of the Nrf2/ARE axis during hyperglycemic condition on vascular cells is paramount. This review focuses on the context-dependent role of Nrf2/ARE signaling on vascular endothelial and smooth muscle cell function during hyperglycemic conditions. This review also highlights improving the Nrf2 system in vascular tissues, which could be a potential therapeutic strategy for vascular dysfunction.

Chronic intake of 4-Methylimidazole induces Hyperinsulinemia and Hypoglycaemia via Pancreatic Beta Cell Hyperplasia and Glucose Dyshomeostasis.

Caramel colours are the preferential food colouring agent globally, reaches wide age groups through eatables. Colas, a sweetened carbonated drink are most common caramel coloured beverage and its consumption is linked with diabetes, obesity, pancreatic cancer and other endocrine disorders. A major by-product produced during caramelization is 4-methylimidazole (4-MEI) that is detected in noteworthy concentrations in colas and other beverages. Previous studies revealed the neurotoxic and carcinogenic potential of 4-MEI in animals at higher doses but the effect of 4-MEI at theoretical maximum daily intake dose on glucose homeostasis is unexplored. Here, mice treated with 4-MEI (32 µg/kg bodyweight/day) for seven weeks exhibited severe hypoglycaemia and hyperinsulinemia mediated by hyperplasia of pancreatic beta cells and induces metabolic alterations. On combinatorial treatment, 4-MEI suppressed the glucogenic potential of non-artificial sweeteners and promotes lipogenesis. Furthermore, increased levels of C-peptide, LDL-cholesterol and triglycerides were observed in the humans with regular intake of 4-MEI containing beverages. In summary, 4-MEI induced pancreatic beta cell hyperplasia and leads to disruption of glucose and lipid homeostasis. This study suggests the need for further assessment and reconsideration of the wide usage of 4-MEI containing caramels as food additives.

Gut microbial degradation of organophosphate insecticides-induces glucose intolerance via gluconeogenesis.

BACKGROUND: Organophosphates are the most frequently and largely applied insecticide in the world due to their biodegradable nature. Gut microbes were shown to degrade organophosphates and cause intestinal dysfunction. The diabetogenic nature of organophosphates was recently reported but the underlying molecular mechanism is unclear. We aimed to understand the role of gut microbiota in organophosphate-induced hyperglycemia and to unravel the molecular mechanism behind this process. RESULTS: Here we demonstrate a high prevalence of diabetes among people directly exposed to organophosphates in rural India (n = 3080). Correlation and linear regression analysis reveal a strong association between plasma organophosphate residues and HbA1c but no association with acetylcholine esterase was noticed. Chronic treatment of mice with organophosphate for 180 days confirms the induction of glucose intolerance with no significant change in acetylcholine esterase. Further fecal transplantation and culture transplantation experiments confirm the involvement of gut microbiota in organophosphate-induced glucose intolerance. Intestinal metatranscriptomic and host metabolomic analyses reveal that gut microbial organophosphate degradation produces short chain fatty acids like acetic acid, which induces gluconeogenesis and thereby accounts for glucose intolerance. Plasma organophosphate residues are positively correlated with fecal esterase activity and acetate level of human diabetes. CONCLUSION: Collectively, our results implicate gluconeogenesis as the key mechanism behind organophosphate-induced hyperglycemia, mediated by the organophosphate-degrading potential of gut microbiota. This study reveals the gut microbiome-mediated diabetogenic nature of organophosphates and hence that the usage of these insecticides should be reconsidered.

Abundant and Altered Expression of PIWI-Interacting RNAs during Cardiac Hypertrophy.

BACKGROUND: The discovery of PIWI-interacting RNAs (piRNAs) has fundamentally changed our understanding of post transcriptional regulation of transposons and other genes. Unlike miRNA and siRNA, the piRNAs are the most abundant but least studied RNA species in mammals. Although the expression of PIWI proteins and piRNAs has long been regarded as germline specific, increasing evidences suggest the expression of piRNAs in somatic cells. METHODS: In this study, the small RNA sequencing executed during induction of cardiac hypertrophy in both in vivo and in vitro conditions were annotated for the expression of piRNAs. The expression of piRNAs was validated by qPCR and RNA immunoprecipitation. In addition, the presence of piRNAs in circulation of myocardial infarction patients was studied by qPCR. RESULTS: We identified an abundant and altered expression of piRNAs during cardiac hypertrophy. The differentially expressed piRNAs was validated by qPCR and RNA immunoprecipitation. The significantly and differentially expressed piRNAs were predicted to target different retrotransposons and mRNAs in the rat genome. The detection of specific piRNA in serum of myocardial infarction patients suggests the potential of piRNA for diagnosis. CONCLUSION: Overall this study is the first to provide a whole-genome analysis of the large repertoire of piRNAs in the cardiac system and this would pave a new path to understanding the molecular aetiology of piRNA and retrotransposons in the physiology and pathology of the cardiac system.

PCDH10 promoter hypermethylation is frequent in most histologic subtypes of mature lymphoid malignancies and occurs early in lymphomagenesis.

