Transcriptomic analysis of the venom gland of the red-headed krait (<Emphasis Type="Italic">Bungarus flaviceps</Emphasis>) using expressed sequence tags

Background  

The Red-headed krait (Bungarus flaviceps, Squamata: Serpentes: Elapidae) is a medically important venomous snake that inhabits South-East Asia. Although the venoms of most species of the snake genus Bungarus have been well characterized, a detailed compositional analysis of B. flaviceps is currently lacking.     

Risks and advantages of using surface laser photogrammetry on free‐ranging marine organisms: a case study on white sharks Carcharodon carcharias

This study employed a non‐lethal measurement tool, which combined an existing photo‐identification technique with a surface, parallel laser photogrammetry technique, to accurately estimate the size of free‐ranging white sharks Carcharodon carcharias. Findings confirmed the hypothesis that surface laser photogrammetry is more accurate than crew‐based estimations that utilized a shark cage of known size as a reference tool. Furthermore, field implementation also revealed that the photographer's angle of reference and the shark's body curvature could greatly influence technique accuracy, exposing two limitations. The findings showed minor inconsistencies with previous studies that examined pre‐caudal to total length ratios of dead specimens. This study suggests that surface laser photogrammetry can successfully increase length estimation accuracy and illustrates the potential utility of this technique for growth and stock assessments on free‐ranging marine organisms, which will lead to an improvement of the adaptive management of the species.     

Separating the wheat from the chaff: a prioritisation pipeline for the analysis of metabolomics datasets

Liquid Chromatography Mass Spectrometry (LC-MS) is a powerful and widely applied method for the study of biological systems, biomarker discovery and pharmacological interventions. LC-MS measurements are, however, significantly complicated by several technical challenges, including: (1) ionisation suppression/enhancement, disturbing the correct quantification of analytes, and (2) the detection of large amounts of separate derivative ions, increasing the complexity of the spectra, but not their information content. Here we introduce an experimental and analytical strategy that leads to robust metabolome profiles in the face of these challenges. Our method is based on rigorous filtering of the measured signals based on a series of sample dilutions. Such data sets have the additional characteristic that they allow a more robust assessment of detection signal quality for each metabolite. Using our method, almost 80% of the recorded signals can be discarded as uninformative, while important information is retained. As a consequence, we obtain a broader understanding of the information content of our analyses and a better assessment of the metabolites detected in the analyzed data sets. We illustrate the applicability of this method using standard mixtures, as well as cell extracts from bacterial samples. It is evident that this method can be applied in many types of LC-MS analyses and more specifically in untargeted metabolomics.

     

Liver-specific Commd1 knockout mice are susceptible to hepatic copper accumulation

Canine copper toxicosis is an autosomal recessive disorder characterized by hepatic copper accumulation resulting in liver fibrosis and eventually cirrhosis. We have identified COMMD1 as the gene underlying copper toxicosis in Bedlington terriers. Although recent studies suggest that COMMD1 regulates hepatic copper export via an interaction with the Wilson disease protein ATP7B, its importance in hepatic copper homeostasis is ill-defined. In this study, we aimed to assess the effect of Commd1 deficiency on hepatic copper metabolism in mice. Liver-specific Commd1 knockout mice (Commd1(Δhep)) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1(Δhep) mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1(Δhep) mice were viable and did not develop an overt phenotype. At six weeks, the liver copper contents was increased up to a 3-fold upon Commd1 deficiency, but declined with age to concentrations similar to those seen in controls. Interestingly, Commd1(Δhep) mice fed a copper-enriched diet progressively accumulated copper in the liver up to a 20-fold increase compared to controls. These copper levels did not result in significant induction of the copper-responsive genes metallothionein I and II, neither was there evidence of biochemical liver injury nor overt liver pathology. The biosynthesis of ceruloplasmin was clearly augmented with age in Commd1(Δhep) mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1(Δhep) mice could be detected. Despite the absence of hepatocellular toxicity in Commd1(Δhep) mice, the changes in liver copper displayed several parallels with copper toxicosis in Bedlington terriers. Thus, these results provide the first genetic evidence for COMMD1 to play an essential role in hepatic copper homeostasis and present a valuable mouse model for further understanding of the molecular mechanisms underlying hepatic copper homeostasis.     

Identification of bacteria with beta-galactosidase activity in faeces from lactase non-persistent subjects

Previous studies suggest that, besides the maldigestion of lactose in the small intestine, the colonic processing of lactose might play a role in lactose intolerance. beta-Galactosidase is the bacterial enzyme which catalyzes the first step of lactose fermentation in the colon. We propose a practical method to differentiate and identify bacteria with beta-galactosidase activity in faeces which combines a colony-lift filter assay with X-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) as substrate for differentiation and the fluorescent in situ hybridization technique for identification. The method was applied to faeces from lactase non-persistent subjects. After 28 subjects had undergone one glucose and two lactose challenges, consistent intolerant (n=5) and tolerant (n=7) groups were defined according to their symptom scores. Of the 28 faecal samples, 80.6% (mean, SD: 12.1, range: 47.8-100%) of the total cultured bacteria were found to possess beta-galactosidase activity, which indicates that the bacterial beta-galactosidase is abundant in the colon. The tolerant and intolerant groups did not differ in the percentage or composition of the bacteria with beta-galactosidase activity or beta-galactosidase activity in faeces. Results suggest that the percentage or composition of the bacteria with beta-galactosidase activity in faeces do not play a role in lactose intolerance.     

