Next-Generation Sequencing and In Vitro Expression Study of ADAMTS13 Single Nucleotide Variants in Deep Vein Thrombosis

BackgroundDeep vein thrombosis (DVT) genetic predisposition is partially known.ObjectivesThis study aimed at assessing the functional impact of nine ADAMTS13 single nucleotide variants (SNVs) previously reported to be associated as a group with DVT in a burden test and the individual association of selected variants with DVT risk in two replication studies.MethodsWild-type and mutant recombinant ADAMTS13 were transiently expressed in HEK293 cells. Antigen and activity of recombinant ADAMTS13 were measured by ELISA and FRETS-VWF73 assays, respectively. The replication studies were performed in an Italian case-control study (Milan study; 298/298 patients/controls) using a next-generation sequencing approach and in a Dutch case-control study (MEGA study; 4306/4887 patients/controls) by TaqMan assays.ResultsIn vitro results showed reduced ADAMTS13 activity for three SNVs (p.Val154Ile [15%; 95% confidence interval [CI] 14–16], p.Asp187His [19%; 95%[CI] 17–21], p.Arg421Cys [24%; 95%[CI] 22–26]) similar to reduced plasma ADAMTS13 levels of patients carriers for these SNVs. Therefore these three SNVs were interrogated for risk association. The first replication study identified 3 heterozygous carriers (2 cases, 1 control) of p.Arg421Cys (odds ratio [OR] 2, 95%[CI] 0.18–22.25). The second replication study identified 2 heterozygous carriers (1 case, 1 control) of p.Asp187His ([OR] 1.14, 95%[CI] 0.07–18.15) and 10 heterozygous carriers (4 cases, 6 controls) of p.Arg421Cys ([OR] 0.76, 95%[CI] 0.21–2.68).ConclusionsThree SNVs (p.Val154Ile, p.Asp187His and p.Arg421Cys) showed reduced ex vivo and in vitro ADAMTS13 levels. However, the low frequency of these variants makes it difficult to confirm their association with DVT.     

Pseudomonas putida A ATCC 12633 oxidizes trimethylamine aerobically via two different pathways

The present study examined the aerobic metabolism of trimethylamine in Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine. In both conditions, the trimethylamine was used as a nitrogen source and also accumulated in the cell, slowing the bacterial growth. Decreased bacterial growth was counteracted by the addition of AlCl₃. Cell-free extracts prepared from cells grown aerobically on tetradecyltrimethylammonium bromide exhibited trimethylamine monooxygenase activity that produced trimethylamine N-oxide and trimethylamine N-oxide demethylase activity that produced dimethylamine. Cell-free extracts from cells grown on trimethylamine exhibited trimethylamine dehydrogenase activity that produced dimethylamine, which was oxidized to methanal and methylamine by dimethylamine dehydrogenase. These results show that this bacterial strain uses two enzymes to initiate the oxidation of trimethylamine in aerobic conditions. The apparent K_m for trimethylamine was 0.7 mM for trimethylamine monooxygenase and 4.0 mM for trimethylamine dehydrogenase, but both enzymes maintain similar catalytic efficiency (0.5 and 0.4, respectively). Trimethylamine dehydrogenase was inhibited by trimethylamine from 1 mM. Therefore, the accumulation of trimethylamine inside Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine may be due to the low catalytic efficiency of trimethylamine monooxygenase and trimethylamine dehydrogenase.     

Micropropagation by explants ofGracilaria chilensisBird, McLachlan and Oliveira

Regeneration studies were carried out on four morphotypes ofGracilaria chilensisfound on the coast of Chile (36–41°S). Vegetative explants were obtained from sections of apical and medial origin and were cultured in five media: filtered autoclaved seawater (FAS), Provasoli enriched seawater (PES), PES + indole-3-acetic acid (PIAA), PES + kinetin (PK) and PK + IAA (PKIAA). Mature female gametophyte and tetrasporophyte explants were obtained from sections of medial origin and were cultured in FAS and PES. Bud differentiation and/or callus formation were the morphogenetic responses to the wounding of the explants in all culture media. Plantlet regeneration was obtained from excised buds and calluses cultured separately.     

