Optimized separation of beta-blockers with multiple chiral centers using capillary electrochromatography-mass spectrometry

This work focuses on the simultaneous analysis of beta-blockers with multiple stereogenic centers using capillary electrochromatography-mass spectrometry (CEC-MS) with a vancomycin stationary phase. The critical mobile phase variables (composition of organic solvents, acid/base ratios) as well as column temperature and electric field strength, effecting enantioresolution and analysis time were first optimized sequentially. Next, to achieve global optimum a multivariate D-optimal design was used. Although multivariate approach did not improve enantioresolution any further, analysis time was significantly reduced. Under optimum CEC-MS conditions, all stereoisomers were resolved with resolution in the range 1.0-3.1 in less than 60 min with an average signal-to-noise (S/N) greater than 1000. The developed CEC-MS method has the potential to emerge as a screening method for analysis of multiple chiral compounds using a single protocol using the same column and mobile phase conditions, thus reducing the operation time and cost.     

<Emphasis Type="Italic">Barracudia australiensis</Emphasis> n. g., n. sp. (Nematoda: Philometridae) from the obtuse barracuda <Emphasis Type="Italic">Sphyraena obtusata</Emphasis> Cuvier (Perciformes: Sphyraenidae) off eastern Australia

Based on light and scanning electron microscopical studies, a new nematode parasite, Barracudia australiensis n. sp. (Philometridae), is described from the gall-bladder of the marine fish (obtuse barracuda) Sphyraena obtusata Cuvier (Sphyraenidae, Perciformes) from off the eastern Pacific coast of Australia, for which a new genus Barracudia n. g. is established. This new genus is mainly characterised by features found in the male: sickle-shaped, ventrally curved spicules, a gubernacum with a broad, dorsally bent distal portion and a markedly dorsoventrally elongated cloacal aperture. Based on these features, Barracudia spp. conspicuously differ from representatives of all other philometrid genera with known males. Philometra philippinensis Quiazon & Yoshinaga, 2013 is transferred to Barracudia as B. philippinensis (Quiazon & Yoshinaga, 2013) n. comb. Barracudia australiensis is the third nominal species of philometrids described from the Sphyraenidae and the 19th species of the Philometridae recorded from fishes in Australian waters.     

Expression and Biochemical Characterization of a Thermophilic Organic Solvent-Tolerant Lipase from Bacillus sp. DR90

The objective of the present study was the isolation, molecular cloning and biochemical characterization of a thermophilic organic solvent-resistant lipase from Bacillus sp. DR90. The lipase gene was expressed in Escherichia coli BL21(DE3) using pET-28a(+) vector. The purification of recombinant lipase was conducted by nickel affinity chromatography and its biochemical properties were determined. The lipase sequence with an ORF of 639 bp contains the conserved pentapeptide Ala-His-Ser-Met-Gly. His-tagged recombinant lipase had a specific activity of 1,126 U/mg with a molecular mass of 26.8 kDa. The cloned lipase was optimally active at pH 8.0 and 75 °C representing high stability in broad ranges of temperature and pH. High performance liquid chromatography was used to determine the major compounds released during the lipase-catalyzed reaction of p-nitrophenyl derivatives as well as the substrate specificity. The purified lipase showed high compatibility towards various organic solvents, surfactants and commercial solid/liquid detergents; therefore the recombinant DR90 lipase could be considered as a probable candidate for future applications, predominantly in detergent processing industries.     

Purification and biochemical characterization of phytocystatin from Brassica alba

Phytocystatins belong to the family of cysteine proteinases inhibitors. They are ubiquitously found in plants and carry out various significant physiological functions. These plant derived inhibitors are gaining wide consideration as potential candidate in engineering transgenic crops and in drug designing. Hence it is crucial to identify these inhibitors from various plant sources. In the present study a phytocystatin has been isolated and purified by a simple two‐step procedure using ammonium sulfate saturation and gel filtration chromatography on Sephacryl S‐100HR from Brassica alba seeds (yellow mustard seeds).The protein was purified to homogeneity with 60.3% yield and 180‐fold of purification. The molecular mass of the mustard seed cystatin was estimated to be nearly 26 000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as by gel filtration chromatography. The stokes radius and diffusion coefficient of the mustard cystatin were found to be 23A° and 9.4 × 10^?7 cm²s^?1 respectively. The isolated phytocystatin was found to be stable in the pH range of 6–8 and is thermostable up to 60 °C. Kinetic analysis revealed that the phytocystatin exhibited non‐competitive type of inhibition and inhibited papain more efficiently (K_i = 3 × 10^?7 M) than ficin (K_i = 6.6 × 10^?7 M) and bromelain (Ki = 7.7 × 10^?7 M respectively). CD spectral analysis shows that it possesses 17.11% alpha helical content. Copyright © 2016 John Wiley & Sons, Ltd.     

Mitochondrial DNA variations in ova and blastocyst: Implications in assisted reproduction

Mitochondrial DNA (mtDNA) of oocyte is critical for its function, embryo quality and development. Analysis of complete mtDNA of 49 oocytes and 18 blastocysts from 67 females opting for IVF revealed 437 nucleotide variations. 40.29% samples had either disease associated or non-synonymous novel or pathogenic mutation in evolutionarily conserved regions. Samples with disease associated mtDNA mutations had low fertilization rate and poor embryo quality, however no difference in implantation or clinical pregnancy rate was observed. Screening mtDNA from oocyte/blastocyst is a simple, clinically reliable method for diagnostic evaluation of female infertility and may reduce risk of mtDNA disease transmission.