Molecular insights into Pashmina fiber production: comparative skin transcriptomic analysis of Changthangi goats and sheep.

Ladakh, one of the highest inhabited regions globally, hosts the unique Changthangi goat, renowned for producing Pashmina, the world's most luxurious natural fiber. In comparison, the fiber derived from Changthangi sheep is considered next only to Pashmina. This research endeavors to compare the skin transcriptome profiles of Changthangi goats and Changthangi sheep, aiming to discern the molecular determinants behind the recognition of Changthangi goats as the source of Pashmina. Drawing upon previously conducted studies, a collective of 225 genes correlated with fiber characteristics were extracted from the differentially expressed genes noticed between the two species (p-value of ≤ 0.05 and a log2 fold change of ≥ 1.5). These genes were analyzed using DAVID software to understand their biological functions and to identify enriched KEGG and Reactome pathways. The protein-protein interaction networks were constructed using Cytoscape, cytoHubba, and STRING to focus on key genes and infer their biological significance. Comparative transcriptome analysis revealed significantly higher expression of genes involved in signaling pathways like Wnt, MAPK, PI3K-Akt, Hedgehog, associated with fiber development and quality in Changthangi goats. These pathways play crucial roles in hair follicle (HF) formation, maintenance of epidermal stem cells, and fiber characteristics. Findings also highlight the enrichment of cell adhesion molecules and ECM-receptor interaction, emphasizing their roles in HF structure, growth, and signaling. This investigation offers an in-depth understanding of the molecular intricacies governing Pashmina production in Changthangi goats, providing valuable insights into their unique genetic makeup and underlying mechanisms influencing the exceptional quality of Pashmina fibers.

Comprehensive evaluation and validation of optimal reference genes for normalization of qPCR data in different caprine tissues.

BACKGROUND: Quantitative real-time PCR (qPCR) is a highly reliable method for validating gene expression data in molecular studies due to its sensitivity, specificity, and efficiency. To ensure accurate qPCR results, it's essential to normalize the expression data using stable reference genes. METHODS: This study aimed to identify suitable reference genes for qPCR studies in goats by evaluating 18 candidate reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) in 10 different caprine tissues (heart, intestine, kidney, liver, lung, muscle, rumen, skin, spleen, and testis). An integrated tool called RefFinder, which incorporates various algorithms like NormFinder, GeNorm, BestKeeper, and ΔCt, was used to assess the stability of expression among these genes. RESULTS: After thorough analysis, ACTB, PPIB, and B2M emerged as the most stable reference genes, while RPL19, RPS15, and RPS9 were found to be the least stable. The suitability of the selected internal control genes was further validated through target gene analysis, confirming their efficacy in ensuring accurate gene expression profiling in goats. CONCLUSION: The study determined that the geometric average of ACTB, PPIB, and B2M creates an appropriate normalization factor for gene expression studies in goat tissues.

Skeletal muscle transcriptomics of sheep acclimated to cold desert and tropical regions identifies genes and pathways accentuating their diversity.

The current study attempts to investigate the differences in gene expression in longissimus thoracis muscles between sheep breeds acclimated to diverse environments. Changthangi sheep inhabits the cold arid plateau of Ladakh, at an altitude above 3000 m with prevalence of rarefied atmosphere. Muzzafarnagri sheep, on the other hand is found in the sub-tropical hot and humid plains at an altitude of about 250 m. Comparative transcriptomics was used to provide a molecular perspective of the differential adaptation of the two breeds. RNA sequencing data was generated from four biological replicates of the longissimus thoracis muscles from both breeds. The common genes expressed in both breeds were involved in muscle contraction and muscle fibre organization. The most significant pathways enriched in Changthangi muscles were glycogen metabolism, reduction of cytosolic Ca++ levels and NFE2L2 regulating anti-oxidant, while those in Muzzafarnagri were extracellular matrix organization and collagen formation. The hub genes identified in Changthangi were involved in hematopoiesis and HIF signaling pathway, suggesting the molecular acclimatization of Changthangi to the high altitude cold desert of Ladakh. The nodal genes discovered in Muzzafarnagri sheep were associated with the extracellular matrix which accentuates its significance in the development, growth and repair of muscles. The observed transcriptomic differences underscore the morphological and adaptive disparity between the two breeds. The candidate genes and pathways identified in this study will form the basis for future research on adaptation to high altitude and body size in small ruminants.

