Immunophenotypic characterization and tenogenic differentiation of mesenchymal stromal cells isolated from equine umbilical cord blood

Mesenchymal stem cells (MSCs) isolated from umbilical cord blood (UCB) in equines have not been well characterized with respect to the expression of pluripotency and mesenchymal markers and for tenogenic differentiation potential in vitro. The plastic adherent fibroblast-like cells isolated from 13 out of 20 UCB samples could proliferate till passage 20. The cells expressed pluripotency markers (OCT4, NANOG, and SOX2) and MSC surface markers (CD90, CD73, and CD105) by RT-PCR, but did not express CD34, CD45, and CD14. On immunocytochemistry, the isolated cells showed expression of CD90 and CD73 proteins, but tested negative for CD34 and CD45. In flow cytometry, CD29, CD44, CD73, and CD90 were expressed by 96.36??±?1.28%, 93.40??±?0.70%, 73.23??±?1.29% and 46.75??±?3.95% cells, respectively. The UCB-MSCs could be differentiated to tenocytes by culturing in growth medium supplemented with 50 ng/ml of BMP-12 by day 10. The differentiated cells showed the expression of mohawk homeobox (Mkx), collagen type I alpha 1 (Col1α1), scleraxis (Scx), tenomodulin (Tnmd) and decorin (Dcn) by RT-PCR. In addition, flow cytometry detected tenomodulin and decorin protein in 95.65?±?2.15% and 96.30?±?1.00% of differentiated cells in comparison to 11.30?±?0.10% and 19.45?±?0.55% cells, respectively in undifferentiated control cells. The findings support the observation that these cells may be suitable for therapeutic applications, including ruptured tendons in racehorses.     

Enzymatic properties of the ferredoxin-dependent nitrite reductase from Chlamydomonas reinhardtii. Evidence for hydroxylamine as a late intermediate in ammonia production

The ferredoxin-dependent nitrite reductase from the green alga Chlamydomonas reinhardtii has been cloned, expressed in Escherichia coli as a His-tagged recombinant protein, and purified to homogeneity. The spectra, kinetic properties and substrate-binding parameters of the C. reinhardtii enzyme are quite similar to those of the ferredoxin-dependent spinach chloroplast nitrite reductase. Computer modeling, based on the published structure of spinach nitrite reductase, predicts that the structure of C. reinhardtii nitrite reductase will be similar to that of the spinach enzyme. Chemical modification studies and the ionic-strength dependence of the enzyme’s ability to interact with ferredoxin are consistent with the involvement of arginine and lysine residues on C. reinhardtii nitrite reductase in electrostatically-stabilized binding to ferredoxin. The C. reinhardtii enzyme has been used to demonstrate that hydroxylamine can serve as an electron-accepting substrate for the enzyme and that the product of hydroxylamine reduction is ammonia, providing the first experimental evidence for the hypothesis that hydroxylamine, bound to the enzyme, can serve as a late intermediate during the reduction of nitrite to ammonia catalyzed by the enzyme.     

Utilization of tannery solid waste for protease production by Synergistes sp. in solid‐state fermentation and partial protease characterization

Synergistes sp. DQ560074 produced a protease in submerged fermentation (SmF) at 400–420 U/mL and in solid‐state fermentation (SSF) at 745–755 U/g. The protease, which belongs to the aspartic protease class, was active over a wide range of pH (5–7) and at high temperatures (25–45°C). The protease is stable and active in various polar protic solvents (50% v/v) like ethanol, isopropanol, n–butanol, in polar aprotic solvents (50% v/v) like acetonitrile, and in non‐polar solvents (50% v/v) such as ethylacetate and toluene, but not in hydrophilic organic solvents (methyl alcohol and acetone). As far as we know, this is the first contribution to the production of a mesophilic protease with solvent stability in SSF using a proteinaceous solid waste.     

Ectopic expression of AmNAC1 from Avicennia marina (Forsk.) Vierh. confers multiple abiotic stress tolerance in yeast and tobacco

Plant Cell, Tissue and Organ Culture (PCTOC) - Abiotic factors like salinity, drought and cold affect agricultural productivity substantially worldwide. NAC (NAM, ATAF1/2 and CUC2) family...     

