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We review the evidence available worldwide on the various challenges in the screening, management, prevention of gestational diabetes mellitus and diabetes in pregnancy. The use of multiple screening and diagnostic tests prescribed by numerous guidelines is challenging for practitioners. Also, sociocultural, demographic and economic challenges affect the prevention and care. Life-course perspectives need to be adopted, as well as an integrated approach in public health care is essential. Tackling these challenges at each phase of life-course, with development and adherence to the country-specific guidelines by practitioners can decrease the burden of gestational diabetes mellitus and diabetes in pregnancy.

Waste generation from groundnut and sugarcane industries are at high alarming rate due to their disposal constraint. These wastes are currently used as a raw material for the production of compost, fuel, alcohol, and paper. Cellulose and its derivatives are being used in many industries like paper, pharmaceutical, and energy owing to its physicochemical properties. Application of cellulose in the leather industry uses cellulose derivatives as finishing chemicals such as nitrocellulose, ethyl cellulose. In the current study, an attempt has been made to find the application of cellulose from groundnut husk and sugarcane bagasse as an auxiliary chemical in leather finishing. Cellulose is extracted from wastes by modified acid hydrolysis method to enhance the yield. Extracted cellulose has been characterized and compared with commercial cellulose sample. From the characterization studies, it confirms that extracted cellulose matched with commercial samples. Extracted cellulose used in leather finishing as an ingredient to upgrade the low-quality leathers, which primarily have grain damages. Cellulose with different concentration of plasticizer has been used in the base coat along with binders and pigments. Finished leathers have been characterized to understand the physical characteristics. From the various analysis, it ascertains that the cellulose has imparted fullness to the finished leather without impacting the aesthetic property. The study provides a better utilization of sugarcane bagasse and groundnut husk wastes in leather industry leading to effective waste management prototype.

Additional file 1. In-depth Interview (IDI) Guide: Health Care Provider- Doctor.

Additional file 2. In-depth Interview (IDI) Guide: Health Care Provider- NURSE.

Volatile organic chemicals (VOCs) are routinely used for processing biological tissue samples in clinical laboratories. Recognizing their serious health and environmental impacts, a few non-volatile green solvents (choline based ionic liquids, ILs) were evaluated as tissue fixatives here. Microscopic evaluation of histo-morphology, fixation and staining quality, and macromolecular integrity (DNA and proteins) were assessed in human eye tissues (sclera, choroid, retinal layers and retinal pigmented epithelium, eyelid and orbit) after IL-fixation. Formalin-fixed tissues were used as standard reference. Microscopic examination revealed favorable histomorphology, tissue fixation and staining characteristics in most tissues immersed in ILs. Time taken to fix, and stability over a period of time (24 h, 48 h, 1 week, 1 month) was also recorded. Electrophoretic analysis revealed stability of cellular proteins and nucleic acids in IL-fixed scleral tissues. Heterogeneity in tissue fixation property relative to the type of ocular tissue, duration of fixation and storage, warrant further design and optimization of ILs to fix biological tissues. The simple cholinium salts based ILs tested here show favorable potential for tissue fixation application, and as an alternative approach to the use of VOCs, towards sustainable biomedical practice.

Kluyveromyces marxianus exoinulinase production in shaker and bio reaction calorimeter has been studied. Biocalorimetric studies revealed enhanced inulinase production due to the linear and exponential feeding of sucrose. Linear fed-batch inulinase production (355 IU/mL) was seventeen-fold greater than batch mode (20.98 IU/mL). Further controlling the specific growth rate by exponential feeding of sucrose gave highest inulinase activity of 728 IU/mL. Exponential feeding strategy yielded highest inulinase activity in the synthetic medium with a reduction in batch time to half that of the linear feeding. This has been substantiated by metabolic and physiological aspects of inulinase production. A Michaelis-Menten type empirical model was developed for fed-batch that can be adopted for controlling specific growth rate based on metabolic heat responses, towards achieving maximum enzyme production and release.

