Prasant Kumar Jena

Layered inorganic nanocomposites: a promising carrier for 5-fluorouracil (5-FU).

We report here the intercalation of 5-fluorouracil (5-FU), an anticancer drug in interlayer gallery of Na(+) clay (Montmorillonite, MMT), with the assistance of biopolymer (chitosan, CS). The X-ray diffraction patterns, thermal and spectroscopic analyses indicated the drug intercalation into the clay interlayer space in support of CS and stabilized in the longitudinal monolayer by electrostatic interaction. In vitro drug release showed controlled release pattern. The genotoxic effect of drug was in vitro evaluated in human lymphocyte cell culture by comet assay, and results indicated significant reduction in DNA damage when drug was intercalated with clay and formulated in composites. The results of in vitro cell viability assay in cancer cells pointed at decreased toxicity of drug when encapsulated in Na(+)-clay plates than the pristine drug. In vivo pharmacokinetics, biodistribution, hepatotoxicity markers, e.g., SGPT and SGOT, and liver/testicular histology in rats showed plasma/tissue drug levels were within therapeutic window as compared to pristine drug. Therefore, drug-clay hybrid and composites can be of considerable value in chemotherapy of cancer with reduced side effects.

Understanding and modulating the Toll like Receptors (TLRs) and NOD like Receptors (NLRs) cross talk in type 2 diabetes.

Obesity and Type 2 diabetes are leading health problems which are characterized by low-grade inflammation with an increase in inflammatory cytokines along with the change in the gut microbiota population. Toll like Receptors (TLRs) and NOD like Receptors (NLRs) are very prominent pathogen recognition receptors, which play a significant role in the innate immune system. Both TLRs and NLRs pathways are mediated through different adaptor proteins; commonly found to activate the NF-kB, which induces the expression of proinflammatory cytokines. It has been suggested that TLRs and NLRs have a significant role in the pathogenesis of inflammation mediated insulin resistance, which further develops metabolic complications. TLRs mediated mechanism for insulin resistance involves activation through TLR ligands such as increased free fatty acids and lipid derivatives from adipocytes as well as the skeletal muscles. Moreover, gut microbiota alteration in the type 2 diabetes also plays a key role by increasing the plasma LPS levels, which specifically activates TLR4 and provokes the inflammation mediated insulin resistance. NOD1 and NOD2 are involved in the pathogenesis of diabetes, possibly through the recognition of the gut microbiota. Gut microbiota modulation by antibiotics plays a crucial role in increasing insulin sensitivity, possibly through the TLRs and NLRs mediated signaling responses, which suggest future therapeutic approaches for obesity, insulin resistance and type 2 diabetes. In this review, we focused on the interdependent role of TLRs and NLRs in metabolic diseases and their cross talk for the pathogenesis of inflammatory diseases.

Purification and partial characterization of a detergent and oxidizing agent stable alkaline protease from a newly isolated Bacillus subtilis VSG-4 of tropical soil

An extracellular detergent tolerant protease producing strain VSG-4 was isolated from tropical soil sample and identified as Bacillus subtilis based on morphological, biochemical characteristics as well as 16S-rRNA gene sequencing. The VSG-4 protease was purified to homogeneity using ammonium sulphate precipitation, dialysis and sephadex G-200 gel permeation chromatography with a 17.4 purification fold. The purified enzyme was active and stable over a broad range of pH (8.0–11.0, optimum at 9.0) and temperature (40°C to 60°C, optimum at 50°C). The thermostability of the enzyme was significantly increased by the addition CaCl2. This enzyme was strongly inhibited by PMSF and DFP, suggesting that it belongs to the serine protease superfamily. The purified VSG-4 alkaline protease showed remarkable stability in anionic (5 mM SDS) and ionic (1% Trion X-100 and 1% Tween 80) detergents. It retained 97±2% and 83.6±1.1% of its initial activity after 1 h preincubation in the presence of 1 % H2O2 and 1 % sodium perborate, respectively. Furthermore, the purified enzyme showed excellent stability and compatibility with some commercial laundry detergents besides its stain removal capacity. Considering these promising properties, VSG-4 protease may find tremendous application in laundry detergent formulations.

Isolation and characterization of probiotic properties of Lactobacilli isolated from rat fecal microbiota.