PCDH10 is epigenetically inactivated in multiple tumor types; however, studies in mature lymphoid malignancies are limited. Here, we have investigated the presence of promoter hypermethylation of the PCDH10 gene in a large cohort of well-characterized subsets of lymphomas. PCDH10 promoter hypermethylation was identified by methylation-specific PCR in 57 to 100% of both primary B- and T-cell lymphoma specimens and cell lines. These findings were further validated by Sequenom Mass-array analysis. Promoter hypermethylation was also identified in 28.6% cases of reactive follicular hyperplasia, more commonly occurring in states of immune deregulation and associated with rare presence of clonal karyotypic aberrations, suggesting that PCDH10 methylation occurs early in lymphomagenesis. PCDH10 expression was down regulated via promoter hypermethylation in T- and B-cell lymphoma cell lines. The transcriptional down-regulation resulting from PCDH10 methylation could be restored by pharmacologic inhibition of DNA methyltransferases in cell lines. Both T- and B-cell lymphoma cell lines harboring methylation-mediated inactivation of PCDH10 were resistant to doxorubicin treatment, suggesting that hypermethylation of this gene might contribute to chemotherapy response.

Distinct involvement of 9p21-24 and 13q14.1-14.3 chromosomal regions in raw betel-nut induced esophageal cancers in the state of Meghalaya, India.

BACKGROUND: Raw betel nut (RBN) chewing is an important contributing factor for esophageal squamous cell carcinoma (ESCC), although associated genomic changes remain unclear. One difficulty in assessing the effects of exclusively RBN induced genetic alterations has been that earlier studies were performed with samples of patients commonly using tobacco and alcohol, in addition to betel-quid. Both CDKN2A (at 9p21) and Rb1 gene (at 13q14.2) are regarded as tumor suppressors involved in the development of ESCC. Therefore, the present study aimed to verify the RBN's ability to induce ESCC and assess the involvement of CDKN2A and Rb1 genes. METHODS: A panel of dinucelotide polymorphic markers were chosen for loss of heterozygosity studies in 93 samples of which 34 were collected from patients with only RBN-chewing habit. Promoter hypermethylation was also investigated. RESULTS: Loss in microsatellite markers D9S1748 and D9S1749, located close to exon 1ß of CDKN2A/ARF gene at 9p21, was noted in 40% ESCC samples with the habit of RBN-chewing alone. Involvement of a novel site in the 9p23 region was also observed. Promoter hypermethylation of CDKN2A gene in the samples with the habit of only RBN-chewing alone was significantly higher (p=0.01) than Rb1 gene, also from the samples having the habit of use both RBN and tobacco (p=0.047). CONCLUSIONS: The data indicate that the disruption of 9p21 where CDKN2A gene resides, is the most frequent critical genetic event in RBN-associated carcinogenesis. The involvement of 9p23 as well as 13q14.2 could be required in later stages in RBN-mediated carcinogenesis.

Effect of coordinated ligands on antiproliferative activity and DNA cleavage property of three mononuclear Cu(II)-terpyridine complexes.

Three mononuclear copper(II) complexes, [Cu(tpy)Cl(2)] 1, [Cu(tpy)(NO(3))(2)(H(2)O)] 2 and [Cu(Ptpy)Cl(2)]·H(2)O·HCl 3 have been synthesised and characterized by various spectroscopic techniques and single crystal X-ray diffraction. Complexes 1 and 3 have five coordinate geometry in solid state, whereas complex 2 has six coordinate geometry. Mass spectral and EPR evidence suggest that in solution all the three complexes exist predominantly as a four coordinate species. Molecular modelling and DNA cleavage studies indicate that complexes 1 and 2 are DNA minor groove binders, whereas 3 is an intercalator. All the three complexes show nuclease activity in the presence of hydrogen peroxide. The three complexes have been found to be cytotoxic towards A549 lung adenocarcinoma cells.

Promoter methylation-mediated inactivation of PCDH10 in acute lymphoblastic leukemia contributes to chemotherapy resistance.

PCDH10 has been implicated as a tumor suppressor, since epigenetic alterations of this gene have been noted in multiple tumor types. However, to date, studies regarding its role in acute and chronic leukemias are lacking. Here, we have investigated the presence of promoter hypermethylation of two CpG islands of the PCDH10 gene by methylation-specific PCR in 215 cases of various subsets of myeloid- and lymphoid-lineage leukemias. We found that PCDH10 promoter hypermethylation was frequent in both B-cell (81.9%) and T-cell (80%) acute lymphoblastic leukemia (ALL), while it was present in low frequency in most subtypes of myeloid leukemias (25.9%) and rare in chronic myeloid leukemia (2.2%). PCDH10 expression was downregulated via promoter hypermethylation in primary ALL samples (N = 4) and leukemia cell lines (N = 11). The transcriptional repression caused by PCDH10 methylation could be restored by pharmacologic inhibition of DNA methyltransferases. ALL cell lines harboring methylation-mediated inactivation of PCDH10 were less sensitive to commonly used leukemia-specific drugs suggesting that PCDH10 methylation might serve as a biomarker of chemotherapy response. Our results demonstrate that PCDH10 is a target of epigenetic silencing in ALL, a phenomenon that may impact lymphoid-lineage leukemogenesis, serve as an indicator of drug resistance and may also have potential implications for targeted epigenetic therapy.

Anomalous behavior of pentacoordinate copper complexes of dimethylphenanthroline and derivatives of terpyridine ligands: Studies on DNA binding, cleavage and apoptotic activity.

Copper(II) complexes with substituted terpyridine ligands, namely [Cu(itpy)(dmp)](NO3)2 (1) and [Cu(ptpy)(dmp)](NO3)2 (2) have been synthesized and characterized. The interaction of the complexes with CT-DNA has been explored using spectroscopic techniques and viscosity. Complexes 1 and 2 bind in the grooves of DNA, interestingly 1 in the minor and 2 in the major groove. Both the complexes have been found to promote DNA cleavage; complex 1 through hydrolytic and 2 oxidative. Complexes 1 and 2 have been found to be cytotoxic and bring about apoptosis of human lung cancer cell line A549.