Background Retinoblastoma (RB) is a childhood retinal malignancy. Effective therapeutic strategies are still being investigated in RB disease management. Here, the anti-cancer effect of shepherdin, a peptido-mimetic inhibiting heat shock protein (HSP90)-Survivin interaction has been analyzed. Methods We analyzed HSP (HSP70/90) and Survivin protein expressions by immunohistochemistry (29 archival tumors), qRT-PCR, FACS and Western analysis (10 un-fixed RB tumors). We also analyzed cellular cytotoxicity and anti-proliferative effect in peptide treated RB cells (Y79, Weri Rb1) and MIO-M1 cells. Results Heterogeneous expressions of HSP70/90 and Survivin with a significant association between HSP70 and HSP90 (r2 = 0.59, p = 0.001) was observed. In RB cells, anti-tumor effects were detected with 0.42 μg/ml of shepherdin at 4 h s of serum starvation. Decreased Survivin, Bcl2, MMP-2 activity with increased Bax, Bim, and Caspase-9 protein expressions were noticed. No significant changes were observed in shepherdin treated non-neoplastic MIO-M1, nor in scramble-peptide treated RB cells. Conclusion The presence of HSPs (HSP70/90) and Survivin reveals multiple cellular mechanisms adopted by RB cells during cancer progression. Serum starvation induced HSP90 whose interactions with Survivin were specifically inhibited by shepherdin. The associated molecular shuffling has been reported. These findings strongly implicate the potential of targeting HSP90-Survivin interaction as an adjuvant therapy in RB management.

Activation of fatty acid synthase (FASN) enzyme in the de novo lipogenic pathway has been reported in various cancers including retinoblastoma (RB), a pediatric ocular cancer. The present study investigates lipogenesis-dependent survival of RB cancer cells and the associated molecular pathways in FASN silenced RB cells. The siRNA-mediated FASN gene knockdown in RB cancer cells (Y79, WERI RB1) repressed FASN mRNA and protein expressions, and decreased cancer cell viability. Global gene expression microarray analysis was performed in optimized FASN siRNA transfected and untransfected RB cells. Deregulation of various downstream cell signaling pathways such as EGFR (n = 55 genes), TGF-beta (n = 45 genes), cell cycle (n = 41 genes), MAPK (n = 39 genes), lipid metabolism (n = 23 genes), apoptosis (n = 21 genes), GPCR signaling (n = 21 genes), and oxidative phosporylation (n = 18 genes) were observed. The qRT-PCR validation in FASN knockdown RB cells revealed up-regulation of ANXA1, DAPK2, and down-regulation of SKP2, SREBP1c, RXRA, ACACB, FASN, HMGCR, USP2a genes that favored the anti-cancer effect of lipogenic inhibition in RB. The expression of these genes in primary RB tumor tissues were correlated with FASN expression, based on their clinico-pathological features. The differential phosphorylation status of the various PI3K/AKT pathway proteins (by western analysis) indicated that the FASN gene silencing indeed mediated apoptosis in RB cells through the PI3K/AKT pathway. Scratch assay clearly revealed that FASN silencing reduced the invading property of RB cancer cells. Dependence of RB cancer cells on lipid metabolism for survival and progression is implicated. Thus targeting FASN is a promising strategy in RB therapy.

Retinoblastoma (RB) is a primary childhood eye cancer. HMGA2 shows promise as a molecule for targeted therapy. The involvement of miRNAs in genome-level molecular dys-regulation in HMGA2-silenced RB cells is poorly understood. Through miRNA expression microarray profiling, and an integrated array analysis of the HMGA2-silenced RB cells, the dysregulated miRNAs and the miRNA-target relationships were modelled. Loop network analysis revealed a regulatory association between the transcription factor ( SOX5) and the deregulated miRNAs imiR-29a, miR-9*, miR-9-3). Silencing of HMGA2 deregulated the vital oncomirs ( miR-7, miR-331, miR-26a, miR-221, miR-17∼92 and miR-106b∼25) in RB cells. From this list, the role of the miR-106b∼25 cluster was examined further for its expression in primary RB tumor tissues (n = 20). The regulatory targets of miR-106b∼25 cluster namely p21 (cyclin-dependent kinase inhibitor) and BIM (pro-apoptotic gene) were elevated, and apoptotic cell death was observed, in RB tumor cells treated with the specific antagomirs of the miR-106b∼25 cluster. Thus, suppression of miR-106b∼25 cluster controls RB tumor growth. Taken together, HMGA2 mediated anti-tumor effect present in RB is, in part, mediated through the miR-106b∼25 cluster.

