Morphologic and proliferative characteristics of goat type a spermatogonia in the presence of different sets of growth factors

PurposeThe present study by using different growth factors was aimed to develop the best practical culture condition for purification of goat undifferentiated SSCs and their colonization under in vitro and in vivo conditions.MethodsThe enzymatically isolated SSCs obtained from one month old goat testes were cultured in DMEM plus FCS supplemented with different sets of growth factors (GDNF, LIF, bFGF, and EGF) for 2 weeks. At the end of each week, the morphological characteristics of cells and colonies alongside with purification rate of undifferentiated type A spermatogonia were evaluated by immunocytochemical staining and flow cytometry.ResultsThe number and size of colonies in treatment groups were significantly (P < 0.01) higher than corresponding values in control group. In immunocytochemical evaluation, the proportion of KIT and PGP9.5 positive cells were significantly (P < 0.001) higher in control and treatment groups, respectively.ConclusionsThe culture medium comprising all four growth factors, especially the one supplemented with the higher concentration of GDNF, was superior to the other groups with respect to the population of undifferentiated type A spermatogonia and its propagation in culture system. Additionally, goat SSCs could colonize within the mouse testis following xenotransplantation.     

Identification of fungal fossils and novel azaphilone pigments in ancient carbonised specimens of <Emphasis Type="Italic">Hypoxylon fragiforme</Emphasis> from forest soils of Châtillon-sur-Seine (Burgundy)

Fungal stromata were recently discovered in association with charcoal and burnt soil aggregates during an archaeological survey in the Châtillon-sur-Seine forest massif. The wood and soil in the samples were dated to the medieval period (between 738 and 1411 AD). Light microscopy and scanning electron microscopy revealed that a few of the stromatal fragments still contained ascospores. Their macromorphological characters were described and secondary metabolite profiles were generated using high performance liquid chromatography with diode array and mass spectrometric detection (HPLC–DAD/MS). The combination of these two data lines then allowed species identification. Most of the fragments were assigned to Hypoxylon fragiforme, the type species of the Hypoxylaceae (Xylariales). Two further species whose stromata grew on the fossil charcoal could be tentatively identified as Jackrogersella cohaerens and (more tentatively) as Hypoxylon vogesiacum. These three species are still commonly encountered in the forests of Central Europe today. Furthermore, the HPLC-HRMS data of H. fragiforme suggested the presence of unknown azaphilone dimers and of further new pigments. These archaeological compounds were compared to fresh stromata of H. fragiforme collected in Germany and subjected to the same analytical protocol. While the major components in both samples were identified as the known mitorubrin type azaphilones and orsellinic acid, the chemical structures of seven novel complex azaphilone pigments, for which we propose the trivial names rutilins C-D and fragirubrins A-E, were elucidated using spectral methods (NMR and CD spectroscopy, high resolution mass spectrometry). It appears that these pigments had indeed persisted for millennia in the fossil stromata.     

In silico Prediction and in vitro Verification of a Novel Multi-Epitope Antigen for HBV Detection

After years of ongoing endeavors for HBV infection prognosis, diagnosis and treatment, it still remains a major health problem worldwide. About 400 million chronic carriers and an annual death rate as high as one million reflects the seriousness of the problem. Developing novel and more effective diagnostic strategies, using in silico approaches and their subsequent empirical verification, will be helpful in providing for blood supply safety, therapeutics efficacy and disease activity assessment. Exploiting various in silico tools a novel multiepitope detection construct was designed which was consisted of eight linked linear immunodominant HBV epitopes. The designed antigen was expressed in Escherichia coli as the host. The detection capability of the designed antigen was tested using Chemiluminescent immunoassay method. Chemiluminescent immunoassay on the expressed antigen revealed that the product may be a credible candidate for simultaneous detection of three main HBV antibodies. All three test samples in two concentrations indicated lower RLU/s in comparison to the positive control which was the direct consequence of HBV antibody detection by the designed antigen. In the present study, employing bioinformatics tools paved the way for rational design of multiepitope antigen in a more cost effective, intelligent and knowledge-based method. The obtained results could be construed as a primary proof of concept that the in silico predictions could be used as primary steps of the biological studies and their subsequent empirical conduction.     

The effect of polymorphisms of gamma-glutamyl hydrolase (GGH) gene on methotrexate-induced toxicity in acute lymphoblastic leukemia

Context: Acute lymphoblastic leukemia patients show differences in methotrexate-induced toxicity after treatment with this anti-cancer drug. Pharmacogenetics is an important determining factor for toxicity diversity. Objective: In this study, the effect of +452 CT and ? 401CT polymorphisms of Gamma-glutamyl hydrolase (GGH) gene on methotrexate serum levels and its associated toxicity in patients with acute lymphoblastic leukemia was assessed. Furthermore, the frequency of the above polymorphisms was investigated for the first time in Iran. Material and methods: The prevalence of these polymorphisms was assessed in 83 Iranian patients with ALL using PCR and RFLP. The relationship between the polymorphism and serum methotrexate levels and its toxicity was estimated by calculating the Odds Ratio. Results: No correlation was found between +452CT polymorphism and serum levels of methotrexate and methotrexate-related toxicity. ?401CT polymorphism was found to be correlated with methotrexate-related toxicity leading to thrombocytopenia (95% CI?=?0.009–0.019, odds ratio?=?0.265) and leukopenia (95% CI?=?0.021–0.042, odds ratio?=?2.182) in consolidation phase of the treatment. Discussion: C allele polymorphism of ?401?C/T allele is a risk factor of leukopenia and thrombocytopenia in patients treated with methotrexate. Moreover, our results suggested that the T allele had a supporting role in prevention of thrombocytopenia. Conclusion: Evaluation of patients for methotrexate-related polymorphism of GGH gene may be useful to determine the appropriate dose of methotrexate and reducing its toxic side effects.