Myxobacteria: biology and bioactive secondary metabolites.

Myxobacteria are Gram-negative eubacteria and they thrive in a variety of habitats including soil rich in organic matter, rotting wood, animal dung and marine environment. Myxobacteria are a promising source of new compounds associated with diverse bioactive spectrum and unique mode of action. The genome information of myxobacteria has revealed many orphan biosynthetic pathways indicating that these bacteria can be the source of several novel natural products. In this review, we highlight the biology of myxobacteria with emphasis on their habitat, life cycle, isolation methods and enlist all the bioactive secondary metabolites purified till date and their mode of action.

Electroporation of Mouse Follicles, Oocytes and Embryos without Manipulating Zona Pellucida.

Electroporation is an effective technique of transfection, but its efficiency depends on the optimization of various parameters. In this study, a simplified and efficient method of gene manipulation was standardized through electroporation to introduce a recombinant green fluorescent protein (GFP) construct as well as RNA-inhibitors in intact mouse follicles, oocytes and early embryos, where various electroporation parameters like voltage, pulse number and pulse duration were standardized. Electroporated preantral follicles were cultured further in vitro to obtain mature oocytes and their viability was confirmed through the localization of a known oocyte maturation marker, ovastacin, which appeared to be similar to the in vivo-derived mature oocytes and thus proved the viability of the in vitro matured oocytes after electroporation. Standardized electroporation parameters, i.e., three pulses of 30 V for 1 millisecond at an interval of 10 s, were applied to manipulate the expression of mmu-miR-26a in preantral follicles through the electroporation of miR inhibitors and mimics. The TUNEL apoptosis assay confirmed the normal development of the electroporated embryos when compared to the normal embryos. Conclusively, for the first time, this study demonstrated the delivery of exogenous oligonucleotides into intact mouse follicles, oocytes and embryos without hampering their zona pellucida (ZP) and further development.

Expression pattern of GLUT 1, 5, 8 and citrate synthase transcripts in buffalo (Bubalus bubalis) preimplantation embryos produced in vitro and derived in vivo.

In vitro-produced (IVP) embryos are reported to be developmentally lesser competent than in vivo-derived (IVD) embryos and supposed to differ in the expression of genes related with glucose metabolism. So, the present study was conducted to analyse the expression pattern of GLUT 1, 5, 8 and citrate synthase (CS) in oocytes and embryos produced in vivo or in vitro in buffalo. IVD embryos were obtained from 18 superovulated buffaloes. IVP embryos were obtained from slaughterhouse-derived oocytes subsequently subjected to in vitro fertilization and culture. Total RNA was isolated from different stages of oocytes (immature and in vitro matured) and embryos (8-16 cell to blastocysts of IVP embryos and morula to blastocysts of IVD embryos). Results demonstrated that the expression of GLUT1, GLUT 8 increased from 8 to 16 cells to blastocyst and was significantly (p < .05) higher in IVP embryos. Expression of both genes was (p < .05) higher in IVD than in IVP blastocysts; though GLUT5 transcripts were not detected at 8- to 16-cell stage IVP embryos, significantly (p < .05) higher transcripts were found at morula and blastocyst stages irrespective of embryo source with significantly (p < .05) higher expression in IVD embryos compared to IVP embryos. No significant difference was observed in citrate synthase expression in embryos at morula stage irrespective of the embryo source while significantly (p < .05) higher transcript level was observed at blastocyst stage with no difference between in vivo and in vitro embryos. It can be concluded that expression of GLUTs and CS is upregulated with progression of embryonic stage and expression is higher in in vivo embryos than in vitro counter parts; thus, it can be said that in vivo-produced embryos are metabolically superior to in vitro embryos.

Barrett's Registry Collaboration of academic centers in Ireland reveals high progression rate of low-grade dysplasia and low risk from nondysplastic Barrett's esophagus: report of the RIBBON network.