Lysyl oxidase (LOX) is a copper dependent amine oxidase which catalyses the cross linking of collagen and elastin towards the maturation of extracellular matrix. The expression and activity of LOX is known to vary under pathological conditions such as tumorigenesis, hyperhomocysteinemia, copper deficiency diseases, pseudoexfoliation syndrome and proliferative diabetic retinopathy. Despite the implication of LOX in many diseases, there is inadequate information about its structure. Therefore, we describe a molecular model of Human Lysyl Oxidase (LOX) with optimal copper orientation in the catalytic cavity for induced fit docking studies with potential modulators. The predicted model was found to be highly plausible as per the stereochemistry checks. Further, Molecular Dynamics (MD) studies also inferred the stability of the predicted structure. We performed Induced Fit Docking (IFD) of LOX modulators to the predicted structure and also validated the molecular interactions in implicit solvent model by calculating Molecular Mechanics Generalized Born Surface Area (MMGBSA). The IFD results strongly reveal that aspartic acid residues in the catalytic cavity as the key players in establishing interactions with small molecules. The insights from this study will aid in better exploration of the structure-function relationship of LOX.

PurposeManagement of Retinoblastoma (RB), a pediatric ocular cancer is limited by drug-resistance and drug-dosage related side effects during chemotherapy. Molecular de-regulation in post-chemotherapy RB tumors was investigated.Materials and MethodscDNA microarray analysis of two post-chemotherapy and one pre-chemotherapy RB tumor tissues was performed, followed by Principle Component Analysis, Gene ontology, Pathway Enrichment analysis and Biological Analysis Network (BAN) modeling. The drug modulation role of two significantly up-regulated genes ( P≤0.05) — Ect2 (Epithelial-cell-transforming-sequence-2), and PRAME (preferentially-expressed-Antigen-in-Melanoma) was assessed by qRT-PCR, immunohistochemistry and cell viability assays.ResultsDifferential up-regulation of 1672 genes and down-regulation of 2538 genes was observed in RB tissues (relative to normal adult retina), while 1419 genes were commonly de-regulated between pre-chemotherapy and post- chemotherapy RB. Twenty one key gene ontology categories, pathways, biomarkers and phenotype groups harboring 250 differentially expressed genes were dys-regulated ( EZH2, NCoR1, MYBL2, RB1, STAMN1, SYK, JAK1/2, STAT1/2, PLK2/4, BIRC5, LAMN1, Ect2, PRAME and ABCC4). Differential molecular expressions of PRAME and Ect2 in RB tumors with and without chemotherapy were analyzed. There was neither up- regulation of MRP1, nor any significant shift in chemotherapeutic IC50, in PRAME over-expressed versus non-transfected RB cells.ConclusionCell cycle regulatory genes were dys-regulated post-chemotherapy. Ect2 gene was expressed in response to chemotherapy-induced stress. PRAME does not contribute to drug resistance in RB, yet its nuclear localization and BAN information, points to its possible regulatory role in RB.

Fatty acid synthase (FASN), a lipogenic multienzyme complex, is overexpressed in the ocular cancer, retinoblastoma, and is strongly correlated with tumor invasion. Dietary nutrients are reported to exert anticancer effects through inhibition of lipid metabolism. Differential gene expression in cultured retinoblastoma cells induced by cerulenin, a chemical inhibitor of FASN, was evaluated by cDNA microarray analysis. Cerulenin treatment resulted in significant upregulation of cytochrome c (CYCS) by 1.2-fold, whereas S-phase kinase-associated protein-2 (SKP2), a negative regulator of cell cycle, and the lipid metabolic genes (PPARA, RXRA, and ACACB) were significantly downregulated by -1.59-, -1.8-, -1.83-, and -1.5-fold, respectively, in comparison with untreated cancer cells. The expressions of key differentially expressed genes were confirmed by quantitative real-time PCR. The altered expression of genes involved in cell proliferation, cell signaling, apoptosis, and cell cycle, correlated with the anticancer effects of cerulenin. FASN inhibition may thus be a potential strategy in retinoblastoma management.

