New method of FACS analyzing and sorting of intact whole ovarian fragments (COPAS) after long time (24 h) cooling to 5 °C before cryopreservation

As recently announced by the American Society for Reproductive Medicine (ASRM), human ovarian tissue cryopreservation is an established option for fertility preservation in prepubertal girls and young women undergoing gonadotoxic treatments for cancer as well as some autoimmune diseases. Proper ovarian tissue assessment before and after cryopreservation is essential to increase success rates. Ovarian fragments from 16 patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following two groups. Pieces of Group 1 (n?=?16) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n?=?16) after operation were cooled to 5 °C for 24 h, then frozen after 24 h pre-cooling to 5 °C, thawed and just after thawing their quality was analyzed. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development and viability of follicles (Calcein-AM and Propidium Iodide) using complex object parametric analyzer and sorter machine (COPAS). Positive effect of cooling of cells to low supra-zero temperatures on their future development after re-warming has been observed. New flow cytometry- technique is suitable for the evaluation and sorting of cryopreserved whole human whole intact ovarian fragments. Long time (24 h) cooling of ovarian tissue to 5 °C before cryopreservation has a trend of a cell viability increasing.

     

Kinetics of adsorption of extended ligands on DNA at small fillings

In the present work, the adsorption kinetics of extended ligands on DNA duplexes at small fillings when molecules of DNA duplexes are on the underlayer within diffusion layer has been investigated. Both diffusion of ligands in solution (diffusion stage) and adsorption of ligands (kinetic stage) are taken into consideration at adsorption of ligands on DNA duplexes. Nonlinear system of differential equations describing adsorption of ligands where not only diffusion stage but also kinetic stage is taken into account, is obtained, moreover the equations allow localizing duplexes in arbitrary place within diffusion layer. Numeric solution of the equations makes possible to investigate the filling kinetics of DNA duplexes by ligands depending on parameters controlling adsorption process. It has been shown that depending on relation between adsorption parameters different kinetic regimes of adsorption – kinetic, complex, and diffusion regimes may be realized.     

MAP Kinase analyser: A tool for plant kinase and substrate analysis

MAPK (Mitogen Activated Protein Kinase) is a Ser/Thr kinase, which plays a crucial role in plant growth and development, transferring the extra cellular stimuli into intracellular response etc. Manual identification of these MAPK in the plant genome is tedious and time taking process. There are number of online servers which predict the P-site (phosphorylation site), find the motifs and domain but there is no specific tool which can identify all them together. In order to identify the P-Site, phosphorylation site consensus sequences and domain of the MAPK in plant genome, we developed a tool, MAP Kinase analyzer. MAP kinase analyzer take protein sequence as input in the fasta format and the output of tool includes: 1) The prediction of the phosphorylation site viz., Serine (S), Threonine (T), and Tyrosine (Y), Contex, Position, Score and phosphorylating kinase as well as the graphical output; 2) Phosphorylation site consensus sequence pattern for different kinases and 3) Domain information about the MAPK's. The MAP kinase analyser tool and supplementary files can be downloaded from http://www.bioinfogbpuat/mapk_OWN_1/.     

Genome-Wide Comparative in silico Analysis of Calcium Transporters of Rice and Sorghum

The mechanism of calcium uptake, translocation and accumulation in Poaceae has not yet been fully understood. To address this issue, we conducted genome-wide comparative in silico analysis of the calcium (Ca2+) transporter gene family of two crop species, rice and sorghum. Gene annotation, identification of upstream cis-acting ele- ments, phylogenetic tree construction and syntenic mapping of the gene family were performed using several bio- informatics tools. A total of 31 Ca2+ transporters, distributed on 9 out of 12 chromosomes, were predicted from rice genome, while 28 Ca2+ transporters predicted from sorghum are distributed on all the chromosomes except chromosome 10 (Chr 10). Interestingly, most of the genes on Chr 1 and Chr 3 show an inverse syntenic relation- ship between rice and sorghum. Multiple sequence alignment and motif analysis of these transporter proteins re- vealed high conservation between the two species. Phylogenetic tree could very well identify the subclasses of channels, ATPases and exchangers among the gene family. The in silico cis-regulatory element analysis suggested diverse functions associated with light, stress and hormone responsiveness as well as endosperm- and meris- tem-specific gene expression. Further experiments are warranted to validate the in silico analysis of the predicted transporter gene family and elucidate the functions of Ca2+ transporters in various biological processes.     

Enhanced proteolysis leads to pre-mature cell death under the influence of elicitor like mycelial components from karnal bunt (tilletia indica) pathogen in wheat callus cultures

Calli raised from mature embryos of susceptible wheat cultivar WH 542 were used in the present study as in vitro bioassay system to study the influence of disease determinant(s) of Karnal bunt (Tilletia indica), a semi-biotrophic fungal pathogen of wheat. Influence of elicitor and conditioned medium (CM) prepared from fungal cultures of T. indica was investigated on induction of programmed cell death (PCD). Induction of PCD was observed as hypersensitive response (HR) in terms of browning at localized regions of callus cultures and induction of proteolytic enzyme(s). Elicitor treated calli showed higher induction of protease activity than untreated and CM-treated cultures, which showed not much change in the activity. It was further substantiated by gel protease assay and activation of caspase-3 like protein(s) in callus cultures that clearly suggested the presence of signaling molecule(s) in the fungal elicitor preparation rather than in conditioned medium. This study further demonstrated that only elicitor preparation possesses such molecule(s), which might be cell wall bound components, rather than secretory in nature as CM was unable to induce PCD in wheat callus cultivars.     

Bacillus cereus produces several nonproteinaceous insecticidal exotoxins

Bacillus cereus is mainly known as a human food-borne opportunistic pathogen. Here, we used biological assays and HPLC to investigate the ability of B. cereus to produce insecticidal exotoxins during the stationary growth phase. None of the 575 B. cereus strains screened produced detectable levels of β-exotoxin I, a small, heat-stable insecticidal nucleotide analogue. However, six out of a subset of 270 B. cereus strains produced several small, nonproteinaceous insecticidal exotoxins different from β-exotoxin I. Thus, B. cereus can secrete a large array of proteinaceous and nonproteinaceous toxins acting on insects and mammals.     

A versatile synthetic route to chiral quinoxaline derivatives from amino acids precursors

Summary The synthesis ofN-protected L-amino acid (3-benzylquinoxalin-2-yl) hydrazide derivatives is reported here. 3-Benzyl-2-hydrazinoquinoxaline was prepared and then coupled withN-Boc-L-amino acids including; Alanine, Valine, Leucine, Phenylalanine, Tyrosine, Serine and Proline in the presence of HBTU as a coupling reagent to provide the expected product with high yield and purity. The products were deprotected by p-toluenesulphonic acid in acetonitrile and then the tosylate salts were evaluated for antibacterial and antifungal activity. Abbreviations: HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide Boc,t-butyloxycarbonyl, DIEA, diisopropylethylamine; DMF, N,N-dimethylformamide; PTSA, p-toluenesulphonic acid, TEA, triethyl amine. Amino acids are abbreviated and designated following the rules of the IUPAC-IUB Commission of BiochemicalNomenclature (J. Biol. Chem., 247 (1972) 977).