Kinetic and functional properties of [3H]ZM241385, a high affinity antagonist for adenosine A2A receptors

We have characterized the binding of [2-(3)H]-4-(2-[7-Amino-2-(2-furyl)-[1,2,4]-triazolo-[2,3-a]-[1,3,5]-triazin-5-ylamino]ethyl)phenol ([(3)H]ZM241385) to adenosine A(2A) receptors in membranes of rat striatum and transfected CHO cells. Saturation experiments showed that [(3)H]ZM241385 binds to a single class of binding sites with high affinity (K(d) = 0.23 nM and 0.14 nM in CHO cell and striatal membranes, respectively). The membranes of CHO cells required pretreatment with adenosine deaminase (ADA) to achieve high-affinity binding, while ADA had no influence on the ligand binding properties in striatal membranes. The binding of [(3)H]ZM241385 was fast and reversible, achieving equilibrium within 20 minutes at all radioligand concentrations. The kinetic analysis of the [(3)H]ZM241385 interaction with A(2A) receptors indicated that the reaction had at least two subsequent steps. The first step corresponds to a fast equilibrium, which also determines the antagonist potency to competitively inhibit CGS21680-induced accumulation of cAMP (first equilibrium constant K(A) = 6.6 nM). The second step corresponds to a slow process of conformational isomerization (equilibrium constant K(i) = 0.03). The combination of the two steps gives the dissociation constant K(d) = 0.20 nM based on the kinetic data, which is in good agreement with the directly measured value. The data obtained shed light on the mechanism of the [(3)H]ZM241385 interaction with adenosine A(2A) receptors from different sources in vitro. The isomerization step of the A(2A) antagonist radioligand binding has to be taken into account for the interpretation of the binding parameters obtained from the various competition assays and explain the discrepancy between antagonist affinity in saturation experiments versus its potency in functional assays.     

Toll-Like Receptor (TLR2 and TLR4) Polymorphisms and Chronic Obstructive Pulmonary Disease

Toll-like receptors (TLRs) participate in the defence against bacterial infections that are common in patients with Chronic Obstructive Pulmonary Disease (COPD). We studied all tagging SNPs in TLR2 and TLR4 and their associations with the level and change over time of both FEV(1) and sputum inflammatory cells in moderate-to-severe COPD. Nine TLR2 SNPs and 17 TLR4 SNPs were genotyped in 110 COPD patients. Associations of SNPs with lung function and inflammatory cells in induced sputum were analyzed cross-sectionally with linear regression and longitudinally with linear mixed-effect models. Two SNPs in TLR2 (rs1898830 and rs11938228) were associated with a lower level of FEV(1) and accelerated decline of FEV(1) and higher numbers of sputum inflammatory cells. None of the TLR4 SNPs was associated with FEV(1) level. Eleven out of 17 SNPs were associated with FEV(1) decline, including rs12377632 and rs10759931, which were additionally associated with higher numbers of sputum inflammatory cells at baseline and with increase over time. This is the first longitudinal study showing that tagging SNPs in TLR2 and TLR4 are associated with the level and decline of lung function as well as with inflammatory cell numbers in induced sputum in COPD patients, suggesting a role in the severity and progression of COPD.     

Separating the wheat from the chaff: a prioritisation pipeline for the analysis of metabolomics datasets

Liquid Chromatography Mass Spectrometry (LC-MS) is a powerful and widely applied method for the study of biological systems, biomarker discovery and pharmacological interventions. LC-MS measurements are, however, significantly complicated by several technical challenges, including: (1) ionisation suppression/enhancement, disturbing the correct quantification of analytes, and (2) the detection of large amounts of separate derivative ions, increasing the complexity of the spectra, but not their information content. Here we introduce an experimental and analytical strategy that leads to robust metabolome profiles in the face of these challenges. Our method is based on rigorous filtering of the measured signals based on a series of sample dilutions. Such data sets have the additional characteristic that they allow a more robust assessment of detection signal quality for each metabolite. Using our method, almost 80% of the recorded signals can be discarded as uninformative, while important information is retained. As a consequence, we obtain a broader understanding of the information content of our analyses and a better assessment of the metabolites detected in the analyzed data sets. We illustrate the applicability of this method using standard mixtures, as well as cell extracts from bacterial samples. It is evident that this method can be applied in many types of LC-MS analyses and more specifically in untargeted metabolomics.     

Algorithmic co-optimization of genetic constructs and growth conditions: application to 6-ACA,a potential nylon-6 precursor

Optimizing bio-production involves strain and process improvements performed as discrete steps. However, environment impacts genotype and a strain that is optimal under one set of conditions may not be under different conditions. We present a methodology to simultaneously vary genetic and process factors, so that both can be guided by design of experiments (DOE). Advances in DNA assembly and gene insulation facilitate this approach by accelerating multi-gene pathway construction and the statistical interpretation of screening data. This is applied to a 6-aminocaproic acid (6-ACA) pathway in Escherichia coli consisting of six heterologous enzymes. A 32-member fraction factorial library is designed that simultaneously perturbs expression and media composition. This is compared to a 64-member full factorial library just varying expression (0.64 Mb of DNA assembly). Statistical analysis of the screening data from these libraries leads to different predictions as to whether the expression of enzymes needs to increase or decrease. Therefore, if genotype and media were varied separately this would lead to a suboptimal combination. This is applied to the design of a strain and media composition that increases 6-ACA from 9 to 48 mg/l in a single optimization step. This work introduces a generalizable platform to co-optimize genetic and non-genetic factors.