Barrett's esophagus (BE) is the main pathological precursor of esophageal adenocarcinoma (EAC). Progression to high-grade dysplasia (HGD) or EAC from nondysplastic BE (NDBE), low-grade dysplasia (LGD) and indefinite for dysplasia (IND) varies widely between population-based studies and specialized centers for many reasons, principally the rigor of the biopsy protocol and the accuracy of pathologic definition. In the Republic of Ireland, a multicenter prospective registry and bioresource (RIBBON) was established in 2011 involving six academic medical centers, and this paper represents the first report from this network. A detailed clinical, endoscopic and pathologic database registered 3,557 patients. BE was defined strictly by both endoscopic evidence of Barrett's epithelium and the presence of specialized intestinal metaplasia (SIM). A prospective web-based database was used to gather information with initial and follow-up data abstracted by a data manager at each site. A total of 2,244 patients, 1,925 with no dysplasia, were included with complete follow-up. The median age at diagnosis was 60.5 with a 2.1:1 male to female ratio and a median follow-up time of 2.7 years (IQR 1.19-4.04), and 6609.25 person years. In this time period, 125 (5.57%) progressed to HGD/EAC, with 74 (3.3%) after 1 year of follow-up and 38 (1.69%) developed EAC, with 20 (0.89%) beyond 1 year. The overall incidence of HGD/EAC was 1.89% per year; 1.16% if the first year is excluded. The risk of progression to EAC alone overall was 0.57% per year, 0.31% excluding the first year, and 0.21% in the 1,925 patients who had SIM alone at diagnosis. Low-grade dysplasia (LGD) progressed to HGD/EAC in 31% of patients, a progression rate of 12.96% per year, 6.71% with the first year excluded. In a national collaboration of academic centers in Ireland, the progression rate for NDBE was similar to recent population studies. Almost one in two who progressed was evident within 1 year. Crucially, LGD diagnosed and confirmed by specialist gastrointestinal pathologists represents truly high-risk disease, highlighting the importance of expertise in diagnosis and management, and providing indirect support for ablative therapies in this context.

The effect of post-synthesis aging on the ligand exchange activity of iron oxide nanoparticles.

HYPOTHESIS: Ligand exchange is a widely-used method of controlling the surface chemistry of nanomaterials. Exchange is dependent on many factors including the age of the core particle being modified. Aging of the particles can impact surface structure and composition, which in turn can affect ligand binding. EXPERIMENTS: To quantify the effects of aging on ligand exchange, we employed a technique to track the exchange of radiolabeled 14C-oleic acid with unlabeled, oleic acid bound to iron oxide nanoparticles. Liquid scintillation counting (LSC) was used to determine the amount of 14C-oleic acid adsorbing to the particles throughout the duration of the exchange for particles aged for 2days, 7days, and 30days. FINDINGS: Results revealed an increase in the total amount of ligands exchanged with aging up to 30days. Kinetic analysis of these results revealed a significant decrease in the overall rate of ligand exchange between 2 and 30days. The change in extent of adsorption with age could suggest increased availability of free binding sites. A follow-up study comparing exchange with oxidized and unoxidized particles suggested this increase in ligand adsorption may be due to changes in the Fe2+/Fe3+ ratio on the surface as the particles aged.

Evaluation of persistence and distribution of intra-dermally administered PKH26 labelled goat bone marrow derived mesenchymal stem cells in cutaneous wound healing model.

The current study was designed to study the persistence and distribution of caprine bone marrow derived mesenchymal stem cells (cBM-MSCs) when administered intra-dermally in experimentally induced cutaneous wounds in rabbits. MSC's from goat bone marrow were isolated and their differentiation potential towards adipogenic and osteogenic lineages were assayed in vitro. The isolated cells were phenotypically analysed using flow cytometry for the expression of MSC specific matrix receptors (CD73, CD105 and Stro-1) and absence of hematopoietic lineage markers. Further, these in vitro expanded MSCs were stained with PKH26 lipophilic cell membrane red fluorescent dye and prepared for transplantation into cutaneous wounds created on rabbits. Five, 2 cm linear full thickness skin incisions were created on either side of dorsal midline of New Zealand white rabbits (n = 4). Four wounds in each animal were implanted intra-dermally with PKH26 labelled cBM-MSCs suspended in 500 µl of Phosphate Buffer Saline (PBS). Fifth wound was injected with PBS alone and treated as negative control. The skin samples were collected from respective wounds on 3, 7, 10 and 14 days after the wound creation, and cryosections of 6 µM were made from it. Fluorescent microscopy of these cryosections showed that the PKH26 labelled transplanted cells and their daughter cells demonstrated a diffuse pattern of distribution initially and were later concentrated towards the wound edges and finally appeared to be engrafted with the newly developed skin tissues. The labelled cells were found retained in the wound bed throughout the period of 14 days of experimental study with a gradual decline in their intensity of red fluorescence probably due to the dye dilution as a result of multiple cell division. The retention of transplanted MSCs within the wound bed even after the complete wound healing suggests that in addition to their paracrine actions as already been reported, they may have direct involvement in various stages of intricate wound healing process which needs to be explored further.

