Access to Affordable Health: A Care Delivery Model of GNRC Hospitals in North-Eastern India.

Introduction: The healthcare delivery system of Assam faces several challenges to provide affordable, accessible and quality care services. GNRC (Guwahati Neurological Research Center) is the first super-speciality hospital to address many of these gaps by delivering integrated affordable healthcare services to the populations of Assam and other parts of North-eastern India. Description & Discussion: This paper describes the implementation of a care delivery model which provides integrated care delivery services through linking hospitals to primary healthcare services, including preventive, promotive, and curative care, along with delivering easily accessible and affordable care to the people of Assam and other parts of North-eastern India. Conclusion: The proposed model is the first innovative approach from North-eastern India, Assam, to deliver affordable, accessible and patient-centric hospital led community-based preventive, promotive, and primary, secondary, and tertiary hospital-based care. It is anticipated that GNRC's "Affordable Health Mission" will help redesign and integrate the way primary, secondary and tertiary healthcare is delivered to the population of Assam in helping patients manage their own health and reduce the numbers that needs to be admitted to secondary care and tertiary care by improving patients' independence and well-being as well as dramatically reducing the cost to the overall health system.

Aquatic Peptide: The Potential Anti-Cancer and Anti-Microbial Activity of GE18 Derived from Pathogenic Fungus Aphanomyces invadans.

Small molecules as well as peptide-based therapeutic approaches have attracted global interest due to their lower or no toxicity in nature, and their potential in addressing several health complications including immune diseases, cardiovascular diseases, metabolic disorders, osteoporosis and cancer. This study proposed a peptide, GE18 of subtilisin-like peptidase from the virulence factor of aquatic pathogenic fungus Aphanomyces invadans, which elicits anti-cancer and anti-microbial activities. To understand the potential GE18 peptide-induced biological effects, an in silico analysis, in vitro (L6 cells) and in vivo toxicity assays (using zebrafish embryo), in vitro anti-cancer assays and anti-microbial assays were performed. The outcomes of the in silico analyses demonstrated that the GE18 peptide has potent anti-cancer and anti-microbial activities. GE18 is non-toxic to in vitro non-cancerous cells and in vivo zebrafish larvae. However, the peptide showed significant anti-cancer properties against MCF-7 cells with an IC50 value of 35.34 µM, at 24 h. Besides the anti-proliferative effect on cancer cells, the peptide exposure does promote the ROS concentration, mitochondrial membrane potential and the subsequent upregulation of anti-cancer genes. On the other hand, GE18 elicits significant anti-microbial activity against P. aeruginosa, wherein GE18 significantly inhibits bacterial biofilm formation. Since the peptide has positively charged amino acid residues, it targets the cell membrane, as is evident in the FESEM analysis. Based on these outcomes, it is possible that the GE18 peptide is a significant anti-cancer and anti-microbial molecule.

PS9, Derived from an Aquatic Fungus Virulent Protein, Glycosyl Hydrolase, Arrests MCF-7 Proliferation by Regulating Intracellular Reactive Oxygen Species and Apoptotic Pathways.

One of the most common diseases in women is breast cancer, which has the highest death globally. Surgery, chemotherapy, hormone treatments, and radiation are the current treatment options for breast cancer. However, these options have several adverse side effects. Recently, peptide-based drugs have gained attention as anticancer therapy. Studies report that peptides from biological toxins such as venom and virulent pathogenic molecules have potential therapeutic effects against multiple diseases, including cancers. This study reports on the in vitro anticancer effect of a short peptide, PS9, derived from a virulent protein, glycosyl hydrolase, of an aquatic fungus, Aphanomyces invadans. This peptide arrests MCF-7 proliferation by regulating intercellular reactive oxygen species (ROS) and apoptotic pathways. Based on the potential for the anticancer effect of PS9, from the in silico analysis, in vitro analyses using MCF-7 cells were executed. PS9 showed a dose-dependent activity; its IC50 value was 25.27-43.28 µM at 24 h. The acridine orange/ethidium bromide (AO/EtBr) staining, to establish the status of apoptosis in MCF-7 cells, showed morphologies for early and late apoptosis and necrotic cell death. The 2,7-dichlorodihydrofluorescein diacetate (DCFDA) staining and biochemical analyses showed a significant increase in reactive oxygen species (ROS). Besides, PS9 has been shown to regulate the caspase-mediated apoptotic pathway. PS9 is nontoxic, in vitro, and in vivo zebrafish larvae. Together, PS9 may have an anticancer effect in vitro.

