Direct Detection and Differentiation of Pathogenic Leptospira Species Using a Multi-Gene Targeted Real Time PCR Approach

Leptospirosis is a growing public and veterinary health concern caused by pathogenic species of Leptospira. Rapid and reliable laboratory tests for the direct detection of leptospiral infections in animals are in high demand not only to improve diagnosis but also for understanding the epidemiology of the disease. In this work we describe a novel and simple TaqMan-based multi-gene targeted real-time PCR approach able to detect and differentiate Leptospira interrogans, L. kirschneri, L. borgpeteresenii and L. noguchii, which constitute the veterinary most relevant pathogenic species of Leptospira. The method uses sets of species-specific probes, and respective flanking primers, designed from ompL1 and secY gene sequences. To monitor the presence of inhibitors, a duplex amplification assay targeting both the mammal β-actin and the leptospiral lipL32 genes was implemented. The analytical sensitivity of all primer and probe sets was estimated to be <10 genome equivalents (GE) in the reaction mixture. Application of the amplification reactions on genomic DNA from a variety of pathogenic and non-pathogenic Leptospira strains and other non-related bacteria revealed a 100% analytical specificity. Additionally, pathogenic leptospires were successfully detected in five out of 29 tissue samples from animals (Mus spp., Rattus spp., Dolichotis patagonum and Sus domesticus). Two samples were infected with L. borgpetersenii, two with L. interrogans and one with L. kirschneri. The possibility to detect and identify these pathogenic agents to the species level in domestic and wildlife animals reinforces the diagnostic information and will enhance our understanding of the epidemiology of leptopirosis.     

Methods for site controlled coupling to carboxymethyldextran surfaces in surface plasmon resonance sensors

In the present study, optimized methods for in-situ ligand immobilization to carboxymethylated dextran coated gold surfaces for use in surface plasmon resonance (SPR) based biosensor devices were developed. Immobilization methods based on selective thiol reactions, either via thiols on the sensor surface or via thiols introduced on the ligand, were shown to give high yields of functionally active material. Methods are also described for linking aldehyde containing ligands to hydrazide modified surfaces and for coupling based on the avidin/biotin concept. The importance of blocking residual active groups (for thiol coupling) and reduction of acid labile hydrazone bonds formed in the aldehyde coupling was demonstrated. The methods were found to work efficiently in combination with conditions suitable for electrostatic preconcentration of ligands to be coupled, a concept we earlier developed for coupling of amine containing ligands (Löfås & Johnsson, 1990).     

Simplifying a life cycle assessment of a mobile phone

Purpose

The possibilities for full life cycle assessment (LCA) of new Information and Communication Technology (ICT) products are often limited, so simplification approaches are needed. The aim of this paper is to investigate possible simplifications in LCA of a mobile phone and to use the results to discuss the possibilities of LCA simplifications for ICT products in a broader sense. Another aim is to identify processes and data that are sensitive to different methodological choices and assumptions related to the environmental impacts of a mobile phone.

Methods

Different approaches to a reference LCA of a mobile phone was tested: (1) excluding environmental impact categories, (2) excluding life cycle stages/processes, (3) using secondary process data from generic databases, (4) using input-output data and (5) using a simple linear relationship between mass and embodied emissions.

Results and discussion

It was not possible to identify one or a few impact categories representative of all others. If several impact categories would be excluded, information would be lost. A precautionary approach of not excluding impact categories is therefore recommended since impacts from the different life cycle stages vary between impact categories. Regarding use of secondary data for an ICT product similar to that studied here, we recommend prioritising collection of primary (specific) data on energy use during production and use, key component data (primarily integrated circuits) and process-specific data regarding raw material acquisition of specific metals (e.g. gold) and air transport. If secondary data are used for important processes, the scaling is crucial. The use of input-output data can be a considerable simplification and is probably best used to avoid data gaps when more specific data are lacking.

Conclusions

Further studies are needed to provide for simplified LCAs for ICT products. In particular, the end-of-life treatment stage need to be further addressed, as it could not be investigated here for all simplifications due to data gaps.     