The objective of the present study was to characterize lactobacilli isolates from the feces of male Wistar rats. Various physiological features of the candidate probiotic isolates were preliminarily investigated, including tolerance to simulated gastric juice and bile salts, antimicrobial activity, antibiotic susceptibility and in vitro aggregation. Based on their morphological and biochemical characteristics, four potential probiotic isolates (CS2, CS3, CS4, and CS7) were screened. The isolates showed good tolerance to stimulated gastric juice and bile salts. CS4 and CS7 exhibited strong antibacterial activities against the pathogens tested as assessed in neutral pH culture supernatants. All lactobacilli isolates were susceptible to all the tested antibiotics, except vancomycin. Moreover, the isolate CS4 and CS7 were found to possess stronger cell surface traits such as hydrophobicity, auto‐aggregation and co‐aggregation capacity. In addition, CS4 and CS7 had greater β‐galactosidase activities than the others. Biochemical tests and 16S rRNA gene sequencing confirmed that CS2, CS3, CS4 and CS7 are Lactobacillus intestinalis PJ2, L. sakei PJ3, L. helveticus PJ4, and L. plantarum PJ7, respectively. Based on the obtained results, L. helveticus PJ4 and L. plantarum PJ7 are ideal in vitro probiotic candidates and require further in vivo evaluation.

Understanding the Role of Heat Shock Protein Isoforms in Male Fertility, Aging and Apoptosis

Heat shock proteins (HSPs) play a role in the homeostasis, apoptosis regulation and the maintenance of the various other physiological processes. Aging is accompanied by a decrease in the resistance to environmental stress, while mitochondria are primary targets in the process of aging, their expression decreasing with age. Mitochondrion also plays a significant role in the process of spermatogenesis. HSPs have been shown to be involved in apoptosis with some of acting as apoptotic inhibitors and are involved in cytoprotection. In this review we discuss the roles of Hsp 27, 60, 70, and 90 in aging and male infertility and have concluded that these particular HSPs can be used as a molecular markers for mitochondrially- mediated apoptosis, aging and male infertility.

Evaluation of Hydro-Alcoholic Extract of Eclipta alba for its Multidrug Resistance Reversal Potential: An In Vitro Study

The development of multidrug resistance (MDR) causes problems in the chemotherapy of human cancer. The present study was designed to evaluate and establish the role of Eclipta alba as MDR reversal agent using multidrug resistant hepatocellular carcinoma cell line (DR-HepG2). To develop DR-HepG2, hepatocellular carcinoma cell line (HepG2) was transfected with 2-Acetylaminofluorene (AAF) and Aflatoxin B1 (AFB). Cytotoxic effects of the Eclipta alba hydroalcoholic extract (EAE) and standard anti-ancer drug Doxorubicin (DOX) were determined in DR-HepG2 and the parental cells HepG2 using MTT assay. The expression level of MDR1 gene and P-glycoprotein (P-gp) level was analyzed by RT-PCR and western blotting. From the present investigation, it was found that EAE (10 and 20 μg/ml) could significantly inhibit cell proliferation in DR-HepG2 whereas DOX (0.5 μg/ml) could not because of enhancement effect of MDR1/P-gp. This study demonstrated for the first time the antiproliferative activities of EAE in multidrug resistant DR-HepG2 cells. The findings revealed that Eclipta alba components are effective inhibitors of MDR1/P-gp.

Influence of Gut Microbiota on Inflammation and Pathogenesis of Sugar RichDiet Induced Diabetes

Type 2 diabetes is characterized by peripheral insulin resistance. Besides immune and inflammatory mechanisms, other pathways involve interaction between gut microbiota and metabolic syndrome. The present study was designed to understand gut microbiota alteration following High Sugar Diet (HSD) and its effect on physiology and gastrointestinal immunology. Male wistar rats were fed with high fructose and HSD for 60 days. Composition of fecal microbiota by DGGE and proinflammatory cytokines in serum was investigated. Expressions of genes such as TLR2, TLR4 and NF-kB in various tissues were also studied. The bacteria coliforms and clostridium level were higher and Lactobacillus was lower in both sugar rich diet fed rats. Highly diverse and densely populated bands were observed in HSD group by DGGE fingerprint. The band profiles of sugar fed group have clustered together. Elevated mRNA expression of TLR2, TLR4, and NF-kB were observed in HSD groups. Increased inflammation was confirmed by blood and tissue biochemical assay and enhanced serum pro-inflammatory cytokines in HSD diet groups. Gut microbiota strongly influenced the metabolic profiling of individuals fed with high calorie intake. The diverse microbial population and increased coliforms and clostridium may affect host gene expression. Targeting TLRs and microbiota could be promising therapeutic approach