Anti-Cancer and Anti-Inflammatory Activities of a Short Molecule, PS14 Derived from the Virulent Cellulose Binding Domain of Aphanomyces invadans, on Human Laryngeal Epithelial Cells and an In Vivo Zebrafish Embryo Model.

In this study, the anti-cancer and anti-inflammatory activities of PS14, a short peptide derived from the cellulase binding domain of pathogenic fungus, Aphanomyces invadans, have been evaluated, in vitro and in vivo. Bioinformatics analysis of PS14 revealed the physicochemical properties and the web-based predictions, which indicate that PS14 is non-toxic, and it has the potential to elicit anti-cancer and anti-inflammatory activities. These in silico results were experimentally validated through in vitro (L6 or Hep-2 cells) and in vivo (zebrafish embryo or larvae) models. Experimental results showed that PS14 is non-toxic in L6 cells and the zebrafish embryo, and it elicits an antitumor effect Hep-2 cells and zebrafish embryos. Anticancer activity assays, in terms of MTT, trypan blue and LDH assays, showed a dose-dependent inhibitory effect on cell proliferation. Moreover, in the epithelial cancer cells and zebrafish embryos, the peptide challenge (i) caused significant changes in the cytomorphology and induced apoptosis; (ii) triggered ROS generation; and (iii) showed a significant up-regulation of anti-cancer genes including BAX, Caspase 3, Caspase 9 and down-regulation of Bcl-2, in vitro. The anti-inflammatory activity of PS14 was observed in the cell-free in vitro assays for the inhibition of proteinase and lipoxygenase, and heat-induced hemolysis and hypotonicity-induced hemolysis. Together, this study has identified that PS14 has anti-cancer and anti-inflammatory activities, while being non-toxic, in vitro and in vivo. Future experiments can focus on the clinical or pharmacodynamics aspects of PS14.

Antiproliferation of MP12 derived from a fungus, Aphanomyces invadans virulence factor, cysteine-rich trypsin inhibitor on human laryngeal epithelial cells, and in vivo zebrafish embryo model.

Peptide-based drug development is an emerging and promising approach in cancer therapeutics. The present study focuses on understanding the mechanism of MP12 peptide (MDNHVCIPLCPP) derived from cysteine-rich trypsin inhibitor protein of virulence factor of pathogenic fungus Aphanomyces invadans. MP12 is involved in antiproliferative activity against the human laryngeal epithelial cell (Hep-2), demonstrated in this study. MP12 sequence showed a significant binding score and has multiple hydrogen bond interactions with the proteins that play a vital role in apoptotic pathways such as Bcl-2, caspase-3, caspase-7, and XIAP. Based on the bioinformatics characterization and molecular docking result, further study was focused on MP12 antiproliferative activity. The peptide showed a dose-dependent inhibition against Hep-2 cell line proliferation, analyzed over MTT and neutral red uptake assays. The IC50 value of the MP12 peptide was calculated based on the antiproliferative property (24.7 ± 0.34 µM). MP12 treated Hep-2 cells showed significant shrinkage in cell morphology compared to untreated cells, inhibiting the cell cycle. The gene expression analysis validated that the MP12 significantly upregulates the caspase-3, caspase-7, and caspase-9 genes. The developmental toxicity study using zebrafish embryos as in vivo model proved that the MP12 is nontoxic. Based on the obtained results, we proposed that the peptide MP12 derived from cysteine-rich trypsin inhibitor protein of virulence molecule of pathogenic fungus have a potential antiproliferative activity. However, further clinical trials need to be focused on the mechanism and therapeutic application against laryngeal cancer.

RM12 similar to substance P from tachykinin of freshwater murrel Channa striatus influence intracellular ROS in vitro fish erythrocytes and developmental toxicity and antioxidant enzymes in vivo zebrafish embryo.

In this study, substance P, an antioxidant peptide of tachykinin, was identified using bioinformatics tools from the earlier established muscle transcriptome of a freshwater murrel Channa striatus and the peptide was named RM12. The antioxidant properties of RM12 were screened using various colorimetric assays. The toxicity of RM12 was experimented using fish erythrocytes, and it is observed that the maximum concentration (320 µM) of RM12 was found to have 15 or 20% of hemolytic activity; however, it was not significant with other tested concentrations (10, 20, 40, 80, and 160 µM). Further, the in vivo antioxidant properties of RM12 were experimented on zebrafish embryo, the intracellular ROS level was estimated by 5 mM H2O2 stress in the zebrafish embryo, and inhibition of apoptosis was evaluated. The antioxidant enzymes were extracted from the H2O2-stressed zebrafish embryo, and the intracellular ROS was eliminated due to RM12. Collectively, the experiment showed that the substance P from the freshwater murrel C. striatus possessed potent antioxidant properties; thus, it can further be focused to develop it as antioxidant molecule in aquaculture organisms.

