Rapid and sensitive static headspace gas chromatography-mass spectrometry method for the analysis of ethanol and abused inhalants in blood

A sensitive and specific method using static headspace gas chromatography coupled with mass spectrometry (GC/MS) has been developed for the quantitative determination of ethanol in biological fluids using n-propanol as internal standard. Gas chromatography was performed in isothermal mode with a GC run time of 2.6 min. The quantification was performed using scan mode abstracting a quantitative ion and a qualifier ion for ethanol and for the internal standard. The method was linear (r(2), 0.999, in the concentration range of 5-200 mg/dl), specific (no interference from methanol acetaldehyde, acetone or from endogenous materials), sensitive (limit of quantification and limit of detection of 0.2 and 0.02 mg/dl, respectively) and robust (less than 5% inter- and intra-assay coefficient of variation). A slightly modified method was also developed for the quantification of five commonly abused inhalants (dichloromethane, ethyl acetate, benzene, toluene and xylene) in blood. The method used a gradient GC program with a run time of 8 min. The quantification was performed using scan mode and integrating the area under the peak using trichloroethane as an internal standard. Without optimization, the method was linear (from 5 to 100 mg/l) and sensitive.     

Characterization of supermacroporous monolithic polyacrylamide based matrices designed for chromatography of bioparticles

Supermacroporous monolithic acrylamide (AAm)-based cryogels were prepared by radical cryo-polymerizaton (polymerization in the moderately frozen system) of AAm with functional monomers and cross-linker N,N'-methylene-bis-acrylamide (MBAAm). Electron microscopy studies revealed supermacroporous structure of the developed cryogels with pore size of 5-100 microm. Cryogel porosity depended on cryo-polymerization conditions. More than 90% of the monolithic bed volume is the interconnected supermacropores filled with water and less than 10% of the monolithic volume is pore walls. The total protein binding capacity (lysozyme in the case of immobilized metal affinity chromatography (IMAC) column and bovine serum albumin (BSA) in the case of anion-exchange (AE) column) was independent of the flow rates till 600 cm/h. Chromatographic behavior of E. coli cells when a cell suspension was applied to ion-exchange cryogel columns depended on both the density of functional ligand and the porosity of the cryogel.     

Detection of thiopental in the steroid fraction of serum from neonates following maternal exposure

To follow up an investigation which studied effects of antenatal dexamethasone therapy on neonatal respiratory performance in multifetal gestations, neonatal serum steroids were determined by HPLC. A major peak (X) whose retention time coincided with that of dexamethasone was observed in many, but not all, serum samples. However, there was no correlation between the neonates whose serum samples displayed this X-peak and the mothers who had actually received the steroid therapy, indicating that the X-substance was not dexamethasone. An alternate mobile phase was employed which separated the X-substance and dexamethasone validating the indication. Among ten clinical conditions of the neonate birth, the X-substance was found to correlate only with the mothers who had the cesarean operation for delivery, suggesting that the substance was not necessarily a steroid. Four anesthetic agents used for cesarean operations were studied; the X-substance was identified as thiopental using a LC/MS technique. This was based on the same retention times, the same negative ions at m/z 240.9 and the same daughter ions at m/z 100.8 between the two substances. Thus, caution must be exercised when HPLC is employed to study serum steroids of patients who have previously been exposed to thiopental. Moreover, recent reports have shown that thiopental affects certain metabolic reactions in the rat; the present findings also suggest a need for further investigations of thiopental effect on neonates.     

Enantioselective syntheses of (R)- and (S)-argentilactone and their cytotoxic activities against cancer cell lines

Concise total syntheses of (R)- and (S)-argentilactone have been developed via enantioselective catalytic allylation (ECA) and ring-closing metathesis pathways (four steps, 39% overall yield and 82-84% ee) from 2-octynal and their in vitro activity against cancer cells is described.     

