Expressed Sequence Tags for Bovine Muscle Satellite Cells,Myotube Formed-Cells and Adipocyte-Like Cells

Background

Muscle satellite cells (MSCs) represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs) and adipocyte-like cells (ALCs), we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs.

Results

A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248) highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2) during myogenesis and hemoglobin subunit alpha2 (HBA2) during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay.

Conclusion

In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.     

Intensified Tuberculosis Case Finding among Malnourished Children in Nutritional Rehabilitation Centres of Karnataka,India: Missed Opportunities

Background

Severe acute malnutrition (SAM) is the most serious form of malnutrition affecting children under-five and is associated with many infectious diseases including Tuberculosis (TB). In India, nutritional rehabilitation centres (NRCs) have been recently established for the management of SAM including TB. The National TB Programme (NTP) in India has introduced a revised algorithm for diagnosing paediatric TB. We aimed to examine whether NRCs adhered to these guidelines in diagnosing TB among SAM children.

Methods

A cross-sectional study involving review of records of all SAM children identified by health workers during 2012 in six tehsils (sub-districts) with NRCs (population: 1.8 million) of Karnataka, India.

Results

Of 1927 identified SAM children, 1632 (85%) reached NRCs. Of them, 1173 (72%) were evaluated for TB and 19(2%) were diagnosed as TB. Of 1173, diagnostic algorithm was followed in 460 (37%). Among remaining 763 not evaluated as per algorithm, tuberculin skin test alone was conducted in 307 (41%), chest radiography alone in 99 (13%) and no investigations in 337 (45%). The yield of TB was higher among children evaluated as per algorithm (4%) as compared to those who were not (0.3%) (OR: 15.3 [95%CI: 3.5-66.3]). Several operational challenges including non-availability of a full-time paediatrician, non-functioning X-ray machine due to frequent power cuts, use of tuberculin with suboptimal strength and difficulties in adhering to a complex diagnostic algorithm were observed.

Conclusion

This study showed that TB screening in NRCs was sub-optimal in Karnataka. Some children did not reach the NRC, while many of those who did were either not or sub-optimally evaluated for TB. This study pointed to a number of operational issues that need to be addressed if this collaborative strategy is to identify more TB cases amongst malnourished children in India.     

Electronic, ductile, phase transition and mechanical properties of Lu-monopnictides under high pressures

The structural, elastic and electronic properties of lutatium-pnictides (LuN, LuP, LuAs, LuSb, and LuBi) were analyzed by using full-potential linearized augmented plane wave within generalized gradient approximation in the stable rock-salt structure (B1 phase) with space group Fm-3m and high-pressure CsCl structure (B2 phase) with space group Pm-3m. Hubbard-U and spin-orbit coupling were included to predict correctly the semiconducting band gap of LuN. Under compression, these materials undergo first-order structural transitions from B1 to B2 phases at 241, 98, 56.82, 25.2 and 32.3 GPa, respectively. The computed elastic properties show that LuBi is ductile by nature. The electronic structure calculations show that LuN is semiconductor at ambient conditions with an indirect band gap of 1.55 eV while other Lu-pnictides are metallic. It was observed that LuN shows metallization at high pressures. The structural properties, viz, equilibrium lattice constant, bulk modulus and its pressure derivative, transition pressure, equation of state, volume collapse, band gap and elastic moduli, show good agreement with available data.

Synthesis,Characterization, and Anti‐Amoebic Activity of N‐(Pyrimidin‐2‐yl)benzenesulfonamide Derivatives

A new series of N‐(pyrimidin‐2‐yl)benzenesulfonamide derivatives, 3a – 3i and 4a – 4i , was synthesized from pyrimidin‐2‐amines, 2a – 2i , with the aim to explore their effects on in vitro growth of Entamoeba histolytica. The chemical structures of the compounds were elucidated by elemental analysis, FT‐IR, ¹H‐ and ¹³C‐NMR, and ESI mass‐spectral data. In vitro anti‐amoebic activity was evaluated against HM1 : IMSS strain of Entamoeba histolytica. The IC₅₀ values were calculated by using the double dilution method. The results were compared with the IC₅₀ value of the standard drug ‘metronidazole’. The selected compounds were tested for their cytotoxic activities by cell‐viability assay using H9C2 cardiac myoblasts cell line, and the results indicated that all the compounds displayed remarkable >80% viabilities to a concentration of 100 μg/ml.     

MicroRNA Regulation of Adipose Derived Stem Cells in Aging Rats

Background

Perturbations in abdominal fat secreted adipokines play a key role in metabolic syndrome. This process is further altered during the aging process, probably due to alterations in the preadipocytes (aka. stromal vascular fraction cells-SVF cells or adipose derived stem cells-ASCs) composition and/or function. Since microRNAs regulate genes involved both in development and aging processes, we hypothesized that the impaired adipose function with aging is due to altered microRNA regulation of adipogenic pathways in SVF cells.