Pharmacological importance of TG12 from tachykinin and its toxicological behavior against multidrug-resistant bacteria Klebsiella pneumonia.

Development of antimicrobial drugs against multidrug-resistant (MDR) bacteria is a great focus in recent years. TG12, a short peptide molecule used in this study was screened from tachykinin (Tac) protein of an established teleost Channa striatus (Cs) transcriptome. Tachykinin cDNA has 345 coding sequence, that denotes a protein contained 115 amino acids; in which a short peptide (TG12) was identified at 83-94. Tachykinin mRNA upregulated in C. striatus treated with Aeromonas hydrophila and Escherichia coli lipopolysaccharide (LPS). The mRNA up-regulation was studied using real-time PCR. The up-regulation tachykinin mRNA pattern confirmed the immune involvement of tachykinin in C. striatus during infection. Further, the identified peptide, TG12 was synthesized and its toxicity was demonstrated in hemolytic and cytotoxic assays using human erythrocytes and human dermal fibroblast cells, respectively. The toxicity study exhibited that the toxicity of TG12 was similar to negative control, phosphate buffer saline (PBS). Moreover, the antibiogram of TG12 was active against Klebsiella pneumonia ATCC 27736, a major MDR bacterial pathogen. Further, the antimicrobial activity of TG12 against pathogenic bacteria was screened using minimum inhibitory concentration (MIC) and anti-biofilm assays, altogether TG12 showed potential activity against K. pneumonia. Fluorescence assisted cell sorter flow cytometer analysis (FACS) and field emission scanning electron microscopy (FESEM) was carried on TG12 with K. pneumonia; the results showed that TG12 significantly reduced K. pneumonia viability as well as TG12 disrupt its membrane. In conclusion, TG12 of CsTac is potentially involved in the antibacterial immune mechanisms, which has a prospectus efficiency in pharma industry against MDR strains, especially K. pneumonia.

TL15 of Arthrospira platensis sulfite reductase scavenges free radicals demonstrated in oxidant induced larval zebrafish (Danio rerio) model.

The antioxidant role of sulfite reductase (SiR) derived from Arthrospira platensis (Ap) was identified through a short peptide, TL15. The study showed that the expression of ApSiR was highly expressed on day ten due to sulfur deprived stress in Ap culture. TL15 peptide exhibited strong antioxidant activity when evaluated using antioxidant assays in a concentration ranging from 7.8 and 125 µM. Further, the cytotoxicity of TL15 peptide was investigated, even at the higher concentration (250 µM), TL15 did not exhibit any toxicity, when tested in vitro using human leucocytes. Moreover, a potential reduction in reactive oxygen species (ROS) production was observed due to the treatment of TL15 peptide (>15.6 µM) to H2O2 exposed leucocytes. For the in vivo assessment of TL15 toxicity and antioxidant ability, experiments were performed in zebrafish (Danio rerio) larvae to analyse the developmental toxicity of TL15 peptide. Results showed that, exposure to TL15 peptide in tested concentrations ranging from 10, 20, 40, and 80 µM, did not affect the development and physiological parameters of the zebrafish embryo/larvae such as morphology, survival, hatching and heart rate. Fluorescent assay was performed using DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) to examine the production of intracellular reactive oxygen species (ROS) in zebrafish treated with TL15 peptide during the embryo-larval stages. Fluorescent images showed that pre-treatment with TL15 peptide to attenuate the H2O2 induced ROS levels in the zebrafish larvae in a dose-dependent manner. Further to uncover the underlying biochemical and antioxidant mechanism, the enzyme activity of superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) levels were studied in zebrafish larvae. TL15 pre-treated groups showed enhanced antioxidant enzyme activity, while the hydrogen peroxide (H2O2) exposed larvae showed significantly diminished activity. Overall results from the study revealed that, TL15 act as a potential antioxidant molecule with dose-specific antioxidant property. Thus, TL15 peptide could be an effective and promising source for biopharmaceutical applications.

Defense involvement of piscidin from striped murrel Channa striatus and its peptides CsRG12 and CsLC11 involvement in an antimicrobial and antibiofilm activity.