Cardiovascular disease (CVD) is a major cause of mortality in both developed and developing countries, owing to significant increase in the intake of high-energy foods, reduced physical activity, and an increase in psychosocial stress, which in turn lead to dysglycemia, hypertension, and dyslipidemia. The incidence of CVD in diabetics is very high that is further aggravated by co-morbidities such as hyperlipidemia and/or hypertension. The purpose of this study is to mathematically model the dynamics of CVD in diabetic population with hyperlipidemia and hypertension. Here, the mathematical model is a system of ordinary differential equations (ODEs). The steady states of the model are computed and their stability is studied. Numerical simulations are performed on the model, and conditions for controlling CVD in diabetics are derived. The results of this analysis suggest that the extent of control of hyperlipidemia and hypertension directly correlates with decrease in CVD development in the diabetic population. Early diagnosis of the modifiable risk factors such as hyperlipidemia and hypertension, followed by effective clinical management to regulate blood lipid levels and blood pressure in diabetics would greatly reduce the burden of cardiovascular complications in diabetic populations.

High-mobility group A2 (HMGA2) protein regulates retinoblastoma (RB) cancer cell proliferation. Here, a stable phosphorothioate-modified HMGA2 aptamer was used to block HMGA2 protein function in RB cells. HMGA2-aptamer internalisation in RB cells (Y79, Weri Rb1) and non-neoplastic human retinal cells (MIO-M1) were optimised. Aptamer induced dose-dependent cytotoxicity in RB cancer cells (0.25-1.5 µM). Increased expression of TGFβ, SMAD4, CDH1, BAX, CASP 3, PARP mRNA and decreased SNAI1, Bcl2 mRNA levels in aptamer-treated RB cells suggests the activation of TGFβ-SMAD4-mediated apoptotic pathway. Synergistic effect with etoposide was observed in aptamer treated RB cells (p value ≤0.05). No significant toxicity was observed in non-neoplastic retinal cells.

Human fatty acid synthase (hFASN), a homo dimeric lipogenic enzyme with seven catalytic domains, is an important clinical target in cancer, metabolic syndrome and infections. Here, molecular modelling and docking methods were implemented to examine the inter-molecular interactions of thioesterase (TE) domain in hFASN with its physiological substrate, and to identify potential chemical inhibitors. TE catalyses the hydrolysis of thioester bond between palmitate and the 4' phosphopantetheine of acyl carrier protein, releasing 16-carbon palmitate. The crystal structure of hFASN TE in two inhibitory conformations (A and B) were geometry-optimized and used for molecular docking with palmitate, orlistat (a known FASN inhibitor) and virtual screening against compounds from National Cancer Institute (NCI) database. Relatively, low binding affinity was observed during the complex formation of palmitate with A (-.164 kcal/mol) and B (-.332 kcal/mol) forms of TE, when compared with orlistat-docked TE (A form: -5.872 kcal/mol and B form: -5.484 kcal/mol), clearly indicating that the native inhibited conformation (crystal structure) was unfavourable for substrate binding. We used these orlistat dual binding modes as positive controls for prioritizing the ligands during virtual screening. From 2, 31,617 molecules in the NCI database, 916 high-scoring compounds (hit ligands) were obtained for A-form and 4582 for B-form of the TE-domain, which were then ranked according to glide docking score, XP H bond score, absorption, distribution, metabolism and excretion and binding free energy (Prime/MM-GBSA). Consequently, two top scoring ligands (NSC: 319661 and NSC: 153166) emerged as promising drug candidates that may be tested in FASN-over-expressing diseases.

Purpose To correlate the differentially expressed miRNAs with clinico-pathological features in uveal melanoma (UM) tumors harbouring chromosomal 3 aberrations among South Asian Indian cohort. Methods Based on chromosomal 3 aberration, UM (n = 86) were grouped into monosomy 3 (M3; n = 51) and disomy 3 (D3; n = 35) by chromogenic in-situ hybridisation (CISH). The clinico-pathological features were recorded. miRNA profiling was performed in formalin fixed paraffin embedded (FFPE) UM samples (n = 6) using Agilent, Human miRNA microarray, 8x15KV3 arrays. The association between miRNAs and clinico-pathological features were studied using univariate and multivariate analysis. miRNA-gene targets were predicted using Target-scan and MiRanda database. Significantly dys-regulated miRNAs were validated in FFPE UM (n = 86) and mRNAs were validated in frozen UM (n = 10) by qRT-PCR. Metastasis free-survival and miRNA expressions were analysed by Kaplen-Meier analysis in UM tissues (n = 52). Results Unsupervised analysis revealed 585 differentially expressed miRNAs while supervised analysis demonstrated 82 miRNAs (FDR; Q = 0.0). Differential expression of 8 miRNAs: miR-214, miR-149*, miR-143, miR-146b, miR-199a, let7b, miR-1238 and miR-134 were studied. Gene target prediction revealed SMAD4, WISP1, HIPK1, HDAC8 and C-KIT as the post-transcriptional regulators of miR-146b, miR-199a, miR-1238 and miR-134. Five miRNAs (miR-214, miR146b, miR-143, miR-199a and miR-134) were found to be differentially expressed in M3/ D3 UM tumors. In UM patients with liver metastasis, miR-149* and miR-134 expressions were strongly correlated. Conclusion UM can be stratified using miRNAs from FFPE sections. miRNAs predicting liver metastasis and survival have been identified. Mechanistic linkage of de-regulated miRNA/mRNA expressions provide new insights on their role in UM progression and aggressiveness.