MeDIP-sequencing for profiling global DNA methylation in buffalo embryos produced by in vitro fertilization.

Assisted reproductive technique like in vitro fertilization has contributed immensely in producing genetically improved livestock. Production of embryos under in vitro conditions can affect global DNA methylation pattern during the course of embryonic development. The present study is aimed at the generation and comparison of global DNA methylome of embryos at 2-cell, 8-cell and blastocyst stage of buffalo embryos produced by in vitro fertilization using MeDIP-Sequencing. It is observed that there is a profound difference in the global DNA methylation profile of IVF embryos at different developmental stages. These differences are manifested throughout the course of embryonic development. Pathways like Wnt signaling pathway, gonadotropin-releasing hormone receptor pathway and integrin signaling were found to be majorly affected by hypermethylation of DNA in IVF embryos throughout the development.

Sequence characterization and comparative expression profile of buffalo WNT10B gene in adult and fetal tissues.

In this study, Wingless-type MMTV (mouse mammary tumor virus) integration site family member (WNT10B) gene was sequence characterized in the Indian water buffalo. Sequence analysis revealed an open reading frame of 1176 nucleotides in buffalo, encoding 391 amino acids long protein. Nineteen nucleotide variations were observed between cattle and buffalo resulting in six amino acid changes. Phylogenetic analysis showed the clustering of ruminant species together. Real-time expression analysis of WNT10B in tissues collected from different organs of fetal and adult buffalo, revealed, the gene being abundantly expressed in the rumen and liver of the fetus. The fetal ovary, heart, kidney, lung, testis and mammary gland showed moderate expression, while in adult tissues, expression was high in the ovary, testis, brain, kidney, small intestine and liver, whereas lower expression was observed in the adult rumen. Significant differences in WNT10B expression levels were found for the brain, small intestine, testes, kidney, heart, rumen, and ovary when adult and fetal tissues were compared. A moderate level of genetic variation was found between cattle and buffalo WNT10B and expression patterns in a variety of tissues in adult buffalo implies that in addition to possible roles in adipogenesis and hematopoiesis, the WNT10B gene might be playing a significant role in other regulatory pathways as well.

Transcriptomic diversity in longissimus thoracis muscles of Barbari and Changthangi goat breeds of India.

The present study is an attempt to examine the differential expression of genes in longissimus thoracis muscles between meat and wool type Indian goat breeds. Barbari goat is considered the best meat breed while Changthangi is famous for its fine fibre quality. RNA sequencing data was generated from four biological replicates of longissimus thoracis muscles of Barbari and Changthangi goats. A clear demarcation could be observed between the breeds in terms of expression of genes associated with lipid metabolism (FASN, SCD, THRSP, DGAT2 and FABP3). Most significant genes with high connectivity identified by gene co-expression network analysis were associated with triacylglycerol biosynthesis pathway in Barbari goat. Highly interactive genes identified in Changthangi goat were mainly associated with muscle fibre type. This study provides an insight into the differential expression of genes in longissimus thoracis muscles between Barbari and Changthangi goats that are adapted to and reared in different agro-climatic regions.

Estimates of genetic parameters for linear body measurements and prediction of body weight in goat.

The objective of this study is to estimate genetic parameters for linear body measurements along with their correlation to live weight with a focus on devising a scale to predict live weights from body measurement. A total of 142,564 records on body measures and live weights were collected from 8701 Jamunapari goats. Genetic parameters were obtained for body length (L), height at withers (H) and heart girth (G) from birth to adult stage by univariate and multivariate analysis using the average information restricted maximum likelihood method. The best model for body measures at birth included the additive effect of animal and dam along with their covariance and maternal environment, whereas for traits measured later in life, the maternal environment was not significant. After accounting for the direct maternal correlation (ram ), the total heritability estimates for linear body measurements (L,H and G) at the preweaning and postweaning stages of growth ranged from 0.14 to 0.20. Significant genetic variability implies further scope for selection. The genetic correlations of live weight at birth, 3, 6, 9 and 12 months with corresponding L,H and G were high in magnitude indicating scope to select animals for higher weight using morphometric measurements. When weighing scales are unavailable in the field, prediction of weight using L and G was recommended [live weight = (0.291 × L) + (0.306 × G) - 16.8]. We recommend the use of body measurements in the Jamunapari goat breeding program owing to their high genetic correlation with corresponding live weights.