Inter retrotransposon based genetic diversity and phylogenetic analysis among the Musa germplasm accessions

In the present investigation, the insertional polymorphisms of retro-elements were studied in the Musa germplasm available at ICAR-NRCB field gene bank using IRAP markers. The maximum number of polymorphic bands were produced by the primer pair Nikita and LTR 6150 (48) followed by LTR 6149 and 3′LTR (47) and minimum of 35 bands were produced by the primer pair Sukkula and LTR 6150. The bands produced were scored as 0 (absent) and 1 (present) and the resultant binary data was subjected to diversity analysis. The dendrogram consisted of two major clusters with members of Eumusa and Rhodochlamys in one indicating their genetic closeness and members of the genus Ensete in another cluster. Results of principal coordinate analysis were congruent to those obtained in hierarchial cluster analysis. The molecular markers used in this study could reveal intra and inter-group diversity among the Musa germplasm accessions with similarity co-efficient ranging from 0.41 to 0.99. IRAP marker system has performed excellently clustering the accessions based on both genomic and subgroup levels. The entire germplasm was found to be robust with no duplications indicating the diverse group of accessions available at ICAR-NRCB field gene bank. It has also exhibited high polymorphism and hence could be effectively used to detect the genetic relatedness among diverse genome of Musa.     

A Fourteen Gene GBM Prognostic Signature Identifies Association of Immune Response Pathway and Mesenchymal Subtype with High Risk Group

Background

Recent research on glioblastoma (GBM) has focused on deducing gene signatures predicting prognosis. The present study evaluated the mRNA expression of selected genes and correlated with outcome to arrive at a prognostic gene signature.

Methods

Patients with GBM (n = 123) were prospectively recruited, treated with a uniform protocol and followed up. Expression of 175 genes in GBM tissue was determined using qRT-PCR. A supervised principal component analysis followed by derivation of gene signature was performed. Independent validation of the signature was done using TCGA data. Gene Ontology and KEGG pathway analysis was carried out among patients from TCGA cohort.

Results

A 14 gene signature was identified that predicted outcome in GBM. A weighted gene (WG) score was found to be an independent predictor of survival in multivariate analysis in the present cohort (HR = 2^.507; B = 0^.919; p<0^.001) and in TCGA cohort. Risk stratification by standardized WG score classified patients into low and high risk predicting survival both in our cohort (p = <0^.001) and TCGA cohort (p = 0^.001). Pathway analysis using the most differentially regulated genes (n = 76) between the low and high risk groups revealed association of activated inflammatory/immune response pathways and mesenchymal subtype in the high risk group.

Conclusion

We have identified a 14 gene expression signature that can predict survival in GBM patients. A network analysis revealed activation of inflammatory response pathway specifically in high risk group. These findings may have implications in understanding of gliomagenesis, development of targeted therapies and selection of high risk cancer patients for alternate adjuvant therapies.     

Sedentary Behaviour and Physical Activity in South Asian Women: Time to Review Current Recommendations?

Objective

Our aims were to describe activity and sedentary behaviours in urban Asian women, with dysglycaemia (diagnosed at recruitment), and without dysglycaemia and examine the relative contribution of these parameters to their glycaemic status.

Methods

2800 urban women (30–45 years) were selected by random cluster sampling and screened for dysglycaemia for a final sample of 272 newly diagnosed, drug naive dysglycaemic and 345 normoglycaemic women. Physical activity and sedentary behaviours were assessed by the International Physical Activity Questionnaire (IPAQ). Demographic data, diet and anthropometry were recorded. Logistic regression analysis assessed contribution of all parameters to dysglycaemia and exposure attributable fractions were calculated.

Results

The mean energy expenditure on walking (2648.5±1023.7 MET-min/week) and on moderate and vigorous physical activity (4342.3±1768.1 MET-min/week) for normoglycemic women and dysglycaemic women (walking;1046.4±728.4 MET-min/week, moderate and vigorous physical activity; 1086.7±1184.4 MET-min/week) was above the recommended amount of physical activity per week. 94.3% of women spent >1000 MET-minutes/week on activity. Mean sitting and TV time for normoglycaemic and dysglycaemic women were 154.3±62.8, 38.4±31.9, 312.6±116.7 and 140.2±56.5 minutes per day respectively. Physical activity and sedentary behaviour contributed to dysglycaemia after adjustment for family history, diet, systolic blood pressure and Body Mass Index. Exposure attributable fractions for dysglycaemia were; lower physical activity: 78%, higher waist circumference: 94%, and TV viewing time: 85%.

Conclusions

Urban South Asian women are at risk of dysglycaemia at lower levels of sedentary behaviour and greater physical activity than western populations, indicating the need for re-visiting current physical activity guidelines for South Asians.     