The metabolic effectiveness of choline lactate in the growth media was investigated relative to conventional carbon source for growing Staphylococcus lentus, a bacterial strain commonly used in bioremediation of industrial effluents and xenobiotic detoxification. Bacterial growth thermodynamics was determined by biocalorimetry. (13)C NMR and FTIR spectroscopic analyses traced the consumption of choline lactate at specific time intervals of bacterial growth. Under aerobic conditions, it is apparent that S. lentus initially metabolized lactate for its energy needs, while the choline cation of the ionic salt seemed to provide its C and N for biosynthetic intermediates for cell structure/function, in the growing bacterial colony. Urea accumulation after 40 h of bacterial growth was recorded. Possible metabolic trajectory of choline lactate consumed during S. lentus growth is suggested here. The theoretical estimation of heats of reaction for the proposed metabolic pathway (455 kJ/mol) was comparable with the experimentally obtained reaction enthalpy (435 kJ/mol), which further validated the proposed metabolic pathway. The biomass and energy profile of bacteria growth in choline media was found to be more favorable than in glucose media. The ionic liquid, choline lactate, offers a metabolically and energetically efficient carbon (and nitrogen) source for growing S. lentus.

Oxidative stress, which refers to the biological damage caused by free radicals produced in excess of innate antioxidant defenses, is indicated in the ocular cancer retinoblastoma (RB). Here we have analysed the differential expression of oxidative stress responsive genes in oxidant-induced RB cells, and in RB tumor tissues.The study included cultured RB cells, and four RB tumor tissues. The reactive oxygen species (ROS) levels in Y79 cells and the RB tumor induced by hydrogen peroxide were quantified by Dichlorofluorescein (DCF) fluorescence assay. We then analysed the gene expression profile of cultured RB cells induced with hydrogen peroxide (400 µM H(2)O(2) for 8 h) by microarray analysis, and the expression of select genes were validated in Y79 cells and RB tumor tissues by real-time PCR analysis.The oxidant-induced RB tumors showed an average increase in ROS levels of 44-fold compared to induced non-neoplastic donor retina. H(2)O(2)-induced RB cell line showed a 3-fold increase in ROS levels. Microarray analysis on RB cell line induced with H(2)O(2) showed differentially regulated genes involved in cellular processes such as: oxidative stress, angiogenesis, lipid metabolism, cell proliferation, and cell signaling pathways. Several up-regulated genes such as SOD, GPX, CAT, CDC25A, CREBBP, JUN, MMP-2, iNOS, CRYAA, RXRA, ACACB and HMGCR were validated by real-time PCR. These results corroborated with the gene expression analysis in RB tumor tissues. Relating the antioxidant gene expression with the clinico-pathologic features of the tumor tissues, we found that the tumor with invasion of choroid, optic nerve and retinal pigment epithelium, had relatively higher ROS levels and minimal antioxidant gene expression, when compared with the tumor with only choroidal invasion.The study suggests active involvement of redox signaling pathways in the pathogenesis of RB. Consideration of oxidative stress components in the clinical management of RB patients is emphasized.

