Isotope dilution LC/MS/MS for the detection of nerve agent exposure in urine

Organophosphorus nerve agents (OPNA), chemically related to and derived from organophosphate insecticides, constitute a clear and present threat to both military and civilian targets. Military regimes and terrorist organizations have demonstrated the will and ability to produce mass casualties by dispersing organophosphorus nerve agents, which, in turn could terrorize populations and overwhelm healthcare systems. A high throughput, robust and sensitive analytical protocol has been developed for the quantitation of the urinary metabolites of sarin (GB), soman (GD), VX, Russian VX (RVX) and cyclohexylsarin (GF) utilizing solid phase extraction (SPE) followed by High Performance Liquid Chromatography (HPLC)-isotope dilution tandem mass spectrometry (LC/MS/MS). The method has demonstrated linearity and reproducibility (1-200 ng/mL) for all analytes and has a Limit of Quantitation (LOQ)< or =0.5 ng/mL for all analytes (S/N> or =10/1). The method was validated by performing 20 individual analyses over 10 days by five scientists with all values falling within two standard deviations of the mean.     

Phylogenetic relationships and generic realignments in the early diverging subtribe Pentziinae (Asteraceae,Anthemideae)

Pentziinae are a subtribe of tribe Anthemideae (Asteraceae), comprising seven almost exclusively southern African endemic genera and c. 60 species. Generic delimitations and relationships in the subtribe are explored using parsimony and Bayesian analyses of nuclear (internal transcribed spacer, ITS) and plastid (ndhF, psbA‐trnH, trnK‐rps16 and rpl32‐trnL) DNA regions, with morphological and fruit anatomical studies. Phylogenetic analyses for 72 accessions (43 species) representing 72% of the subtribe indicate that several of the genera are not monophyletic as currently circumscribed. Pentzia tortuosa differs from its congeners in its inconspicuously three‐ribbed fruits and in several other distinctive morphological characters. In the phylogenetic trees based on the nuclear dataset, P. tortuosa is recovered in the perennial clade with its congeners, but in the plastid data set, it is strongly placed among the early diverging lineages comprising annual taxa with three‐ribbed fruits. Even with the exclusion of P. tortuosa, Pentzia remains paraphyletic by the inclusion of at least Rennera and possibly also Cymbopappus and Marasmodes. A possible sister relationship between Marasmodes and Pentzia, however, cannot be excluded. The four Rennera specise are therefore here transferred to Pentzia, whereas Marasmodes and Cymbopappus are retained pending further investigation. Among the annual lineages, Foveolina is also found to be polyphyletic, with F. dichotoma (the type species) and F. tenella clearly allied with Oncosiphon, and the anomolous species, F. burchellii, recovered with Myxopappus in the nuclear trees. Both Myxopappus species share the disciform capitula and heteromorphic fruits with Foveolina burchellii (characters previously overlooked in Myxopappus and reported here for the first time). © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 633–647.     

Monoclonal antibodies reactive with the T cell receptor zeta chain: production and characterization using a new method

We have developed a novel method for the production and characterization of monoclonal antibodies reactive with lineage-restricted intracellular Ag. Using this technique, we have produced a panel of antibodies that react specifically with permeabilized T lymphocytes but not with permeabilized B lymphocytes or native T cells. One of these antibodies, designated TIA-2, was found to react with greater than 98% of peripheral blood T lymphocytes. Immunoblotting experiments showed TIA-2 to recognize a 32 kd protein that was reduced to 16 kDa in the presence of 2-mercaptoethanol. Immunoprecipitates analyzed on non-reducing/reducing diagonal polyacrylamide gels showed the homodimeric structure recognized by TIA-2 to be associated with additional structures whose pattern closely resembled that of the T cell receptor complex. When immunoprecipitates formed using antibodies reactive with CD3 epsilon were immunoblotted with TIA-2, the homodimeric TCR zeta chain was specifically recognized. Using TIA-2 as a TCR zeta specific reagent, we show that whole cell expression of this TCR subunit is dramatically reduced following exposure to mAb reactive with CD3. mAb reactive with activating epitopes of CD2 were also capable of down-modulating the expression of TCR zeta, but to a lesser degree. Exposure to Con A or IL-2, on the other hand, did not reduce the whole cell expression of TCR zeta. Given the central importance of TCR zeta in the expression of a functionally competent Ag receptor, its reduced expression in response to certain activating stimuli is likely to play an important role in regulating T cell responsiveness.     

