Transformation of Rhodococcus Pigment Production Hydroxylase (PPH) gene into Camelina sativa: an alternative marker for the detection of transgenic plants.

Production of transgenic plants with desired agronomic and horticultural traits has gained great importance to fulfill demands of the growing population. Genetic transformation is also a fundamental step to study basics of plant sciences. Different transformation protocols have been developed and used which are reliable and efficient. These protocols used antibiotic or herbicide resistance genes incorporated along with gene of interest to identify transformed plants from non-transformed ones. These marker genes may pose a threat to human and environment. Use of visual markers enables direct and easier observation of transformed plants with more precision. In current study a gene cassette with 'pigment production hydroxylase (PPH) gene under fiber specific promoter (GhSCFP) and downstream Nos-terminator was designed. After checking the structural and functional efficiency of codon optimized gene using bioinformatics tools, the cassette was sent for chemical synthesis from commercial source. The pigment gene cassette (PPH_CEMB), cloned in pCAMBIA-1301, was transformed into Agrobacterium through electroporation. Agrobacterium-mediated floral dip method was used to transform Camelina sativa inflorescence. After seed setting a total of 600 seed were observed for change in color and out of these, 19 seeds developed a reddish-brown coloration, while the remaining 581 seeds remained yellow. The transformation efficiency calculated on basis of color change was 1.0%. PCR analysis of leaves obtained after sowing reddish seeds confirmed the transformation of pigment production gene, while no PCR amplification was observed in leaves of plants from wild type seeds. From the results it is evident that Agrobacterium-mediated transformation of C. sativa inflorescence is very efficient and environment friendly technique not only for detection of transformed plants but also to study basic cellular processes.

Statistical modeling for bioconvective tangent hyperbolic nanofluid towards stretching surface with zero mass flux condition.

This article presents the implementation of a numerical solution of bioconvective nanofluid flow. The boundary layer flow (BLF) towards a vertical exponentially stretching plate with combination of heat and mass transfer rate in tangent hyperbolic nanofluid containing microorganisms. We have introduced zero mass flux condition to achieve physically realistic outcomes. Analysis is conducted with magnetic field phenomenon. By using similarity variables, the partial differential equation which governs the said model was converted into a nonlinear ordinary differential equation, and numerical results are achieved by applying the shooting technique. The paper describes and addresses all numerical outcomes, such as for the Skin friction coefficients (SFC), local density of motile microorganisams (LDMM) and the local number Nusselt (LNN). Furthermore, the effects of the buoyancy force number, bioconvection Lewis parameter, bioconvection Rayleigh number, bioconvection Pecelt parameter, thermophoresis and Brownian motion are discussed. The outcomes of the study ensure that the stretched surface has a unique solution: as Nr (Lb) and Rb (Pe) increase, the drag force (mass transfer rate) increases respectively. Furthermore, for least values of Nb and all the values of Nt under consideration the rate of heat transfer upsurges. The data of SFC, LNN, and LDMM have been tested utilizing various statistical models, and it is noted that data sets for SFC and LDMM fit the Weibull model for different values of Nr and Lb respectively. On the other hand, Frechet distribution fits well for LNN data set for various values of Nt.

Effects of household bleach on sputum smear microscopy to concentrate acid fast bacilli for the diagnosis of pulmonary tuberculosis.

Tuberculosis (TB) is one of the major public health problem among contagious diseases in Pakistan. TB diagnosis mainly depends on sputum smear microscopy. The main objective of this study was to evaluate the effects of household bleach on sputum smear microscopy to concentrate acid fast bacilli for the diagnosis of pulmonary tuberculosis. Sputum specimens of 200 suspected TB patients were collected for the study. Smears were prepared from the purulent part of sputum sample before and after bleach treatment, heat fixed and stained with the ZN technique. The obtained data were analyzed by chi-squared test using SPSS software. Out of 200 isolates, 22 (11%) patients had positive smears for acid fast bacilli (AFB) by direct ZN staining. After treatment with household bleach (NaOCL) and centrifugation, the number of AFB positive patients were increased from 22 (11%) to 37 (18.5%). The bleach-concentration method for sputum samples significantly increased the TB detection rate as compared to direct sputum smear microscopy. Thus, a shift from direct sputum microscopy to bleach-concentration technique should be considered a better method for detection of AFB in sputum through smear microscopy.

Studies on N-acetylneuraminic acid aldolase.

N-Acetylneuraminic acid aldolase from Clostridium perfringens was irreversibly inactivated by 1mm-bromopyruvate with a half-life of 4.2min at pH7.2 and 37 degrees C. The rate of inactivation was diminished in the presence of pyruvate but not with N-acetyl-d-mannosamine, indicating that the inhibitor acted at, or close to, the pyruvate-binding site. The apparent K(i) for bromopyruvate, calculated from the variation of half-life with inhibitor concentration, was 0.46mm, compared with a competitive K(i) 3.0mm for pyruvate. Incubation of the enzyme with radioactive bromopyruvate gave a radioactive, enzymically inactive, protein in which the bromopyruvate had alkylated cysteine residues. Incubation of the enzyme with radioactive pyruvate, followed by reduction with sodium borohydride, led to inactivation of the enzyme and binding of the pyruvate to the protein by reduction of a Schiff's base initially formed with the in-amino group of a lysine residue; only one-twentieth as many pyruvyl residues were bound by this method, showing that bromopyruvate is not specific for the active site. After protection of the enzyme active site with pyruvate, treatment with unlabelled bromopyruvate and dialysis, the enzyme retained 72% activity. When this treated enzyme was separately incubated with radioactive bromopyruvate, or radioactive pyruvate followed by sodium borohydride, the ratio of radioactive pyruvyl residues bound by the two methods was 2.3:1. After reduction and hydrolysis of the bromopyruvate-treated enzyme, the only detectable radioactive amino acid derivative was chromatographically and electrophoretically identical with S-(3-lactic acid)-cysteine. The enzyme was fully active in the presence of EDTA and was not stimulated by bivalent metal ions. It was strongly inhibited by silver and mercuric ions. The apparent molecular weight, determined by Sephadex chromatography, was 250000. A mechanism of action is proposed for the enzyme. Bromopyruvate reacts rapidly at pH6.0 with thiol-containing amino acids. Cysteine appears to react anomalously.