Functional assay for shiga-like toxin via detection by antibody capture and multivalent galabiose binding

A functional detection assay was developed for Escherichia coli secreted shiga-like toxin based on antibody capture and visualization with a multivalent galabiose ligand. It was possible to detect verotoxin in medically relevant E. coli samples in a dose dependent fashion. This method is a new step towards measuring functional protein levels in complex mixtures, which can be used for diagnostic purposes in a clinical setting.     

Simultaneous quantification of emtricitabine and tenofovir nucleotides in peripheral blood mononuclear cells using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry

Emtricitabine (FTC) and tenofovir (TFV) are widely used antiviral agents that require intracellular phosphorylation to become active. This article describes the development and validation of an assay for the simultaneous quantification of FTC mono-, di- and triphosphate (FTC-MP, -DP and -TP), TFV and TFV mono- and diphosphate (TFV-MP and -DP) in peripheral blood mononuclear cells. Reference compounds and internal standards were obtained by thermal degradation of FTC-TP, TFV-DP, stable isotope-labeled TFV-DP and stable isotope-labeled cytosine triphosphate. Cells were lysed in methanol:water (70:30, v/v) and the extracted nucleotides were analyzed using weak anion-exchange chromatography coupled with tandem mass spectrometry. Calibration ranges in PBMC lysate from 0.727 to 36.4, 1.33 to 66.4 and 1.29 to 64.6 nM for FTC-MP, FTC-DP and FTC-TP and from 1.51 to 75.6, 1.54 to 77.2 and 2.54 to 127 nM for TFV, TFV-MP and TFV-DP, respectively, were validated. Accuracies were within ?10.3 and 16.7% deviation at the lower limit of quantification at which the coefficients of variation were less than 18.2%. At the other tested levels accuracies were within ?14.3 and 9.81% deviation and the coefficients of variation lower than 14.7%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully applied to clinical samples.     

Limnological and ecological sensitivity of Rwenzori mountain lakes to climate warming

An increasing number of studies forecast that anthropogenic climate change poses serious consequences for the biodiversity and ecosystem functioning of high-elevation mountain lakes, through a series of both direct and indirect effects. The impacts of future climate warming on alpine ecosystems are of particular concern, given that warming is expected to be most pronounced at high elevations around the globe. Here, we evaluate the limnological and ecological sensitivity of high-elevation lakes in the Rwenzori Mountains (Uganda-D. R. Congo) to climate change. This is done by comparing the species assemblages of larval chironomid remains deposited recently in lake sediments with those deposited at the base of short cores (dated to within or shortly after the Little Ice Age) in 16 lakes. Chironomid-based reconstructions of mean annual air temperature (MATemp) are made using a variety of inference models (with transfer functions based on weighted averaging, weighted-averaging partial least squares, and a weighted modern analogue technique), and two different calibration data sets, one covering the full regional temperature gradient and one comprising only high-elevation Rwenzori lakes and ponds. The reconstructed historical temperature change ranges between a cooling of −2.03°C and a warming of +3.22°C (with n = 16 lakes × 3 models × 2 calibration data sets). However, excluding the atypical mid-elevation lake Mahoma (2,990 m altitude), we find a three-to-one ratio of cases of inferred warming against inferred cooling, and of the 24 Δ MATemp values exceeding 0.60°C, 23 are positive and only one is negative. Chironomid-inferred temperature changes mostly fall within the error range of the regional temperature inference models. A generalized linear mixed model analysis of the combined result from all lakes (except Mahoma) nevertheless indicates significantly warmer MATemp (on average +0.38 ± 0.11°C) at present compared to between ~85 and ~645 years ago. Inferred temperature changes are independent of whether lakes are located in glaciated or non-glaciated catchments, and of the age of the core base, suggesting that at least part of the signal is due to relatively recent, anthropogenic warming. The direction of faunal change at the lakes in relation to established species–environment relationships suggests that part of the observed shifts in species composition reflect lake-specific evolution in habitat features other than temperature, such as nutrients, pH or oxygen regime, which in our present calibration data set co-vary with temperature to a greater or lesser extent. The fairly uniform and marked historical warming trend in Rwenzori lakes documented by this study highlights their ecological vulnerability and their value as early warning systems for detecting the limnological and ecological effects of global warming.     