Microbiology of a NaCl stalactite ‘salticle’ in Triassic halite

Large regions of Earth's surface are underlain by salt deposits that evaporated from ancient oceans and are populated by extreme halophilic microbes. Some of these halophiles may have been preserved over geological timescales within hypersaline fluid inclusions, but ingresses of water and/or anthropogenic activities can lead to the formation of alternative habitats, including NaCl stalactites or other speleothems. While the microbiology of ancient evaporites has been well studied, the ecology of these recently formed structures is less-well understood. Here, the microbiology of a NaCl stalactite (‘salticle’) in a Triassic halite mine is characterized. The specific aims were to determine the presence of fluid inclusions, determine the microbial structure of the salticle compared with a nearby brine-pool and surficial soil, and characterize the ecophysiological capabilities of this unique ecosystem. The salticle contained fluid inclusions, and their microbiome was composed of Euryarchaetota, Proteobacteria, and Actinobacteria, with Haloarchaea in greater abundance than brine-pool or soil microbiomes. The salticle metagenome exhibited a greater abundance of genes involved in osmoregulation, anaerobic respiration, UV resistance, oxidative stress, and stress-protein synthesis relative to the soil microbiome. We discuss the potential astrobiological implications of salticles as enclosed salt-saturated habitats that are protected from ionizing radiation and have a stable water activity.     

Design and validation of a dynamic flow perfusion bioreactor for use with compliant tissue engineering scaffolds

In tissue engineering, flow perfusion bioreactors can be used to enhance nutrient diffusion while mechanically stimulating cells to increase matrix production. The goal of this study was to design and validate a dynamic flow perfusion bioreactor for use with compliant scaffolds. Using a non-permanent staining technique, scaffold perfusion was verified for flow rates of 0.1-2.0 mL/min. Flow analysis revealed that steady, pulsatile and oscillatory flow profiles were effectively transferred from the pump to the scaffold. Compared to static culture, bioreactor culture of osteoblast-seeded collagen-GAG scaffolds led to a 27-34% decrease in cell number but stimulated an 800-1200% increase in the production of prostaglandin E(2), an early-stage bone formation marker. This validated flow perfusion bioreactor provides the basis for optimisation of bioreactor culture in tissue engineering applications.     

220- and 130-kDa MLCKs have distinct tissue distributions and intracellular localization patterns

To better understand thedistinct functional roles of the 220- and 130-kDa forms of myosin lightchain kinase (MLCK), expression and intracellular localization weredetermined during development and in adult mouse tissues. Northernblot, Western blot, and histochemical studies show that the 220-kDaMLCK is widely expressed during development as well as in several adultsmooth muscle and nonmuscle tissues. The 130-kDa MLCK is highlyexpressed in all adult tissues examined and is also detectable duringembryonic development. Colocalization studies examining thedistribution of 130- and 220-kDa mouse MLCKs revealed that the 130-kDaMLCK colocalizes with nonmuscle myosin IIA but not with myosin IIB orF-actin. In contrast, the 220-kDa MLCK did not colocalize with eithernonmuscle myosin II isoform but instead colocalizes with thickinterconnected bundles of F-actin. These results suggest that in vivo,the physiological functions of the 220- and 130-kDa MLCKs are likely tobe regulated by their intracellular trafficking and distribution.

     

Synthesis,pharmacology and pharmacokinetics of 3-(4-aryl-piperazin-1-ylalkyl)-uracils as uroselective alpha1A-antagonists

Predominance in the urethra and prostate of the alpha(1A)-adrenoceptor subtype, which is believed to be the receptor mediating noradrenaline induced smooth muscle contraction in these tissues, led to the preparation of alpha(1A)-selective antagonists to be tested as uroselective compounds for the treatment of benign prostatic hyperplasia. Thus, a number of selective alpha(1A)-adrenoceptor antagonists were synthesized and assayed in vitro for potency and selectivity. Dog pharmacokinetic parameters of 12 (RO700004) and its metabolite 40 (RO1104253) were established. The relative selectivity of intravenously administered 12, 40 and standard prazosin to inhibit hypogastric nerve stimulation-induced increases in intraurethral prostatic pressure versus phenylephrine-induced increases in diastolic blood pressure in anesthetized dogs was 76, 71 and 0.6, respectively.