Small GTP binding protein Ras contributes to norepinephrine-induced mitogenesis of vascular smooth muscle cells(1)

Norepinephrine stimulates release of arachidonic acid from tissue lipids. Arachidonic acid metabolites generated through the lipoxygenase and cytochrome P-450 pathways but not cyclooxygenase stimulate mitogen activated protein (MAP) kinase activity and proliferation of vascular smooth muscle cells (VSMC). Moreover, norepinephrine has been shown to activate the Ras/MAP kinase pathway through generation of cytochrome P-450 metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE). The purpose of this study was to investigate the contribution of Ras in norepinephrine-induced mitogenesis in aortic VSMC. Farnesylation of Ras by farnesyl transferase is required for its full activation. Norepinephrine-induced DNA synthesis, as measured by [(3)H]-thymidine incorporation, was attenuated by inhibitors of Ras farnesyl transferase FPT III and BMS-191563. These agents also inhibited 20-HETE-stimulated [(3)H]-thymidine incorporation. In cells transiently transfected with dominant negative Ras (RasN17), norepinephrine, and 20-HETE-induced proliferation of VSMC was attenuated. Both norepinephrine and 20-HETE increased localization of Ras to plasma membrane and MAP kinase activity; FPT III attenuated these effects. These data suggest that VSMC proliferation induced by norepinephrine and 20-HETE is mediated by Ras/MAP kinase pathway.     

Analysis of chromosomal aberration frequencies in the peripheral blood lymphocytes of smokers exposed to uranyl compounds

One hundred fifteen smokers working in a nuclear fuel manufacturing facility were analysed for various types of chromosomal aberrations. They experienced exposure for a period of 1-25 years. Their age ranges from 23 to 52 years. A total of 94 smokers and 118 non-smokers who were not exposed to uranyl compounds or to any other known mutagens and belong to the same age group formed the control subjects. The results showed that there is a significant increase in the frequency of chromosomal aberrations in the exposed smokers when compared to the control smokers. In the control group, the smokers showed a high frequency of chromosomal aberrations when compared to non-smokers suggesting clastogenic effect of smoking. Chromosomal aberrations observed in the exposed smokers could be due to the cumulative effect of both smoking and exposure to uranyl compounds.     

Are plant formins integral membrane proteins?

Background  

The formin family of proteins has been implicated in signaling pathways of cellular morphogenesis in both animals and fungi; in the latter case, at least, they participate in communication between the actin cytoskeleton and the cell surface. Nevertheless, they appear to be cytoplasmic or nuclear proteins, and it is not clear whether they communicate with the plasma membrane, and if so, how. Because nothing is known about formin function in plants, I performed a systematic search for putative Arabidopsis thaliana formin homologs.     

Gene editing the phytoene desaturase alleles of Cavendish banana using CRISPR/Cas9

Bananas are a staple food source and a major export commodity worldwide. The Cavendish dessert banana is a triploid AAA genome type and accounts for around 47% of global production. Being essentially sterile, genetic modification is perhaps the only pathway available to improve this cultivar. In this study, we used the CRISPR/Cas9 gene editing system to deliver a self-cleaving polycistronic guide RNA (gRNA) designed to target exon 1 of the Phytoene desaturase (PDS) gene in the Cavendish cultivar “Williams”. Genotyping of 19 independent events showed a 100% PDS modification rate primarily in the form of insertions (1–105 nt) or deletions (1–55 nt) (indels) at the predicted cleavage site. Tri-allelic disruptive modifications were observed in 63% of plants and resulted in both albinism and dwarfing. Pale green (16%) and wildtype green (21%) phenotypes generally correlated with in-frame indels in at least one of the three PDS alleles. Editing efficiency was dependent on both target site selection and Cas9 abundance. This is the first report of a highly effective CRISPR/Cas9 modification system using a polycistronic gRNA in Cavendish banana. Such an editing platform will be of considerable utility for the development of disease resistance and novel agro-traits in this commercially important cultivar into the future.