Investigation of Chitosan for Prevention of Diabetic Progression Through Gut Microbiota Alteration in Sugar Rich Diet Induced Diabetic Rats

Sugar rich diet induces inflammation and insulin resistance mainly through gut microbiota alteration. Gut microflora dysbiosis increases plasma lipopolysaccharide and reduces short chain fatty acids to impair the insulin signaling cascades by different molecular pathways to progress into diabetes. Chitosan based formulations have major significance in insulin delivery system due to their ability to protect the insulin from enzymatic degradation and its efficient inter-epithelial transport. This study was designed to investigate the effect of chitosan administration on gut microflora mediated signaling pathways to prevent the diet induced diabetes. Male wistar rats were divided into non-diabetic group with a normal diet (CD), diabetic group with high sucrose diet (HSD) and treatment group with HSD and chitosan (60 mg/kg). After 8 weeks of the study, significant alterations in two major gut dominant microbial phyla i.e Firmicutes and Bacteroides and four dominant microbial species i.e. Lactobacilli, Bifidobacteria, Escherichia and Clostridia were observed in HSD group compared to CD. This microbial dysbiosis in dominant phyla was significantly prevented in chitosan administrated HSD group. Chitosan administration had also reduced the HSD induced activation of Toll like receptors and Nod like receptors signaling pathways compared to HSD control group to reduce the inflammation. These suggest that chitosan can prevent the progression of Type 2 Diabetes through gut microbiota alteration, reducing endotoxin and microbes mediated inflammation.

In Vivo Evaluation of Eclipta alba Extract as Anticancer and Multidrug Resistance Reversal Agent

The present study investigates the anticancer and multidrug resistance (MDR) reversal potential of hydro-alcoholic Eclipta alba extract (EAE) through in vivo experiments. Diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) were used for liver cancer induction in animal model, whereas for MDR induction, AAF was used. The level of antioxidant enzymes was studied in serum along with biochemical parameters. Cancer and MDR-induced liver cells have higher levels of reactive oxygen species (ROS) and, in turn, are responsible for the maintenance of the cancer phenotype. Treatment with EAE declines the ROS level and revealed the ROS scavenging properties. Alfa feto protein levels were found to increase significantly in cancer-induced animals confirming induction and progression of liver cancer, EAE treatment was found to bring back the altered levels within normal range indicating the therapeutic effect of plant extract over liver cancer. Zymogram showed the inhibition of MMPs and RT-PCR analysis revealed that the mRNA expression of nuclear factor-kB was markedly decreased upon EAE treatment. Further, our results showed that EAE could significantly inhibit mdr1 gene encode P-glycoprotein expression. Our data suggest that EAE is a novel anticancer and potent MDR reversal agent and may be a potential adjunctive agent for tumor chemotherapy.

Partial Purification and Characterization of a Bacteriocin DT24 Produced by Probiotic Vaginal Lactobacillus brevis DT24 and Determination of its Anti-Uropathogenic Escherichia coli Potential

The emergence of antibiotic resistance has increased the interest for finding new antimicrobials in the past decade. Probiotic Lactic acid bacteria producing antimicrobial proteins like bacteriocin can be excellent agents for development as novel therapeutic agents and complement to conventional antibiotic therapy. Uropathogenic Escherichia coli, most causative agent of Urinary tract infection, has developed resistance to various antibiotics. In the present investigation, antibacterial substance like bacteriocin (Bacteriocin DT24) produced by probiotic Lactobacillus brevis DT24 from vaginal sample of healthy Indian woman was partially purified and characterized. It was efficiently working against various pathogens, that is, Uropathogenic E. coli, Enterococcus faecium, Enterococcus faecalis and Staphylococcus aureus. The antimicrobial peptide was relatively heat resistant and also active over a broad range of pH 2–10. It has been partially purified by ammonium sulfate precipitation and gel filtration chromatography and checked on reverse-phase high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacteriocin DT24 was approximately 7-kDa protein. The peptide is inactivated by proteolytic enzymes, trypsin and lipase but not when treated with catalase, α-amylase and pepsin. It showed bacteriostatic mode of action against uropathogenic E. coli. Such characteristics indicate that this bacteriocin-producing probiotic may be a potential candidate for alternative agents to control urinary tract infections and other pathogens.