Comparative study on characterization and wound healing potential of goat (Capra hircus) mesenchymal stem cells derived from fetal origin amniotic fluid and adult bone marrow.

Caprine amniotic fluid (cAF) and bone marrow cells (cBM) were isolated, expanded and phenotypically characterized by mesenchymal stem cells (MSCs) specific cell surface markers. Both cell types were compared for multilineage differentiation potential by flow cytometry using specific antibodies against lineage specific markers. Furthermore, in vitro expanded cAF-MSCs showed higher expression of trophic factors viz. VEGF and TGF-ß1 as compared to cBM-MSCs. Full-skin thickness excisional wounds created on either side of the dorsal midline (thoracolumbar) of New Zealand White rabbits were randomly assigned to subcutaneous injection of either fetal origin cAF-MSCs (n=4) or adult cBM-MSCs (n=4) or sterile PBS (control, n=4). The rate of wound closure was found faster (p<0.05) in cAF-MSCs treated wounds as compared with cBM-MSCs and PBS treated wounds especially on 21st day post-skin excision. Histomorphological examination of the healing tissue showed that wound healing was improved (p<0.05) by greater epithelialization, neovascularization and collagen development in cAF-MSCs as compared to cBM-MSCs and PBS treated wounds.

Effect of insulin supplementation on in vitro maturation of pre-antral follicles from adult and pre-pubertal mice.

This study was aimed to determine the impact of insulin concentrations on in vitro pre-antral follicle growth, survival, antrum formation rate, and retrieval of mature oocytes in mice. Mice pre-antral follicle growth were recorded on days 2, 4, 6, 8, 10, and 12 in α-modified essential media (α-MEM) supplemented with insulin concentrations of 6, 8, and 10 µg/ml along with 10% FBS, 100 mIU/ml follicle stimulating hormone, 10 mIU/ml luteinizing hormone, 100 µg/ml penicillin, and 50 µg/ml streptomycin. After 12 d of growth in vitro, follicles were allowed to mature for 16-18 h in α-MEM supplemented with 1.5 IU/ml human chorionic gonadotrophin (hCG) and 5 ng/ml epidermal growth factor (EGF). The initial diameter (54.86 ± 2.5 µm) of mice oocyte progressively increased in all the three insulin concentration groups and attained a maximum size on day 12 (71.90 ± 2.8 µm). Supplementation with higher concentrations of insulin (both 8 and 10 µg/ml) significantly enhanced antrum formation without effecting the oocyte diameter and percent retrieval of mature oocyte in all the three concentration groups. Both in vitro cultured as well as in vivo collected follicles and oocytes showed similar localization and expression of oocyte maturation markers SAS1B and GDF9. Insulin concentration of 8 µg/ml was found to be optimal for in vitro follicle culture of adult mice (42-49 d). Optimized follicle culture conditions were also assessed successfully with pre-pubertal mice (12-14 d); however, adult mice showed higher follicle survival, antrum formation, and more mature oocytes production in comparison to pre-pubertal mice.

Expression of mRNA encoding IGF-I, IGF-II, type-I,and II IGF-receptors and IGF-binding proteins-1-4 during ovarian follicular development in buffalo (Bubalus bubalis).

The present study was designed to investigate the expression pattern of IGF-I, IGF-II, type-I and II IGF-receptors, and IGFBP-1-4 in different stages of buffalo ovarian preantral follicles (PFs), antral follicles (AFs), ovulatory follicles (OFs), and immature (IM) and in vitro matured (MO) oocytes. Buffalo ovaries were collected from local abattoir, PFs (200-250 µm), AFs (1-3 mm), and OFs (5-8 mm) were isolated by mechanical method. PFs, AFs, OFs, and oocytes were lysed to release mRNA, reverse transcribed, and then subjected to RT-PCR, whereas protein were localized through immunohistochemistry. Relative expression of mRNA transcripts was clearly seen for IGF-II, type-I and II IGF-receptors, and IGFBP-1-4 in all the stages of developing follicles and oocytes. We were unable to detect mRNA and protein expression of IGF-1 in any of the oocytes or follicles at any stage of the development. IGF-II and both IGF receptors mRNA expression were found higher (P < 0.05) in PFs compared to AFs and OFs. Expression of IGFBP-1 and 2 in PFs, as well as IGFBP-3 and 4 in AFs, was found with higher (P < 0.05) levels. The expression results were further confirmed by localization of IGF-II, type-I and II IGF-receptors, and IGFBP-1-4 proteins. In conclusion, IGF-II appears to be the only ligand that is endogenously expressed by all the follicular stages and oocytes, which may act in an autocrine manner through the Type-1 IGF receptor. Expression of IGFBP-1-4 and IGF-II suggests the possible role of these genes in recruitment, growth, proliferation, and steroidogenic responses during developmental phases of buffalo ovarian follicles.

