Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.     

Evidence for Inbreeding and Genetic Differentiation among Geographic Populations of the Saprophytic Mushroom Trogia venenata from Southwestern China

During the past 40 years, more than 400 Sudden Unexplained Deaths (SUDs) have occurred in Yunnan, southwestern China. Epidemiological and toxicological analyses suggested that a newly discovered mushroom called Trogia venenata was the leading culprit for SUDs. At present, relatively little is known about the genetics and natural history of this mushroom. In this study, we analyzed the sequence variation at four DNA fragments among 232 fruiting bodies of T. venenata collected from seven locations. Our ITS sequence analyses confirmed that all the isolates belonged to the same species. The widespread presence of sequence heterozygosity within many strains at each of three protein-coding genes suggested that the fruiting bodies were diploid, dikaryotic or heterokaryotic. Within individual geographic populations, we found significant deviations of genotype frequencies from Hardy-Weinberg expectations, with the overall observed heterozygosity lower than that expected under random mating, consistent with prevalent inbreeding within local populations. The geographic populations were overall genetically differentiated. Interestingly, while a positive correlation was found between population genetic distance and geographic distance, there was little correlation between genetic distance and barium concentration difference for the geographic populations. Our results suggest frequent inbreeding, geographic structuring, and limited gene flow among geographic populations of T. venenata from southwestern China.     

Structural Insight of a Trimodular Halophilic Cellulase with a Family 46 Carbohydrate-Binding Module

Cellulases are the key enzymes used in the biofuel industry. A typical cellulase contains a catalytic domain connected to a carbohydrate-binding module (CBM) through a flexible linker. Here we report the structure of an atypical trimodular cellulase which harbors a catalytic domain, a CBM46 domain and a rigid CBM_X domain between them. The catalytic domain shows the features of GH5 family, while the CBM46 domain has a sandwich-like structure. The catalytic domain and the CBM46 domain form an extended substrate binding cleft, within which several tryptophan residues are well exposed. Mutagenesis assays indicate that these residues are essential for the enzymatic activities. Gel affinity electrophoresis shows that these tryptophan residues are involved in the polysaccharide substrate binding. Also, electrostatic potential analysis indicates that almost the entire solvent accessible surface of CelB is negatively charged, which is consistent with the halophilic nature of this enzyme.     

The metalloproteinase ADAM‐12 regulates bronchial epithelial cell proliferation and apoptosis

Abstract. Objectives: The ADAMs (a disintegrin and metalloproteinase) enzymes compose a family of membrane‐bound proteins characterized by their multi‐domain structure and ADAM‐12 expression is elevated in human non‐small cell lung cancers. The aim of this study was to investigate the roles played by ADAM‐12 in critical steps of bronchial cell transformation during carcinogenesis. Materials and methods: To assess the role of ADAM‐12 in tumorigenicity, BEAS‐2B cells were transfected with a plasmid encoding human full‐length ADAM‐12 cDNA, and then the effects of ADAM‐12 overexpression on cell behaviour were explored. Treatment of clones with heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF) neutralizing antibodies as well as an EGFR inhibitor allowed the dissection of mechanisms regulating cell proliferation and apoptosis. Results: Overexpression of ADAM‐12 in BEAS‐2B cells promoted cell proliferation. ADAM‐12 overexpressing clones produced higher quantities of HB‐EGF in their culture medium which may rely on membrane‐bound HB‐EGF shedding by ADAM‐12. Targeting HB‐EGF activity with a neutralizing antibody abrogated enhanced cell proliferation in the ADAM‐12 overexpressing clones. In sharp contrast, targeting of amphiregulin, EGF or transforming growth factor‐α failed to influence cell proliferation; moreover, ADAM‐12 transfectants were resistant to etoposide‐induced apoptosis and the use of a neutralizing antibody against HB‐EGF activity restored rates of apoptosis to be similar to controls.Conclusions: ADAM‐12 contributes to enhancing HB‐EGF shedding from plasma membranes leading to increased cell proliferation and reduced apoptosis in this bronchial epithelial cell line.     

Effects of larval competition on survival and growth in Mediterranean fruit flies

1. Larval success was compared when one, two, or three egg clutches were laid in kumquat fruits (≈ 10 ml in volume) either successively on the same day or at the rate of one clutch per day. 2. Increased clutch density was associated with a significant decrease in larval survival rate and non‐significant decreases in larval growth rate and pupal mass. 3. Larval and pupal parameters showed significantly larger variance when clutches were laid on successive days than on the same day, suggesting a competitive advantage for older larvae over younger larvae. 4. The results suggest that, in small fruit, reduced fitness due to larval competition may act against possible fitness benefits due to social facilitation among adult females, hence reducing the likelihood of non‐linear population dynamics caused by processes such as the Allee effect.