Fitting random regression models with Legendre polynomial and B-spline to model the lactation curve for Indian dairy goat of semi-arid tropic.

The present investigation aimed at genetic evaluation of tropical Indian dairy Jamunapari goat using random regression models (RRM) for the estimation of genetic parameters in the first three lactations across test days (TD) and also to come out with a pragmatic breeding plan in the nucleus. Variations in the lactation curves were modelled using 67,172 TD milk yield (TDMY) records. To obtain adequate and parsimonious models for the estimation of genetic parameters, orthogonal Legendre Polynomials (LP) and B-splines (BS) were compared. The analysis was carried out using a single-trait RRM approach. Average TDMY was 0.72, 0.81 and 0.79 kg in 1st to 3rd parities that also had 4th TD peak yield in common. BS function resulted in robust genetic parameters and a smoother curve for lactation as compared to LP. Maternal effects were evaluated and then dropped from the final model, owing to no significant contribution to the genetic variance. The best RRM was a quadratic BS function with six knots for the mean trend, curves of additive genetic, animal permanent environmental (c2 ) and 22 classes of residual variance. Additive variances and heritability (h2 ) estimates were higher in the early lactation. For first parity, the estimates of h2 varied between 0.19 to 0.35 across TD. Moderate h2 estimate suggests further scope for selection using desirable combinations of TD over the lactation. We observed a very high variance due to c2 across TD in three lactations. Genetic correlations were positive and larger between adjacent TDMY and weakened for distant TDMY. Looking into the robust estimates of genetic parameters and better fitting of lactation curve, we suggest the use of B-spline function for regular genetic evaluation of Jamunapari goat.

Influences of maternal factors on the estimate of genetic parameters for goat feed efficiency traits.

The objective of the present study was to estimate the genetic parameters for the feed efficiency traits in Barbari goats. The data records of 9332 progenies born to 413 sires and 2580 dams were collected with respect to the average daily weight gain (ADG), i.e., ADG1 (birth to weaning), ADG2 (weaning to 6 months), ADG3 (6 to 12 months), as well as derived trait Kleiber ratio (KR), i.e., KR1 (ADG1/3MW0.75), KR2 (ADG2/6MW0.75), and KR3 (ADG3/12MW0.75). The data were corrected for fixed covariates like period of kidding, the season of birth, sex, type of birth, and parity. Univariate and multivariate animal models with an average information function of restricted maximum likelihood (REML) were used to estimate genetic factors for these traits. The best model was evaluated based on the likelihood ratio test. The direct heritability estimates were 0.21 ± 0.03, 0.17 ± 0.03, 0.23 ± 0.04, 0.22 ± 0.04, 0.16 ± 0.04, and 0.26 ± 0.04 for ADG1, ADG2, ADG3, KR1, KR2, and KR3, respectively. However, they were inflated due to high and negative estimates of covariance between direct animal and maternal genetic effects. Moderate estimates of heritability augur the scope for improvement for feed efficiency traits. The maternal genetic effects (m2) significantly contributed to 3-12% of the total phenotypic variance. The realized heritability of mass selection, which takes into account direct and maternal genetic variance together, shows a low to moderate estimate of genetic variance for ADG and KR. The genetic correlation ranged from - 0.48 ± 0.11 (ADG1-KR3) to 0.95 ± 0.00 (ADG1-KR1), phenotypic correlation ranged from - 0.28 ± 0.01 (ADG2-KR3) to 0.94 ± 0.01 (ADG1-KR1), maternal genetic correlation ranged from - 0.22 (KR2-KR3) to 0.96 (ADG1-KR1) and - 0.69 (ADG1-KR3) to 0.95 (ADG1-KR1) for the maternal permanent environment, respectively. Kids can be indirectly chosen for higher feed efficiency since ADG and their associated KR have substantial genetic correlations. It is suggested that the KR should be used as a selection criterion for Barbari goats for improving feed efficiency.