Microtubule-associated protein 1 light chain 3 (LC3) interacts with Bnip3 protein to selectively remove endoplasmic reticulum and mitochondria via autophagy

Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. Bnip3 is an atypical BH3-only protein that is known to cause mitochondrial dysfunction and cell death. Interestingly, Bnip3 can also protect against cell death by inducing mitochondrial autophagy. The mechanism for this process, however, remains poorly understood. Bnip3 contains a C-terminal transmembrane domain that is essential for homodimerization and proapoptotic function. In this study, we show that homodimerization of Bnip3 is also a requirement for induction of autophagy. Several Bnip3 mutants that do not interfere with its mitochondrial localization but disrupt homodimerization failed to induce autophagy in cells. In addition, we discovered that endogenous Bnip3 is localized to both mitochondria and the endoplasmic reticulum (ER). To investigate the effects of Bnip3 at mitochondria or the ER on autophagy, Bnip3 was targeted specifically to each organelle by substituting the Bnip3 transmembrane domain with that of Acta or cytochrome b(5). We found that Bnip3 enhanced autophagy in cells from both sites. We also discovered that Bnip3 induced removal of both ER (ERphagy) and mitochondria (mitophagy) via autophagy. The clearance of these organelles was mediated in part via binding of Bnip3 to LC3 on the autophagosome. Although ablation of the Bnip3-LC3 interaction by mutating the LC3 binding site did not impair the prodeath activity of Bnip3, it significantly reduced both mitophagy and ERphagy. Our data indicate that Bnip3 regulates the apoptotic balance as an autophagy receptor that induces removal of both mitochondria and ER.     

Enterotoxigenic Bacteroides fragilis in Hungary

Bacteroides fragilis, which constitutes about 1% of the colonic microflora in humans, is the most frequent anaerobic species involved in abscesses, soft-tissue infections and bacteraemias. Additionally, enterotoxigenic strains of B. fragilis have been demonstrated to be associated with diarrhoea in domestic animals and humans. Enterotoxigenic strains of B. fragilis derived from stool specimens and from infectious processes produce a toxin which induces a cytotoxic response in HT-29 colon carcinoma cells. These findings prompted us to investigate the prevalence of enterotoxigenic strains of B. fragilis isolated from various clinical specimens in Hungary. A total of 134 strains were collected from different clinical settings: 74 from infectious processes, 20 from stools of healthy subjects and 40 from the faeces of patients with diarrhoea where no other enteric pathogen could be isolated. Cell culture assays with HT-29 cells were performed on the filtered culture supernatants of the isolated strains. Of the 134 strains, 34 (25.3%) proved toxin-positive. The presence of free toxin was also observed in 20 of 50 (40%) of the faeces of adults with diarrhoea.     

Effects Of A Post-Weaning Cafeteria Diet In Young Rats: Metabolic Syndrome,Reduced Activity And Low Anxiety-Like Behaviour

Among adolescents, overweight, obesity and metabolic syndrome are rapidly increasing in recent years as a consequence of unhealthy palatable diets. Animal models of diet-induced obesity have been developed, but little is known about the behavioural patterns produced by the consumption of such diets. The aim of the present study was to determine the behavioural and biochemical effects of a cafeteria diet fed to juvenile male and female rats, as well as to evaluate the possible recovery from these effects by administering standard feeding during the last week of the study. Two groups of male and female rats were fed with either a standard chow diet (ST) or a cafeteria (CAF) diet from weaning and for 8 weeks. A third group of males (CAF withdrawal) was fed with the CAF diet for 7 weeks and the ST in the 8th week. Both males and females developed metabolic syndrome as a consequence of the CAF feeding, showing overweight, higher adiposity and liver weight, increased plasma levels of glucose, insulin and triglycerides, as well as insulin resistance, in comparison with their respective controls. The CAF diet reduced motor activity in all behavioural tests, enhanced exploration, reduced anxiety-like behaviour and increased social interaction; this last effect was more pronounced in females than in males. When compared to animals only fed with a CAF diet, CAF withdrawal increased anxiety in the open field, slightly decreased body weight, and completely recovered the liver weight, insulin sensitivity and the standard levels of glucose, insulin and triglycerides in plasma. In conclusion, a CAF diet fed to young animals for 8 weeks induced obesity and metabolic syndrome, and produced robust behavioural changes in young adult rats, whereas CAF withdrawal in the last week modestly increased anxiety, reversed the metabolic alterations and partially reduced overweight.     