Depletion of 14-3-3 protein exacerbates cardiac oxidative stress, inflammation and remodeling process via modulation of MAPK/NF-?B signaling pathways after streptozotocin-induced diabetes mellitus

Diabetic cardiomyopathy is associated with increased oxidative stress and inflammation. Mammalian 14-3-3 proteins are dimeric phosphoserine-binding proteins that participate in signal transduction and regulate several aspects of cellular biochemistry. The aim of the study presented here was to clarify the role of 14-3-3 protein in the mitogen activated protein kinase (MAPK) and nuclear factor-kB (NF-κB) signaling pathway after experimental diabetes by using transgenic mice with cardiac-specific expression of a dominant-negative 14-3-3 protein mutant (DN 14-3-3). Significant p-p38 MAPK activation in DN 14-3-3 mice compared to wild type mice (WT) after diabetes induction and with a corresponding up regulation of its downstream effectors, p-MAPK activated protein kinase 2 (MAPKAPK-2). Marked increases in cardiac hypertrophy, fibrosis and inflammation were observed with a corresponding up-regulation of atrial natriuretic peptide, osteopontin, connective tissue growth factor, tumor necrosis factor α, interleukin (IL)-1β, IL-6 and cellular adhesion molecules. Moreover, reactive oxygen species, left ventricular expression of NADPH oxidase subunits, p22 phox, p67 phox, and Nox4, and lipid peroxidation levels were significantly increased in diabetic DN 14-3-3mice compared to diabetic WT mice. Furthermore, myocardial NF-κB activation, inhibitor of kappa B-α degradation and mRNA expression of proinflammatory cytokines were significantly increased in DN 14-3-3 mice compared to WT mice after diabetes induction. In conclusion, our data suggests that depletion of 14-3-3 protein induces cardiac oxidative stress, inflammation and remodeling after experimental diabetes induction mediated through p38 MAPK, MAPKAPK-2 and NF-κB signaling.     

Fourier transform infrared microspectroscopy identifies protein propionylation in histone deacetylase inhibitor treated glioma cells

Histone deacetylase inhibitors (HDIs) have attracted considerable attention as potential drug molecules in tumour biology. In order to optimise chemotherapy, it is important to understand the mechanisms of regulation of histone deacetylase (HDAC) enzymes and modifications brought by various HDIs. In the present study, we have employed Fourier transform infrared microspectroscopy (FT‐IRMS) to evaluate modifications in cellular macromolecules subsequent to treatment with various HDIs. In addition to CH₃ (methyl) stretching bands at 2872 and 2960 cm^–1, which arises due to acetylation, we also found major changes in bands at 2851 and 2922 cm^–1, which originates from stretching vibrations of CH₂ (methylene) groups, in valproic acid treated cells. We further demonstrate that the changes in CH₂ stretching are concentration‐dependent and also induced by several other HDIs. Recently, HDIs have been shown to induce propionylation besides acetylation [1]. Since propionylation involves CH₂ groups, we hypothesized that CH₂ vibrational frequency changes seen in HDI treated cells could arise due to propionylation. As verification, pre‐treatment of cells with propionyl CoA synthetase inhibitor resulted in loss of CH₂ vibrational changes in histones, purified from valproic acid treated cells. This was further proved by western blot using propionyl‐lysine specific antibody. Thus we demonstrate for the first time that propionylation could be monitored by studying CH₂ stretching using IR spectroscopy and further provide a platform for monitoring HDI induced multiple changes in cells. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Contributions of the Epstein-Barr virus EBNA1 protein to gastric carcinoma

Approximately 10% of gastric carcinomas (GC) are comprised of cells latently infected with Epstein-Barr virus (EBV); however, the mechanism by which EBV contributes to the development of this malignancy is unclear. We have investigated the cellular effects of the only EBV nuclear protein expressed in GC, EBNA1, focusing on promyelocytic leukemia (PML) nuclear bodies (NBs), which play important roles in apoptosis, p53 activation, and tumor suppression. AGS GC cells infected with EBV were found to contain fewer PML NBs and less PML protein than the parental EBV-negative AGS cells, and these levels were restored by silencing EBNA1. Conversely, EBNA1 expression was sufficient to induce the loss of PML NBs and proteins in AGS cells. Consistent with PML functions, EBNA1 expression decreased p53 activation and apoptosis in response to DNA damage and resulted in increased cell survival. In addition, EBNA1 mutants unable to bind CK2 kinase or ubiquitin-specific protease 7 had decreased ability to induce PML loss and to interfere with p53 activation. PML levels in EBV-positive and EBV-negative GC biopsy specimens were then compared by immunohistochemistry. Consistent with the results in the AGS cells, EBV-positive tumors had significantly lower PML levels than EBV-negative tumors. The results indicate that EBV infection of GC cells leads to loss of PML NBs through the action of EBNA1, resulting in impaired responses to DNA damage and promotion of cell survival. Therefore, PML disruption by EBNA1 is one mechanism by which EBV may contribute to the development of gastric cancer.