Akt Phosphorylation Orchestrates T11TS Mediated Cell Cycle Arrest in Glioma Cells.

The novel anti-neoplastic glycopeptide T11TS retards glioma both in in-vitro clinical samples and in-vivo models. This study investigates the correlation between altering the glioma microenvironment with glioma arrest and death. Flow cytometry, immunoblotting, ELISA, and co-immunoprecipitation were employed to investigate glioma cell arrest and death. Results include a decline in phosphorylation of Akt and attenuation of p21 phosphorylation (Thr145,Ser146) and disassociation of p-Akt-Mdm2 and p-Akt-BAD facilitating death by Akt>BAD. T11TS influence phosphorylation patterns in two focal axes Akt>p21 and Akt>Mdm2>p53. The current article provides crucial insight in deciphering the mechanism of T11TS induced glioma cell arrest and death.

Low oxygen tension potentiates proliferation and stemness but not multilineage differentiation of caprine male germline stem cells.

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O2) environment, whereas, their in-vitro expansion is typically performed under normoxia (20-21% O2). The comparative information about the effects of low and normal O2 levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (ß-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O2) and normoxia (21% O2). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p < 0.05) improved, thus, yielding a significantly (p < 0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O2 conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and ß-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

Media switching at different time periods affects the reprogramming efficiency of buffalo fetal fibroblasts.

Many contrasting reports are available on generation of bovine induced pluripotent stem cells (iPSCs) employing different timelines and culture conditions which signifies reprogramming process varies between species and cell types. The present study determines an optimum time period required to re-initiate reprogramming events in buffalo fibroblasts after introduction of exogenous genes (OCT4, SOX2, KLF4 and c-MYC) by lentiviral vector. The reprogramming efficiency is cumulative result of many factors including culture conditions and addition of growth factors in culture media. In our study, we observed when stem cell culture conditions were provided Day 5 post-transduction, it results in maximum reprogramming efficiency in comparison when same conditions were provided too early or on later days. The putative iPSCs were expanded on feeder layer for 15 passages and found positive for alkaline phosphatase and pluripotency markers (OCT4, SOX2, KLF4, c-MYC, UTF, TELOMERASE, FOXD3, REX1, STAT3, NUCLEOSTAMIN and TRA1-81). Also, they produced embryoid bodies showing expression for ectodermal (NF68, MOBP), mesodermal (ASA, BMP4) and endodermal (GATA4, AFP) markers to confirm their pluripotent nature. Our results suggest that reprogramming is accompanied by time dependent events and providing stem cell culture conditions at definite time during reprogramming can help in generation of iPSCs with greater efficiency.

First complete genome sequence of garlic virus X infecting Allium sativum- G282 from India.

The present report communicates the first full genome sequencing of the Garlic virus X from northern India. The total genome size of Garlic virus X (MK503771) reported in this study is 8458 bp ssRNA. The full genome sequence analysis showed the close relationship of Garlic virus X from India to that of from China, Korea, Australia and Spain. The full genome sequence based study of Indian Garlic virus X reveals the geographical relationship of this virus in India and global origin which may assists in development of control strategy for this virus.

Nutritional Status of Children with Biliary Atresia and the Role of Portoenterostomy to Improve Nutrition.