Methodology and Principal Findings

Alterations in mRNA and proteins associated with adipogenic differentiation (ERK5 and PPARg) but not osteogenic (RUNX2) pathways were observed in SVF cells isolated from visceral adipose tissue with aging (6 to 30 mo) in female Fischer 344 x Brown Norway Hybrid (FBN) rats. The impaired differentiation capacity with aging correlated with altered levels of miRNAs involved in adipocyte differentiation (miRNA-143) and osteogenic pathways (miRNA-204). Gain and loss of function studies using premir or antagomir-143 validated the age associated adipocyte dysfunction.

Conclusions and Significance

Our studies for the first time indicate a role for miRNA mediated regulation of SVF cells with aging. This discovery is important in the light of the findings that dysfunctional adipose derived stem cells contribute to age related chronic diseases.     

Domperidone treatment attenuates stress-induced suppression of reproduction in viviparous mosquitofish Gambusia affinis

The aim of this study was to determine the effect of stress on reproduction and the possible involvement of dopaminergic systems in the reproductive stress response in the mosquitofish Gambusia affinis. Exposure of fish to aquaculture stressors (four 10 min episodes of stress, each corresponding to a different stressor such as handling, chasing, frequent netting and low water levels), for a period of 30 days caused reduction in the mean numbers of stage I–IV follicles associated with lower number of pregnant females and embryos in most of the developmental stages compared with experimental controls. Besides, increase in the intensity of labelling and the per cent area of tyrosine hydroxylase (TH; a rate-limiting enzyme in the biosynthetic pathway of catecholamines)- immunoreactive (ir) neurons was observed in the preoptic area (POA) and the nucleus preopticus (NPO) regions of the brain concomitant with reduction in the labelling of gonadotropin releasing hormone–immunoreactive (GnRH-ir) fibres in the proximal pars distalis (PPD) of the pituitary gland in stressed fish compared with experimental controls. Treatment of domperidone (DOM) caused an increase in the number of stage II and V follicles and promoted pregnancy rate concomitant with an increase in the number of embryos at various developmental stages compared with those of experimental controls. Similar treatment to stressed fish caused an increase in the number of stages I–V follicles compared with those in stress alone group. The GnRH fibres showed increased immunolabelling in stress + DOM treated fish compared with stress alone fish. On the other hand, TH-immunoreactivity in the POA and the NPO regions was reduced in stress + DOM treated fish compared with stress-alone group. These results suggest that stress inhibits follicular development and subsequent hatching success through the suppression of GnRH and that the inhibition appears to be mediated through dopamine, for the first time in a viviparous fish.     

Rev3, the catalytic subunit of Polζ, is required for maintaining fragile site stability in human cells

It has been long speculated that mammalian Rev3 plays an important, yet unknown role(s) during mammalian development, as deletion of Rev3 causes embryonic lethality in mice, whereas no other translesion DNA synthesis polymerases studied to date are required for mouse embryo development. Here, we report that both subunits of Polζ (Rev3 and Rev7) show an unexpected increase in expression during G₂/M phase, but they localize independently in mitotic cells. Experimental depletion of Rev3 results in a significant increase in anaphase bridges, chromosomal breaks/gaps and common fragile site (CFS) expression, whereas Rev7 depletion primarily causes lagging chromosome defect with no sign of CFS expression. The genomic instability induced by Rev3 depletion seems to be related to replication stress, as it is further enhanced on aphidicolin treatment and results in increased metaphase-specific Fanconi anemia complementation group D type 2 (FANCD2) foci formation, as well as FANCD2-positive anaphase bridges. Indeed, a long-term depletion of Rev3 in cultured human cells results in massive genomic instability and severe cell cycle arrest. The aforementioned observations collectively support a notion that Rev3 is required for the efficient replication of CFSs during G₂/M phase, and that the resulting fragile site instability in Rev3 knockout mice may trigger cell death during embryonic development.     

Carboxyl terminal domain basic amino acids of mycobacterial topoisomerase I bind DNA to promote strand passage

Bacterial DNA topoisomerase I (topoI) carries out relaxation of negatively supercoiled DNA through a series of orchestrated steps, DNA binding, cleavage, strand passage and religation. The N-terminal domain (NTD) of the type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn²⁺ finger motifs in the CTD. The Zn²⁺ finger motifs were found to be essential in Escherichia coli topoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial topoI lacks Zn²⁺ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. We also show that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the catalytic step. Although the NTD binds to DNA in a site-specific fashion to carry out DNA cleavage and religation, the basic residues in CTD bind to non-scissile DNA in a sequence-independent manner to promote the crucial strand passage step during DNA relaxation. The loss of Zn²⁺ fingers from the mycobacterial topoI could be associated with Zn²⁺ export and homeostasis.