Sunny hours and variations in the prevalence of asthma in schoolchildren according to the International Study of Asthma and Allergies (ISAAC) Phase III in Spain

The objective of this study was to estimate the relationship between the prevalence of asthma in schoolchildren aged 6?C7?years and 13?C14?years and the mean annual sunny hours (MASH) in Spain, and to explore predictive models for asthma prevalence. The prevalence of asthma was obtained from the International Study of Asthma and Allergies (ISAAC) Phase III 2002?C2003, and climate and socio-economic variables from official sources. Nine centres were studied and a further four centres, two of which are in ISAAC, to test the predictive models. Logistic regression was used to estimate adjusted prevalence rates of asthma for each centre, and multiple regression models to study the effects of MASH and other meteorological and socio-economic variables. The adjusted prevalence rate of asthma decreased 0.6% [95% confidence interval (CI) 0.4?C0.8%] for the 6-7?years group and 1.1% (95% CI 0.8?C1.3%) for the 13?C14?years group with an increase in the MASH of 100?h. Relative humidity was negatively associated with asthma in the older age group, and gross province product per capita (GPP) was positively associated with asthma in the younger age group. The predictive models, which included MASH, gender, relative humidity, and GPP, anticipated prevalence rates of asthma without significant differences between the levels observed and those expected in 9 of the11 measurements carried out. The results indicate that sunny hours have a protective effect on the prevalence of asthma in schoolchildren.     

Raman and FTIR microscopic imaging of colon tissue: a comparative study

Colon tissue constitutes a valid model for the comparative analysis of soft tissue by Raman and Fourier transform infrared (FTIR) imaging because it contains four major tissue types such as muscle tissue, connective tissue, epithelium and nerve cells. Raman microscopic images were recorded in the mapping mode using 785 nm laser excitation and a step size of 10 μm from three regions within a thin section that encompassed mucus, mucosa, submucosa, and longitudinal and circular muscle layers. FTIR microscopic images that were composed of 4, 8 and 9 individual images of 4096 spectra each were recorded from the same regions using a FTIR spectrometer coupled to a microscope with a focal plane array detector. Furthermore, Raman microscopic images were recorded at a step size of 2.5 μm from three ganglia that belong to the myenteric plexus. The results are discussed with respect to lateral resolution, spectral resolution, acquisition time and sensitivity of both modalities. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A fuzzy anomaly indicator for environmental monitoring at continental scale

Environmental status assessment and monitoring can be performed by the integration of multi-source datasets at continental and global scales. We propose a methodology for the development of a new anomaly indicator (AI) which can highlight the occurrence of anomalous conditions in a synthetic fashion by analysis of a set of spatial input data. Anomalous conditions are defined relative to long-term average assumed as normal or reference status of the vegetated land surface. The indicator is defined according to fuzzy set theory which is a powerful means of handling uncertain and imprecise knowledge of environmental systems. The indicator integrates, in an innovative way, the anomaly scores of a set of contributing factors extracted from the analysis of historical time series, mainly of Earth observations data. These time series are used to automatically derive the fuzzy membership functions that quantify the contribution of each factor to the final indicator. No reference data and expert knowledge are strictly required for the implementation of the AI although the methodology allows customization where this type of information is available. The method was tested over the African continent for the period 1996–2002; monthly AI values were derived with input datasets of vegetation phenology and rainfall estimates. The output AI continental maps bring new information by integrating multiple factors and they highlight patterns of anomalous conditions of the status of the environment. The analysis of the correlation with the El Niño Southern Oscillation (ENSO) shows that the AI is able to identify the effects of this phenomenon and its spatio-temporal dynamics. The 1997–1998 and 2000–2001 ENSO events are clearly highlighted by the highest AI values in specific regions of the continent. The indicator proposed is a valuable tool which can help guide in depth and detailed investigations of environmental conditions at local scale.