Molecular characterization and xenogenic application of Wharton's jelly derived caprine mesenchymal stem cells.

Aim of the present study was in vitro expansion and characterization of caprine wharton's jelly derived mesenchymal stem cells (cWJ-MSCs) to investigate their tissue healing potential in xenogenic animal model. Plastic adherent fibroblastoid cell populations with distinctive homogeneous morphology were isolated from caprine Wharton's jelly explants. These Wharton's jelly derived cells were found positive for the surface markers CD-73, STRO-1 and CD-105, whereas they were negative for hematopoetic stem cell marker CD-34. In vitro cultured cWJ-MSCs also showed differentiation properties into osteogenic, adipogenic and chondrogenic lineages as demonstrated by von Kossa, Oil Red-O and Alcian blue staining respectively, which was further confirmed and quantified by flow cytometric analysis. Furthermore, these well characterized cWJ-MSCs were evaluated for the wound-healing potential in full-thickness skin wounds in rabbit model for 28 days. Caprine WJ- MSCs treated skin wounds showed significantly (P < 0.05) higher percentage of wound contraction especially at the 21(st) day post transplantation when compared to PBS treated control group animals. Further, we observed better healing potential of cWJ-MSCs in terms of histo-morphological evaluation, epithelialisation and collagenization with matured vascularization stage by day 28 as compared to control. In conclusion, cWJ- MSCs provide an alternative inexhaustible source of mesenchymal stem cells and also unravel new perspectives pertaining to the therapeutic use of these cells in different species.

Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices.

BACKGROUND AND OBJECTIVES: Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. METHODS AND RESULTS: Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA- 4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. CONCLUSIONS: Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.

Impact of gonadotropin supplementation on the expression of germ cell marker genes (MATER, ZAR1, GDF9, and BMP15) during in vitro maturation of buffalo (Bubalus bubalis) oocyte.

The present study was designed to investigate whether gonadotropins [follicle-stimulating hormone (FSH) and luteinizing hormone (LH)] and buffalo follicular fluid (bFF) supplementation in maturation medium influences the transcript abundance of germ cell marker genes [maternal antigen that embryos require (MATER), Zygote arrest 1 (ZAR1), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15)] mRNA in buffalo (Bubalus bubalis) oocytes. Buffalo ovaries were collected from local abattoir, oocytes were aspirated from antral follicles (5-8 mm) and matured in vitro using two different maturation regimens, viz, group A: gonadotropin (FSH and LH) and group B: non-gonadotropin-supplemented maturation medium containing 20% buffalo follicular fluid (bFF). mRNA was isolated from immature (330) and in vitro matured oocytes from both the groups (A, 320; B, 340), and reverse transcribed using Moloney murine leukemia virus reverse transcriptase. Expression levels of MATER, ZAR1, GDF9, and BMP15 mRNA transcripts were analyzed in oocytes of both maturation groups as well as immature oocytes using real-time PCR. QPCR results showed that GDF9 and BMP15 transcripts were significantly (p<0.05) influenced with gonadotropins and bFF supplementation during in vitro maturation of buffalo oocyte; however, MATER and ZAR1 transcripts were not influenced with gonadotropins and bFF supplementation in vitro. These results indicated that the expression levels of MATER, ZAR1, GDF9, and BMP15 mRNA were varied differentially during in vitro maturation of buffalo oocyte and were found to be gonadotropins (FSH and LH) or bFF dependent for GDF9 and BMP15.

Co-culture of buffalo (Bubalus bubalis) preantral follicles with antral follicles: a comparative study of developmental competence of oocytes derived from in vivo developed and in vitro cultured antral follicles.