BACKGROUND: Nutritional depletion and growth stunting are present in patients with biliary atresia; "normal" nutrient and vitamin supplementation fail to correct these deficiencies. Children with this condition form the largest group for possible liver transplantation in the future; hence, stress should be laid on close attention to their nutrition. METHODS: Twenty-five patients with biliary atresia as cases and 25 age-matched children as controls were enrolled in the study from November 2010 to June 2012. Preoperatively, patients underwent standard investigations and anthropometric measurement (weight, height, and head circumference) assessment. Nutritional status (assessed with standard growth chart) was compared with control population, and children were divided into poor nutritional status and good nutritional status. Kasai's portoenterostomy was performed in all patients, and comparison was done between preoperative nutritional status with postoperative status of children and also between hepatic iminodiacetic acid (HIDA) scan-positive (patent bilioenteric pathway) children with HIDA scan-negative children. Postoperatively, after 12 weeks, the same anthropometric measurements were taken again, growth velocity (GV) was assessed, and children were divided into poor, average, and good GV. RESULTS: Nutritional status of children with biliary atresia was significantly poor than that of control group. Postoperatively, children had better nutritional status than preoperative nutritional status, especially in HIDA scan-positive children. GV was also significantly better in those children in whom postoperative HIDA scan was positive. CONCLUSION: Children with biliary atresia have poor nutritional status in comparison to normal population and require multifaceted approach to achieve adequate nutrition. Establishment of a patent bilioenteric pathway in these children improves their nutritional status and GV.

Biochemical and functional attributes of raw and boiled potato flesh and peel powders for suitability in food applications.

Four different potato cultivars, namely, Kufri Chipsona 1 and Kufri Frysona (processing purpose), Kufri Jyoti and Kufri Bahar (table purpose) were converted into flesh and peel powder (raw and after boiling) and studied for their respective biochemical and functional attributes to get an idea of possible dynamics of their utilization in different food formulation as bioadditives. The 16 variants of powder obtained retained less than 10% moisture content and demonstrated 'very good' to 'fair' flowability. Peel powders recorded a higher total mineral, fiber, phenolic contents and total antioxidant activity than the flesh powders which were significantly affected by boiling. Among raw and boiled flesh powders, highest reducing and total sugars were recorded for Kufri Bahar while least was observed in Kufri Chipsona 1. Colour coordinate showed that boiling imparts brightness to flesh powder while peel powder got darkened. Boiling of the tubers resulted in an increase in the resistant starch (~ 29% maximum) and flavour (~ 180% maximum) component. Peel exhibited a total glycoalkaloid content in the range of 0.75 (Kufri Frysona) to 1.7 mg/100 g (Kufri Bahar) that is well within the acceptable limits. Rheological study of the flesh powders revealed a reduction of about 11-18 °C in pasting temperature and about 87-90% in peak viscosity, setback, breakdown value and final viscosity upon boiling. This study revealed that the traditional processing method such as boiling can significantly modify the techno-functional characteristics of potato flesh and peel powders which can further govern their end use in various food formulations.

T11TS immunotherapy augments microglial and lymphocyte protective immune responses against Cryptococcus neoformans in the brain.

Cryptococcus neoformans, the encapsulated yeast acquired through inhalation, remains localized in lungs, but harbours the CNS in immunocompromised individuals. Several treatment regimes have failed combating this disease totally, but long-term usage of drugs leads to organ damage. As T11-target structure (T11TS) has documented profound immune potentiation, we aimed to investigate the role of microglia, pivotal immune cells of brain in ameliorating cryptococcosis, with T11TS immunotherapy. Murine model with C neoformans infection was prepared by intraperitoneal injection and the brains of rats examined 7 days post-infections for histopathology by PAS and Alcian blue staining corroborated with organ fungal burden evidencing restorative T11TS action on Cryptococcal meningitis. Immunotherapy with three doses of T11TS, a CD2 ligand, in C neoformans infected rats, upregulates toll-like receptors 2, -4 and -9 of microglia, indicating increased phagocytosis of the fungus. Flowcytometric analysis revealed increased numbers of T11TS treated brain infiltrating CD4+ and CD8+ T-lymphocytes along with increased MHC I and MHC II on microglia, activating the infiltrating lymphocytes aiding the killing mechanism. Present study also indicated that T11TS increased production of Th1 inflammatory cytokines conducive to fungal elimination while the inhibitory Th2 cytokines were dampened. This preclinical study is first of its kind to show that T11TS effected profound immune stimulation of microglial activity of C neoformans infected rats eradicating residual fungal burden from the brain and can be a useful therapeutic strategy in fighting against this deadly disease.