Clathrin Hub Expression Dissociates the Actin-Binding Protein Hip1R from Coated Pits and Disrupts Their Alignment with the Actin Cytoskeleton

The actin cytoskeleton has been implicated in the maintenance of discrete sites for clathrin-coated pit formation during receptor-mediated endocytosis in mammalian cells, and its function is intimately linked to the endocytic pathway in yeast. Here we demonstrate that staining for mammalian endocytic clathrin-coated pits using a monoclonal antibody against the AP2 adaptor complex revealed a linear pattern that correlates with the organization of the actin cytoskeleton. This vesicle organization was disrupted by treatment of cells with cytochalasin D, which disassembles actin, or with 2,3-butanedione monoxime, which prevents myosin association with actin. The linear AP2 staining pattern was also disrupted in HeLa cells that were induced to express the Hub fragment of the clathrin heavy chain, which acts as a dominant-negative inhibitor of receptor-mediated endocytosis by direct interference with clathrin function. Additionally, Hub expression caused the actin-binding protein Hip1R to dissociate from coated pits. These findings indicate that proper function of clathrin is required for coated pit alignment with the actin cytoskeleton and suggest that the clathrin–Hip1R interaction is involved in the cytoskeletal organization of coated pits.     

Simple,Rapid and Inexpensive Quantitative Fluorescent PCR Method for Detection of Microdeletion and Microduplication Syndromes

Because of economic limitations, the cost-effective diagnosis of patients affected with rare microdeletion or microduplication syndromes is a challenge in developing countries. Here we report a sensitive, rapid, and affordable detection method that we have called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR). Our procedure is based on the finding of genomic regions with high homology to segments of the critical microdeletion/microduplication region. PCR amplification of both using the same primer pair, establishes competitive kinetics and relative quantification of amplicons, as happens in microsatellite-based Quantitative Fluorescence PCR. We used patients with two common microdeletion syndromes, the Williams-Beuren syndrome (7q11.23 microdeletion) and the 22q11.2 microdeletion syndromes and discovered that MQF-PCR could detect both with 100% sensitivity and 100% specificity. Additionally, we demonstrated that the same principle could be reliably used for detection of microduplication syndromes, by using patients with the Lubs (MECP2 duplication) syndrome and the 17q11.2 microduplication involving the NF1 gene. We propose that MQF-PCR is a useful procedure for laboratory confirmation of the clinical diagnosis of microdeletion/microduplication syndromes, ideally suited for use in developing countries, but having general applicability as well.     

A global quantitative synthesis of local and landscape effects on wild bee pollinators in agroecosystems

Bees provide essential pollination services that are potentially affected both by local farm management and the surrounding landscape. To better understand these different factors, we modelled the relative effects of landscape composition (nesting and floral resources within foraging distances), landscape configuration (patch shape, interpatch connectivity and habitat aggregation) and farm management (organic vs. conventional and local‐scale field diversity), and their interactions, on wild bee abundance and richness for 39 crop systems globally. Bee abundance and richness were higher in diversified and organic fields and in landscapes comprising more high‐quality habitats; bee richness on conventional fields with low diversity benefited most from high‐quality surrounding land cover. Landscape configuration effects were weak. Bee responses varied slightly by biome. Our synthesis reveals that pollinator persistence will depend on both the maintenance of high‐quality habitats around farms and on local management practices that may offset impacts of intensive monoculture agriculture.