The present study was undertaken to examine whether the presence of antral follicles (AFs) affects the survival, growth and steroidogenesis of preantral follicles (PFs) and compare the maturation and developmental competence of buffalo oocytes derived from in vivo developed and in vitro cultured AFs. Two experiments were carried out. In experiment I, PFs (200-250 µm) were isolated and cultured with or without AFs (3-5 mm) in TCM-199 medium that contained 10% fetal bovine serum (FBS), 1% insulin transferin selenium (ITS), 20 ng/ml epidermal growth factor (EGF), 0.5 µg/ml follicle-stimulating hormone (FSH) and 100 ng/ml insulin-like growth factor (IGF)-I. In experiment II, in vitro developmental competence was compared for the cumulus-oocyte complexes (COCs) recovered from in vivo developed and in vitro cultured AFs. Survival, growth, development of antrum, accumulation of estradiol and progesterone was (P < 0.05) higher when PFs were co-cultured with AFs. Developmental competence of both types of follicular oocytes did not differ significantly in terms of maturation and cleavage rate, but morula and blastocyst production rate were (P < 0.05) higher with in vivo developed AFs as compared with the in vitro cultured antral follicular oocytes. In conclusion, co-culture of PFs with AFs supports long-term survival and growth of buffalo PFs and this co-culture system plays a dual role for in vitro production of embryos as well as understanding the relationship between developing PFs and AFs.

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells.

Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.

In vivo differentiation potential of buffalo (Bubalus bubalis) embryonic stem cell.

Embryonic stem cells (ESCs) derived from inner cell mass (ICM) of mammalian blastocyst are having indefinite proliferation and differentiation capability for any type of cell lineages. In the present study, ICMs of in vitro-derived buffalo blastocysts were cultured into two different culture systems using buffalo fetal fibroblast as somatic cell support and Matrigel as synthetic support to obtain pluripotent buffalo embryonic stem cell (buESC) colonies. Pluripotency of the ESCs were characterised through pluripotency markers whereas, their differentiation capability was assessed by teratoma assay using immuno-compromised mice. Cumulus ooccyte complexes from slaughter house-derived ovaries were subjected to in vitro maturation, in vitro fertilization and in vitro culture to generate blastocysts. Total 262 blastocysts were derived through IVEP with 11.83 % (31/262) hatching rate. To generate buESCs, 15 ICMs from hatched blastocysts were cultured on mitomycin-C-treated homologous fetal fibroblast feeder layer, whereas the leftover 16 ICMs were cultured on extra-cellular matrix (Matrigel). No significant differences were observed for primary ESCs colony formation between two culture systems. Primary colonies as well as passaged ESCs were characterised by alkaline phosphatase staining, karyotyping and expression of transcription-based stem cell markers, OCT-4 and cell surface antigens SSEA-4 and TRA-1-60. Batch of ESCs found positive for pluripotency markers and showing normal karyotype after fifteenth passage were inoculated into eight immuno-compromised mice through subcutaneous and intramuscular route. Subcutaneous route of inoculation was found to be better than intramuscular route. Developed teratomas were excised surgically and subjected to histological analysis. Histological findings revealed presence of all the three germinal layer derivatives in teratoma sections. Presence of germinal layer derivatives were further confirmed by reverse transcriptase-polymerase chain reaction for the presence of differentiation markers like nerve cell adhesion molecule, fetal liver kinase-1 and alpha-feto protein for ectoderm, mesoderm and endoderm, respectively.

Expression and quantification of Oct-4 gene in blastocyst and embryonic stem cells derived from in vitro produced buffalo embryos.

The POU-domain transcription factor Pou5f1 (Oct-4) is involved in transcriptional regulation during early embryonic development and cell differentiation. Despite highly conserved genomic organization of Oct-4 gene in mammals, expression pattern of Oct-4 is highly variable in different species. In the present study, expression pattern of Oct-4 in buffalo blastocyst, trophoectoderm (TE), and embryonic stem cells (ESCs) was investigated. For the derivation and characterization of buffalo ESCs, inner cell masses (ICMs) were isolated from 18 hatched and 21 expanded in vitro produced buffalo blastocyst and cultured over mitomycin-C-treated buffalo fetal fibroblast feeder layer. Alkaline phosphatase (AP) activity, SSEA-1 and 4, TRA 1-60 and 1-81, and Oct-4 proteins were localized in ICM, TE, and ESCs. Quantification of Oct-4 was done by amplifying a transcript of 125 base pairs by real-time polymerase chain reaction. Primary cell colony formation was higher (P < 0.05) in hatched blastocyst (83.33%, 15/18) compared to mechanically isolated ICMs from expanded blastocyst (52.38%, 11/21). Undifferentiated buffalo ESCs were positive for AP and expressed Oct-4, SSEA-1 and 4, TRA-1-60, and TRA-1-81 proteins. Oct-4 transcripts and proteins were detected in the ICM, TE cells and were invariably present in ESCs; however, expression level of Oct-4 transcript were significantly higher in ICM and ESCs as compared to TE cells. In conclusion, expression of Oct-4 is not only restricted to the ICM and ESCs but its expression was also detected in TE cells suggesting that instead of using Oct-4 as a single marker, it is better to have other flanking molecular markers for the identification of buffalo pluripotent embryonic stem cells.