T11TS Treatment Augments Apoptosis of Glioma Associated Brain Endothelial Cells, Hint Toward Anti-Angiogenic Action in Glioma.

Malignant glioma continues to be a clinical challenge with an urgent need for developing curative therapeutic intervention. Apoptosis induction in tumor-associated endothelial cells represent a central mechanism that counteracts angiogenesis in glioma and other solid tumors. We previously demonstrated that intraperitoneal administration of sheep erythrocyte membrane glycopeptide T11-target structure (T11TS) in rodent glioma model inhibits PI3K/Akt pathway and Raf/MEK/ERK signaling in glioma-associated brain endothelial cells. In the present study, we investigated whether T11TS treatment influence apoptosis signaling in vivo in glioma-associated brain endothelial cells. Annexin-V/PI staining showed that T11TS treatment in glioma-induced rats increases apoptosis of glioma-associated endothelial cells within glioma milieu compared to brain endothelial cells in glioma induced and control groups. Flowcytometric JC-1 assay revealed that T11TS administration triggers loss of mitochondrial membrane potential in glioma-associated brain endothelial cells. Flowcytometry, immunoblotting, and in situ immunofluoresecnt imaging were employed to investigate the effect of T11TS on apoptotic regulatory proteins in brain endothelial cells. T11TS treatment-upmodulated expression of p53, Bax, Fas, FasL, and FADD in glioma associated endothelial cells and downregulated Bcl-2 protein. T11TS therapy induced cytochrome-c release into cytosol, activated caspase -9, 8, 3, and cleaved Bid in glioma associated brain endothelial cells. The study demonstrates that T11TS induces apoptosis in glioma-associated brain endothelial cells via p53 accumulation and activation of intrinsic as well as Fas-dependent extrinsic pathway. The pro-apoptotic action of T11TS on glioma-associated endothelial cells provides crucial insight into how T11TS exerts its anti-angiogenic function in glioma. J. Cell. Physiol. 232: 526-539, 2017. © 2016 Wiley Periodicals, Inc.

Metal homeostasis in bacteria: the role of ArsR-SmtB family of transcriptional repressors in combating varying metal concentrations in the environment.

Bacterial infections cause severe medical problems worldwide, resulting in considerable death and loss of capital. With the ever-increasing rise of antibiotic-resistant bacteria and the lack of development of new antibiotics, research on metal-based antimicrobial therapy has now gained pace. Metal ions are essential for survival, but can be highly toxic to organisms if their concentrations are not strictly controlled. Through evolution, bacteria have acquired complex metal-management systems that allow them to acquire metals that they need for survival in different challenging environments while evading metal toxicity. Metalloproteins that controls these elaborate systems in the cell, and linked to key virulence factors, are promising targets for the anti-bacterial drug development. Among several metal-sensory transcriptional regulators, the ArsR-SmtB family displays greatest diversity with several distinct metal-binding and nonmetal-binding motifs that have been characterized. These prokaryotic metolloregulatory transcriptional repressors represses the expression of operons linked to stress-inducing concentrations of metal ions by directly binding to the regulatory regions of DNA, while derepression results from direct binding of metal ions by these homodimeric proteins. Many bacteria, e.g., Mycobacterium tuberculosis, Bacillus anthracis, etc., have evolved to acquire multiple metal-sensory motifs which clearly demonstrate the importance of regulating concentrations of multiple metal ions. Here, we discussed the mechanisms of how ArsR-SmtB family regulates the intracellular bioavailability of metal ions both inside and outside of the host. Knowledge of the metal-challenges faced by bacterial pathogens and their survival strategies will enable us to develop the next generation drugs.

Cumulus cell-conditioned medium supports embryonic stem cell differentiation to germ cell-like cells.