Fatty acid synthase (FASN), a multi-enzyme complex, is involved in lipid biosynthesis. FASN is over-expressed in different types of cancers and is being widely investigated for its role in cancer progression, diagnosis and therapy. Here, three inhibitors targeting different domains of FASN--cerulenin, triclosan and orlistat--were evaluated for their anti-proliferative efficacy in ocular cancer, retinoblastoma (RB) cells and their toxicity (if any) in normal cells. FASN inhibitors were tested in cultured retinoblastoma Y79 cells, normal fibroblast (3T3) and Muller glial (MIOM1) cells. Cell viability was determined by MTT-based assay, and IC(50) (50% inhibitory concentration) of the FASN inhibitors was calculated in neoplastic and non-neoplastic cells. The IC(50) after 48 and 96 hr of incubation with the three anti-FASN agents showed that cerulenin, triclosan and orlistat inhibited retinoblastoma cell proliferation in a dose- and time-dependent manner. The cancer cells exhibited differential dose- and time-dependent response/sensitivities to cerulenin, triclosan and orlistat. The 48-hr neoplastic IC(50) dosages were, however, not toxic to the normal cells. These findings were confirmed by phase-contrast microscopic assessment of cell morphology. Therapeutic index (TI) was calculated as a ratio of the IC(50) normal cells, to the IC(50) neoplastic cells. Relative to normal MIOM1 cells, TI was 9.18 for cerulenin, while 5.32 for triclosan and 1.72 for orlistat. The TI computed relative to 3T3 cells was 28.64, 7.10 and 2.58 for cerulenin, triclosan and orlistat, respectively. DNA fragmentation analysis suggests that FASN inhibitors induced apoptotic DNA damage in retinoblastoma cells. Thus, FASN inhibition can be an effective strategy in retinoblastoma therapy.

Purpose: To understand the disease mechanism and to identify the potential tumor markers that would help in therapeutics, comparative proteomic analysis of 29 retinoblastoma (RB) tumors was performed using 14 non-neoplastic retinas (age ranged from 45 to 89 years) as control tissues. Experimental design: 2-DE and MALDI-TOF-TOF MS/MS were used to identify differentially expressed proteins. Results: Twenty-seven distinct differentially expressed proteins were identified, including 16 upregulated 11 downregulated proteins. Significantly, higher mRNA levels of apolipoprotein A1 (po0.001), transferrin (TF; po0.001), CRABP2 (po0.001), a-crystallin A (CRYAA; po0.001) were observed in RBs when compared with normal retinas and hence are consistent with the proteomic data. Immunohistochemistry was also performed for selected proteins on paraffin RB blocks to confirm protein expression. RB with invasion showed significantly higher expression by 2-DE-MS/MS analysis of CRABP2 (po0.001), peroxiredoxin 6 (p 50.025), apolipoprotein A1 (po0.001), recoverin (po0.001). Conclusions and clinical relevance: Thus, this study provides a dynamic protein profile of RB tumors, which could provide clues to study the mechanisms of RB oncogenesis and possibly be developed as potential biomarkers for prognosis and therapy.

Retinoblastoma (RB) is the most common primary intraocular malignancy of infancy and childhood, where tumor invasion into the choroid, optic nerve, and/or orbit are risk factors for metastasis. Here we have correlated oxidative stress with the clinicopathologic characteristics of retinoblastoma.Tumor samples were processed for histopathologic examination. Malondialdehyde, a biomarker of oxidative stress, was immunohistochemically analyzed in 34 archival retinoblastoma tumor specimens, which included 17 tumors that did not have any invasion of the choroid, optic nerve, and/or orbit, and another 17 tumors that had some form of invasion of the choroid, optic nerve, and/or orbit. Lipid peroxidation levels were biochemically measured in another cohort of retinoblastoma tissue samples (n = 16), and correlated clinicopathologically.Malondialdehyde immunostaining was positive in all 34/34 (100%) tumors, and their corresponding clinicopathologic features were recorded. Malondialdehyde levels were significantly higher in tumors with invasion of the choroid, optic nerve, and orbit, when compared with tumors with no invasion (p0.05). No significant correlation was noted between malondialdehyde immunoreactivity and the differentiation/laterality of the tumors. Lipid peroxidation levels increased significantly in tumors with invasion of the choroid, optic nerve, and orbit than in tumors without invasion (p0.05).RB with invasion of the choroid, optic nerve, and/or orbit strongly correlates with increased oxidative stress.

