Determination of free and total S-phenylmercapturic acid by HPLC/MS/MS in the biological monitoring of benzene exposure

Urinary S-phenylmercapturic acid (SPMA) is a biomarker suggested by the American Conference of Governmental Industrial Hygienists (ACGIH) for assessing occupational exposure to benzene. A possible cause of the miscorrelation between environmental monitoring and biological monitoring for benzene exposure, which many authors complain about, is the existence of a urinary metabolite that turns into SPMA by acid hydrolysis. Forty urine samples were tested to determine which concentration value would correspond to the ACGIH Biological Exposure Index (BEI) of 25 µg g^-1 creatinine if exposure assessment was based on the determination of SPMA after quantitative hydrolysis of its precursor. An aliquot of each sample was hydrolysed with 9 M H₂SO₄, a second one was brought to pH 2 and a third one was used as it was (free SPMA). SPMA was determined by high-performance liquid chromatography/tandem mass spectrometric technique (HPLC/MS/MS) using an internal standard. The analytical method was validated in the range 0.5-50 µg l^-1. The average SPMA in pH 2 samples is 45-60% of the total, while free SPMA varies from 1% to 66%. The hydrolysis of pre-SPMA reduces the likelihood of variability in the results by reducing pH differences in urine samples and increasing the amount of measured SPMA. The BEI limit value would be about 50 µg g^-1 creatinine.     

Solution structure of ApaG from Xanthomonas axonopodis pv. citri reveals a fibronectin-3 fold

ApaG proteins are found in a wide variety of bacterial genomes but their function is as yet unknown. Some eukaryotic proteins involved in protein-protein interactions, such as the human polymerase delta-interacting protein (PDIP38) and the F Box A (FBA) proteins, contain ApaG homology domains. We have used NMR to determine the solution structure of ApaG protein from the plant pathogen Xanthomonas axonopodis pv. citri (ApaG(Xac)) with the aim to shed some light on its molecular function. ApaG(Xac) is characterized by seven antiparallel beta strands forming two beta sheets, one containing three strands (ABE) and the other four strands (GFCC'). Relaxation measurements indicate that the protein has a quite rigid structure. In spite of the presence of a putative GXGXXG pyrophosphate binding motif ApaG(Xac) does not bind ATP or GTP, in vitro. On the other hand, ApaG(Xac) adopts a fibronectin type III (Fn3) fold, which is consistent with the hypothesis that it is involved in mediating protein-protein interactions. The fact that the proteins of ApaG family do not display significant sequence similarity with the Fn3 domains found in other eukaryotic or bacterial proteins suggests that Fn3 domain may have arisen earlier in evolution than previously estimated.     

A species-specific qPCR assay provides novel insight into range expansion of the Mediterranean monk seal (Monachus monachus) by means of eDNA analysis

Biodiversity and Conservation - The monk seal is the most endangered pinniped worldwide and the only one found in the Mediterranean, where its distribution and abundance have suffered a drastic...     

Prediction of stability changes upon mutation in an icosahedral capsid

Identifying the contributions to thermodynamic stability of capsids is of fundamental and practical importance. Here we use simulation to assess how mutations affect the stability of lumazine synthase from the hyperthermophile Aquifex aeolicus, a T = 1 icosahedral capsid; in the simulations the icosahedral symmetry of the capsid is preserved by simulating a single pentamer and imposing crystal symmetry, in effect simulating an infinite cubic lattice of icosahedral capsids. The stability is assessed by estimating the free energy of association using an empirical method previously proposed to identify biological units in crystal structures. We investigate the effect on capsid formation of seven mutations, for which it has been experimentally assessed whether they disrupt capsid formation or not. With one exception, our approach predicts the effect of the mutations on the capsid stability. The method allows the identification of interaction networks, which drive capsid assembly, and highlights the plasticity of the interfaces between subunits in the capsid. Proteins 2015; 83:1733–1741. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc     

Oviposition substrate in Asian tiger mosquito surveillance: Do the sizes matter?

Ovitraps are regarded as a reliable system to monitor Aedes albopictus dynamics. However, the dimensions of the oviposition substrate are not standardized, and no studies have investigated which should be the most effective sizes. In this study, the effect of paddle sizes in tiger mosquito egg collection was evaluated. Egg count and density on the wide surfaces and margins of different‐sized oviposition substrates have been evaluated in two studies (A and B). In study A, a total of 29,995 Ae. albopictus eggs was counted in 250 classic oviposition substrates. Eggs were found on both wide surfaces (53.1%) and margins (46.9%). Egg density was significantly larger in margins compared to wide surfaces. Overall in study B, 983 Ae. albopictus eggs were collected. According to paddle sizes, 51.8% of eggs were on large and 48.2% on small paddles. Mean egg density of wide surfaces was significantly larger in small paddles (0.25 eggs/cm²) compared to large paddles (0.06 eggs/cm²). Results indicate that wider oviposition substrates do not mean larger number of Ae. albopictus eggs. Indeed, on paddles four times thinner than others, the number of eggs counted was not statistically different. These findings suggest that small paddles may be routinely employed in ovitraps, thus allowing savings of materials and money.     

Alpha sarcoglycan is required for FGF-dependent myogenic progenitor cell proliferation in vitro and in vivo

Mice deficient in α-sarcoglycan (Sgca-null mice) develop progressive muscular dystrophy and serve as a model for human limb girdle muscular dystrophy type 2D. Sgca-null mice suffer a more severe myopathy than that of mdx mice, the model for Duchenne muscular dystrophy. This is the opposite of what is observed in humans and the reason for this is unknown. In an attempt to understand the cellular basis of this severe muscular dystrophy, we isolated clonal populations of myogenic progenitor cells (MPCs), the resident postnatal muscle progenitors of dystrophic and wild-type mice. MPCs from Sgca-null mice generated much smaller clones than MPCs from wild-type or mdx dystrophic mice. Impaired proliferation of Sgca-null myogenic precursors was confirmed by single fiber analysis and this difference correlated with Sgca expression during MPC proliferation. In the absence of dystrophin and associated proteins, which are only expressed after differentiation, SGCA complexes with and stabilizes FGFR1. Deficiency of Sgca leads to an absence of FGFR1 expression at the membrane and impaired MPC proliferation in response to bFGF. The low proliferation rate of Sgca-null MPCs was rescued by transduction with Sgca-expressing lentiviral vectors. When transplanted into dystrophic muscle, Sgca-null MPCs exhibited reduced engraftment. The reduced proliferative ability of Sgca-null MPCs explains, at least in part, the severity of this muscular dystrophy and also why wild-type donor progenitor cells engraft efficiently and consequently ameliorate disease.