ARS2 is a conserved eukaryotic gene essential for early mammalian development

Determining the functions of novel genes implicated in cell survival is directly relevant to our understanding of mammalian development and carcinogenesis. ARS2 is an evolutionarily conserved gene that confers arsenite resistance on arsenite-sensitive Chinese hamster ovary cells. Little is known regarding the function of ARS2 in mammals. We report that ARS2 is transcribed throughout embryonic development and is expressed ubiquitously in mouse and human tissues. The mouse ARS2 protein is predominantly localized to the nucleus, and this nuclear localization is ablated in ARS2-null embryos, which in turn die around the time of implantation. After 24 h of culture, ARS2-null blastocysts contained a significantly greater number of apoptotic cells than wild-type or heterozygous blastocysts. By 48 h of in vitro culture, null blastocysts invariably collapsed and failed to proliferate. These data indicate ARS2 is essential for early mammalian development and is likely involved in an essential cellular process. The analysis of data from several independent protein-protein interaction studies in mammals, combined with functional studies of its Arabidopsis ortholog, SERRATE, suggests that this essential process is related to RNA metabolism.     

Extragastric manifestations of Helicobacter pylori infection: other Helicobacters

The finding that Helicobacter pylori is the main cause of gastritis and peptic ulcer disease has opened a new era in the gastrointestinal world. Today there is evidence that H. pylori may also play a role in different nongastric diseases, opening the new "extragastric manifestations of H. pylori infection" field. Concerning this, several studies have been published in the last year. The most convincing data arise from those investigating idiopathic thrombocytopenic purpura and sideropenic anemia, while there is also an increasing evidence for a possible association with atherosclerotic disease. Furthermore, the discovery of a number of other novel Helicobacter species has stimulated the research in different extragastric diseases, in which an infectious hypothesis is plausible. In particular, several species have been studied for a potential role in different liver and intestinal diseases with interesting findings.     

Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity

Today it is generally accepted that there are several endocytic mechanisms, both the clathrin-dependent one and mechanisms which operate without clathrin and with different requirements when it comes to dynamin, small GTP-binding proteins of the Rho family and specific lipids. It should be noted that clathrin-independent endocytosis can occur even when the cholesterol level in the membrane has been reduced to so low levels that caveolae are gone and clathrin-coated membrane areas are flat. Although new investigators in the field take it for granted that there is a multitude of entry mechanisms, it has taken a long time for this to become accepted. However, more work needs to be done, because one can still ask the question: How many endocytic mechanisms does a cell have, what are their function, and how are they regulated? This article describes some of the history of endocytosis research and attempts to give an overview of the complexity of the mechanisms and their regulation.     

The distribution of the coalescence time and the number of pairwise nucleotide differences in the "isolation with migration" model

This paper is concerned with the “isolation with migration” model, where a panmictic ancestral population gave rise to a symmetric n-island model, time τ ago. Explicit analytical expressions are derived for the probability density function of the coalescence time of a pair of genes sampled at random from the same subpopulation or from different subpopulations, and for the probability distribution of the number of pairwise nucleotide differences.     

Submicroscopic duplications of the hydroxysteroid dehydrogenase HSD17B10 and the E3 ubiquitin ligase HUWE1 are associated with mental retardation

Submicroscopic copy-number imbalances contribute significantly to the genetic etiology of human disease. Here, we report a novel microduplication hot spot at Xp11.22 identified in six unrelated families with predominantly nonsyndromic XLMR. All duplications segregate with the disease, including the large families MRX17 and MRX31. The minimal, commonly duplicated region contains three genes: RIBC1, HSD17B10, and HUWE1. RIBC1 could be excluded on the basis of its absence of expression in the brain and because it escapes X inactivation in females. For the other genes, expression array and quantitative PCR analysis in patient cell lines compared to controls showed a significant upregulation of HSD17B10 and HUWE1 as well as several important genes in their molecular pathways. Loss-of-function mutations of HSD17B10 have previously been associated with progressive neurological disease and XLMR. The E3 ubiquitin ligase HUWE1 has been implicated in TP53-associated regulation of the neuronal cell cycle. Here, we also report segregating sequence changes of highly conserved residues in HUWE1 in three XLMR families; these changes are possibly associated with the phenotype. Our findings demonstrate that an increased gene dosage of HSD17B10, HUWE1, or both contribute to the etiology of XLMR and suggest that point mutations in HUWE1 are associated with this disease too.