Expression of nitric oxide synthase isoforms in different stages of buffalo (Bubalus bubalis) ovarian follicles: effect of nitric oxide on in vitro development of preantral follicle.

The present study was designed to investigate the expression of nitric oxide synthase (NOS) isoforms in buffalo ovarian preantral (PFs), antral (AFs) and ovulatory (OFs) follicles (Experiment 1); effect of NO on in vitro survival and growth of PFs (Experiment 2) and NOS activity in immature oocytes by NADPH-diaphorase test (Experiment 3). In Experiment 1, NOS isoforms (neuronal, inducible and endothelial) were localized immunohistochemically; mRNA and protein expression was analyzed by semi-quantitative RT-PCR and western blot, respectively. In Experiment 2, PFs were isolated by micro-dissection method from buffalo ovaries and cultured in 0 (control), 10(-3), 10(-5), 10(-7) and 10(-9) M sodium nitroprusside (SNP). PFs were further cultured with 10(-5) M SNP + 1.0 mM N(ω)-nitro-L-arginine methyl ester (L-NAME) or 1.0 µg/ml hemoglobin (Hb) to examine the reversible effect of SNP. Immunohistochemical studies demonstrated that inducible nitric oxide synthase (iNOS) immunoreactivity was predominantly localized in granulosa and theca cells whereas, neuronal (nNOS) and endothelial (eNOS) nitric oxide synthase in the theca, granulosa and cumulus cells of PFs, AFs and OFs. The amount of mRNA as well as protein of nNOS and eNOS was found similar between different stages of follicles. In contrast, higher level of iNOS mRNA was observed in OFs and protein in the AFs. Higher doses of SNP (10(-3), 10(-5), 10(-7) M) inhibited (P < 0.05) while, lower dose of SNP (10(-9) M) stimulated (P < 0.05) the survival, growth, and antrum formation of PFs. The inhibitory effects of SNP were reversed by Hb, while L-NAME was not found effective. In conclusion, expression of NOS isoforms mRNA and protein in PFs, AFs, and OFs and NOS enzyme activity in immature follicular oocytes suggest a role for NO during ovarian folliculogenesis in buffalo. NO plays a dual role on growth and survival of PFs depending on its concentration in the culture medium.

Origin of the dynamics of the spin state in undoped BaFe2As2: Mössbauer studies.

Fe-As based superconductors provide a good system for understanding the relationship between magnetism and superconductivity. Considerable efforts have been expended in understanding the magnetic behavior of the parent compound, BaFe2As2. However, it had not been realized that traces of adsorbed O2 by the material bring about drastic changes in its magnetic behavior. O2 is known to trap electrons, forming O2(-). Guided by this discovery, we observe in the absence of O2 a dynamic transition between intermediate and low spin states of Fe and hysteresis effects, and in the presence of O2 trapping of the magnetic state. These observations are likely to have a bearing on the role of magnetism in superconductivity.

Mössbauer studies of the superconducting cobalt/nickel-doped BaFe2As2. Whither go the injected electron(s)?

Mössbauer studies of cobalt- and nickel-doped BaFe(2)As(2) show that the s-electron density at the (57)Fe nuclei, as measured by the isomer shift, is the same as that for the parent BaFe(2)As(2). Apparently, the electron population of the d shell, which shields the s-electron density at the nuclei, remains unchanged. We invoke the involvement of p-orbital hybridization with the d orbital in Fe-As bonding. Furthermore, the shrinkage of the lattice on substitution enhances the As-As sp hybridization, providing a path for the migration of additional electrons. The proposed mechanism is consistent with Hall coefficient and thermoelectric effect measurements.