Cumulus cells provide cellular interactions and growth factors required for oogenesis. In vitro studies of oogenesis are limited primarily because of the paucity of their source, first trimester fetal gonads, and the small number of germ lineage precursor cells present within these tissues. In order to understand this obscure but vitally important process, the present study was designed to direct differentiation of embryonic stem (ES) cells into germ lineage cells. For this purpose, buffalo ES cells were differentiated, as embryoid bodies (EBs) and monolayer adherent cultures, in the presence of different concentrations of cumulus-conditioned medium (CCM; 10%, 20% and 40%) for different periods of culture (4, 8 and 14 days) to identify the optimum differentiation-inducing concentration and time. Quantitative polymerase chain reaction analysis revealed that 20%-40% CCM induced the highest expression of primordial germ cell-specific (deleted in Azoospermia- like (Dazl), dead (Asp-Glu-Ala-Asp) box polypeptide 4 (Vasa also known as DDX4) and promyelocytic leukemia zinc finger protein (Plzf)); meiotic (synaptonemal complex protein 3 (Sycp3), mutl homolog I (Mlh1), transition protein 1/2 (Tnp1/2) and protamine 2 (Prm2); spermatocyte-specific boule-like RNA binding protein (Boule) and tektin 1 (Tekt1)) and oocyte-specific growth differentiation factor 9 (Gdf9) and zona pellucida 2 /3 (Zp2/3)) genes over 8-14 days in culture. Immunocytochemical analysis revealed expression of primordial germ cell (c-KIT, DAZL and VASA), meiotic (SYCP3, MLH1 and PROTAMINE 1), spermatocyte (ACROSIN and HAPRIN) and oocyte (GDF9 and ZP4) markers in both EBs and monolayer differentiation cultures. Western blotting revealed germ lineage-specific protein expression in Day 14 EBs. The significantly lower (P<0.05) concentration of 5-methyl-2-deoxycytidine in differentiated EBs compared to undifferentiated EBs suggests that methylation erasure may have occurred. Oocyte-like structures obtained in monolayer differentiation stained positive for ZONA PELLUCIDA protein 4 and progressed through various embryo-like developmental stages in extended cultures.

Evaluation of the mobile nurse training (MNT) intervention - a step towards improvement in intrapartum practices in Bihar, India.

BACKGROUND: Evidence shows that improving the quality of intrapartum care is critical for maternal survival. However, a significant rise in the proportion of facility-based births over the last decade in India - attributable to a cash transfer program - has not resulted in a corresponding reduction in maternal mortality, thanks, in part, to low-skilled care at facilities. The current study evaluated a mobile knowledge-based intervention aimed at improving quality of care by mentoring in-service staff nurses at public obstetric facilities. METHODS: An independent evaluation team conducted baseline and post-intervention assessments at every facility using a mix of methods that included training assessments and Direct Observation of Deliveries. The assessment involved passive observation of pregnant women from the time of their admission at the facility and recording the obstetric events and delivery-related practices on a pre-formatted checklist-based tool. Maternal practices were classified into positive and negative ones and scored. Linear regression analysis was used to evaluate the association of MNT intervention with summary scores for positive, negative and overall practice scores. We evaluated retention of intervention effect by comparing the summary scores at baseline, immediately following intervention and 1 year after intervention. RESULTS: In both unadjusted and adjusted analyses, the intervention was found to be significantly associated with improvement in positive practice score (Unadjusted: parameter estimate (ß) = 16.90; 95% confidence interval (CI) = 15.20, 18.60. Adjusted: ß = 13.14; 95% CI = 10.97, 15.32). The intervention was also significantly associated with changes in negative practice score, which was reverse coded to represent positive change (Unadjusted: ß = 11.66; 95% CI = 10.06, 13.27. Adjusted: ß = 2.99; 95% CI = 1.35, 4.63), and overall practice score (Unadjusted: ß = 15.74; 95% CI = 14.39, 17.08; Adjusted: ß = 10.89; 95% CI = 9.18, 12.60). One year after the intervention, negative practices continued to improve, albeit at a slower rate; positive labor practices and overall labor practice remained higher than the baseline but with some decline over time. CONCLUSIONS: Findings suggest that in low resource settings, interventions to strengthen quality of human resources and care through mentoring works to improve intrapartum maternal care.