PurposeRetinoblastoma (RB), a primary pediatric intraocular tumor, arises from primitive retinal layers. Several novel molecular strategies are being developed for the clinical management of RB. miRNAs are known to regulate cancer-relevant biological processes. Here, the role of selected miRNAs, namely, miR-532-5p and miR-486-3p, has been analyzed for potential therapeutic targeting in RB.MethodsA comprehensive bioinformatic analysis was performed to predict the posttranscriptional regulators (miRNAs) of the select panel of genes [Group 1: oncogenes (HMGA2, MYCN, SYK, FASN); Group 2: cancer stem cell markers (TACSTD, ABCG2, CD133, CD44, CD24) and Group 3: cell cycle regulatory proteins (p53, MDM2)] using Microcosm, DIANALAB, miRBase v 18, and REFSEQ database, and RNA hybrid. The expressions of five miRNAs, namely, miR-146b-5p, miR-532-5p, miR-142-5p, miR-328, and miR-486-3p, were analyzed by qRT-PCR on primary RB tumor samples (n = 30; including 17 invasive RB tumors and 13 noninvasive RB tumors). Detailed complementary alignment between 5’ seed sequence of differentially expressed miRNAs and the sequence of target genes was determined. Based on minimum energy level and piCTAR scores, the gene targets were selected. Functional roles of these miRNA clusters were studied by using mimics in cultured RB (Y79, Weri Rb-1) cells in vitro. The gene targets (SYK and FASN) of the studied miRNAs were confirmed by qRT-PCR and western blot analysis. Cell proliferation and apoptotic studies were performed.ResultsNearly 1948 miRNAs were identified in the in silico analysis, From this list, only 9 upregulated miRNAs (miR-146b-5p, miR-305, miR-663b, miR-299, miR-532-5p, miR-892b, miR-501, miR-142-5p, and miR-513b) and 10 downregulated miRNAs (miR-1254, miR-328, miR-133a, miR-1287, miR-1299, miR-375, miR-486-3p, miR-720, miR-98, and miR-122*) were found to be common with the RB serum miRNA profile. Downregulation of five miRNAs (miR-146b-5p, miR-532-5p, miR-142-5p, miR-328, and miR-486-3p) was confirmed experimentally. Predicted common oncogene targets (SYK and FASN) of miR-486-3p and miR-532-5p were evaluated for their mRNA and protein expression in these miRNA mimic-treated RB cells. Experimental overexpression of these miRNAs mediated apoptotic cell death without significantly altering the cell cycle in RB cells.ConclusionKey miRNAs in RB pathogenesis were identified by an in silico approach. Downregulation of miR-486-3p and miR-532-5p in primary retinoblastoma tissues implicates their role in tumorigenesis. Prognostic and therapeutic potential of these miRNA was established by the miRNA mimic strategy.

In the hypercholesterolemic state, the net result of combined oxidative and nitrosative stress is a pro-inflammatory phenotype that is manifested as increased adhesion molecule expression, enhanced leucocyte trafficking, and increased vascular permeability. The present work explores the inflammatory aspects of hypercholesterolemic atherogenesis, and also evaluates the role of a low-molecular-weight heparin derivative (LMWH), Certoparin, on a biochemical basis.Two groups of male Wistar rats were fed an atherogenic diet (normal rat chow plus 4% cholesterol, 1% cholic acid and 0.5% thiouracil-CCT diet) for 2 weeks. While one was left untreated, the other was administered LMWH (300 microg/day/rat commencing on day 8 and continued for a week). Increased concentrations of plasma C-reactive protein and fibrinogen and cardiac TNF-alpha indicated severe inflammation in the atherogenic diet fed rats. In comparison, these biochemical indices of inflammation diminished significantly in the LMWH treated group (p0.001). Significant depletion of thiols, along with accentuated activities of the glutathione metabolising was observed in the cardiac and hepatic tissues of the untreated atherogenic rats, indicating heightened oxidative response. Tissue damage was marked by elevated levels of plasma and tissue hexose, hexosamine, hexuronic acid and sialic acid, which were reversed towards normalcy on LMWH administration. The activities of lysosomal enzymes (N-acetyl glucosaminidase, beta-glucuronidase, beta-galactosaminidase and cathepsin-D) showed a marked increase in the CCT-diet fed rats, while LMWH treated rats showed normal activities (p0.001). The osmotic fragility test revealed that the untreated hyperlipidemic rat erythrocytes were significantly fragile at high salt concentrations, while the response was normalized in the LMWH treated group (p0.05). Further, hypercholesterolemia induced downregulation of physiological nitric oxide levels was corrected upon treatment with heparin-derivative.The results of this work highlight the inflammatory changes in atherogenic conditions and that the low-molecular-weight heparin derivative affords substantial protection.