In this study, we have evaluated bioinformatics characterization and antimicrobial role of two piscidin (Pi) peptide identified from the established transcriptome of striped murrel Channa striatus (Cs). The identified CsPi cDNA contains 256 nucleotides encode a protein with 70 amino acids in length which has two antimicrobial peptides and named CsRG12 and CsLC11. The gene expression analysis with various immune stimulants indicated an induced expression pattern of CsPi. Antibiogram showed that CsRG12 and CsLC11 was active against Staphylococcus aureus ATCC 33592, a major multi-drug resistant (MDR) bacterial pathogen and Bacillus cereus ATCC 2106. The minimum inhibitory concentration (MIC) and antibiofilm assays were conducted to observe the activity of pathogenic bacteria with these derived antimicrobial peptides. Flow cytometry analysis noticed that the CsRG12 and CsLC11 disrupt the membrane formation of S. aureus and B. cereus, which was further assured by scanning electron microscopic (SEM) images that bleb formation leads to disruption around the bacterial membrane. Overall, it is reported that CsPi is involved in innate immunity as the gene expression plays a remarkable role in up and down regulation during infection. In addition, the involvement of peptides in antibiofilm formation and bacterial membrane disruption support its immune character. This study leads to a possibility for the development of therapeutics in aquaculture biotechnology.

Antioxidant molecular mechanism of adenosyl homocysteinase from cyanobacteria and its wound healing process in fibroblast cells.

An antioxidant molecule namely, adenosyl homocysteinase (AHc) was identified from the earlier constructed transcriptome database of Spirulina, where it was cultured in a sulphur deprived condition. From the AHc protein, a small peptide NL13 was identified using bioinformatics tools and was predicted to have antioxidant property. Further, the peptide was synthesised and its antioxidant mechanism was addressed at molecular level. NL13 was subjected to various antioxidant assays including DPPH assay, HARS assay, SARS Assay, NO assay and ABTS assay, where NL13 exhibited significant (P < 0.05) potential antioxidant activity compared to its antioxidant control, Trolox. Cytotoxicity was performed on Human whole blood and the cell viability was performed on VERO fibroblast cells. In both assays, it was found that NL13 did not exhibit any cytotoxic effect towards the cells. Further, the intracellular ROS was performed on Multimode reader followed by imaging on fluorescence microscope which showed scavenging activity even at lower concentration of NL13 (31.2 µM). An effective wound healing property of NL13 on VERO cells was confirmed by analysing the cell migration rate at two different time intervals (24 and 48 h). Overall, the study shows that NL13 peptide scavenges the intracellular oxidative stress.

Determination of individual type 2 diabetes risk profile in the North East Indian population & its association with anthropometric parameters.

Background & objectives: Diabetes genomics research has illuminated single nucleotide polymorphism (SNP) in several genes including, fat mass and obesity associated (FTO) (rs9939609 and rs9926289), potassium voltage-gated channel subfamily J member 11 (rs5219), SLC30A 8 (rs13266634) and peroxisome proliferator-activated receptor gamma 2 (rs1805192). The present study was conducted to investigate the involvement of these polymorphisms in conferring susceptibility to type 2 diabetes (T2D) in the North East Indian population, and also to establish their association with anthropometric parameters. Methods: DNA was extracted from blood samples of 155 patients with T2D and 100 controls. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing. To confirm the association between the inheritance of SNP and T2D development, logistic regression analysis was performed. Results: For the rs9939609 variant (FTO), the dominant model AA/(AT+TT) revealed significant association with T2D [odds ratio (OR)=2.03, P=0.021], but was non-significant post correction for multiple testing (P=0.002). For the rs13266634 variant (SLC30A 8), there was considerable but non-significant difference in the distribution pattern of genotypic polymorphisms between the patients and the controls (P=0.004). Significant association was observed in case of the recessive model (CC+CT)/TT (OR=4.56 P=0.001), after adjusting for age, gender and body mass index. In addition, a significant association (P=0.001) of low-density lipoprotein (mg/dl) could be established with the FTO (rs9926289) polymorphism assuming dominant model. Interpretation & conclusions: The current study demonstrated a modest but significant effect of SLC30A8 (rs13266634) polymorphisms on T2D predisposition. Considering the burgeoning prevalence of T2D in the Indian population, the contribution of these genetic variants studied, to the ever-increasing number of T2D cases, appears to be relatively low. This study may serve as a foundation for performing future genome-wide association studies (GWAS) involving larger populations.

Effects of Resveratrol and Mangiferin on PPARγ and FALDH Gene Expressions in Adipose Tissue of Streptozotocin-Nicotinamide-Induced Diabetes in Rats.

Type 2 diabetes (T2D) is characterized by insufficient insulin secretion by the pancreatic beta cells and insulin resistance in liver, skeletal muscle, and white adipose tissue. Adipose tissue plays a major role in glucose homeostasis and lipid metabolism. Dietary antioxidants such as resveratrol and mangiferin may offer some protection against the early stage of diabetes mellitus. Therefore, an attempt has been made to investigate the effects of resveratrol and mangiferin on biochemical parameters and molecular mechanism of PPARγ and FALDH gene expression in adipose tissue of streptozotocin- (STZ-) nicotinamide- (NA-) induced diabetic rats. Albino Wister rats were randomly divided into five groups: control rats (Group 1), diabetic control rats (Group 2), diabetic rats given resveratrol (40 mg/kg body weight per day; Group 3), diabetic rats given mangiferin (40 mg/kg body weight per day; Group 4), diabetic rats given glibenclamide (0.6 mg/kg body weight per day; Group 5). Serum biochemical parameters-total cholesterol (TC), total triglyceride (TG), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, glycosylated hemoglobin (HbA1c), urea, and uric acid were analyzed. We found that the oral administration of resveratrol and mangiferin to STZ-NA-induced diabetic rats for 30 days showed the significant protective effect on all the biochemical parameters. A significant reduction in blood glucose and HbA1c levels was observed in rats treated with 40 mg/kg body weight per day of resveratrol or mangiferin. Moreover, both these antioxidants showed significant enhancement of PPARγ and FALDH gene expression in rat adipose tissue compared to control rats.

Modulatory effect of baicalein on gene expression and activity of antioxidant enzymes in streptozotocin-nicotinamide induced diabetic rats

Oxidative stress plays the central role in the pathogenesis and progression of diabetic complications. The present study aims to investigate the beneficial effect of oral administration of flavone baicalein in streptozotocin-nicotinamide (STZ-NA) induced diabetic rats by measuring oxidative stress markers, antioxidant enzyme activities and expression analysis of antioxidant genes. Experimental diabetes was induced by a single intraperitoneal (i.p.) injection of STZ (55 mg /kg b.wt), 15 min after the i.p. administration of NA. At the end of the experimental period, thiobarbituric acid reactive substances (TBARS), activities of antioxidant enzymes and expression levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GPx) were measured in diabetic rats along with serum biochemical parameters namely total cholesterol (TC), total triglyceride (TG), aspartate transaminase (AST) alanine transaminase (ALT) and glycosylated hemoglobin (HbA1c). Oral administration of baicalein (40 mg/kg b.wt/day) demonstrated a significant ameliorative effect on all studied biochemical and oxidative stress parameters. Biochemical findings were corroborated by qPCR expression analysis which showed significant upregulation of antioxidant genes in diabetic rats. These results suggest that baicalein supplementation may reduce diabetes and its complications by suppressing oxidative stress and enhancing gene expression and antioxidant enzyme activities in diabetic rats.

Comparative analysis of different dietary antioxidants on oxidative stress pathway genes in L6 myotubes under oxidative stress.

Enhanced oxidative stress plays an important role in the progression and onset of diabetes and its complications. Strategies or efforts meant to reduce the oxidative stress are needed which may mitigate these pathogenic processes. The present study aims to investigate the in vitro ameliorative potential of nine antioxidant molecules in L6 myotubes under oxidative stress condition induced by 4-hydroxy-2-nonenal and also to comprehend the gene expression patterns of oxidative stress genes upon the supplementation of different antioxidants in induced stress condition. The study results demonstrated a marked increase in the level of malondialdehyde and protein carbonyl content with a subsequent increase in the free radicals that was reversed by the pretreatment of different dietary antioxidant. From the expression analysis of the oxidative stress genes, it is evident that the expression of these genes is modulated by the presence of antioxidants. The highest expression was found in the cells treated with Insulin in conjugation with an antioxidant. Resveratrol is the most potent modulator followed by Mangiferin, Estragole, and Capsaicin. This comparative analysis ascertains the potency of Resveratrol along with Insulin in scavenging the reactive oxygen species (ROS) generated under induced stress conditions through antioxidant defense mechanism against excessive ROS production, contributing to the prevention of oxidative damage in L6 myotubes.