Human alpha‐ and beta‐NRXN1 isoforms rescue behavioral impairments of Caenorhabditis elegans neurexin‐deficient mutants

Neurexins are cell adhesion proteins that interact with neuroligin and other ligands at the synapse. In humans, mutations in neurexin or neuroligin genes have been associated with autism and other mental disorders. The human neurexin and neuroligin genes are orthologous to the Caenorhabditis elegans genes nrx‐1 and nlg‐1, respectively. Here we show that nrx‐1‐deficient mutants are defective in exploratory capacity, sinusoidal postural movements and gentle touch response. Interestingly, the exploratory behavioral phenotype observed in nrx‐1 mutants was markedly different to nlg‐1‐deficient mutants; thus, while the former had a ‘hyper‐reversal’ phenotype increasing the number of changes of direction with respect to the wild‐type strain, the nlg‐1 mutants presented a ‘hypo‐reversal’ phenotype. On the other hand, the nrx‐1‐ and nlg‐1‐defective mutants showed similar abnormal sinusoidal postural movement phenotypes. The response of these mutant strains to aldicarb (acetylcholinesterase inhibitor), levamisole (ACh agonist) and pentylenetetrazole [gamma‐aminobutyric (GABA) receptor antagonist], suggested that the varying behavioral phenotypes were caused by defects in ACh and/or GABA inputs. The defective behavioral phenotypes of nrx‐1‐deficient mutants were rescued in transgenic strains expressing either human alpha‐ or beta‐NRXN‐1 isoforms under the worm nrx‐1 promoter. A previous report had shown that human and rat neuroligins were functional in C. elegans. Together, these results suggest that the functional mechanism underpinning both neuroligin and neurexin in the nematode are comparable to human. In this sense the nematode might constitute a simple in vivo model for understanding basic mechanisms involved in neurological diseases for which neuroligin and neurexin are implicated in having a role.     

Post-translational processing of modular xylanases from <Emphasis Type="Italic">Streptomyces</Emphasis> is dependent on the carbohydrate-binding module

Xylanases are very often modular enzymes composed of one or more catalytic domains and carbohydrate-binding modules (CBMs) connected by a flexible linker region. Usually, when these proteins are processed they lose their carbohydrate-binding capacity. Here, the role of the linker regions and cellulose- or xylan-binding domains in the processing of Xys1L from Streptomyces halstedii JM8 and Xyl30L from Streptomyces avermitilis UAH30 was studied. Xys1 variants with different linker lengths were tested, these being unable to avoid protein processing. Moreover, several fusion proteins between the Xys1 and Xyl30 domains were obtained and their proteolytic stability was studied. We demonstrate that CBM processing takes place even in the complete absence of the linker sequence. We also show that the specific carbohydrate module determines this cleavage in the proteins studied.     

Proteomic tools for the analysis of transient interactions between metalloproteins

Metalloproteins play major roles in cell metabolism and signalling pathways. In many cases, they show moonlighting behaviour, acting in different processes, depending on the physiological state of the cell. To understand these multitasking proteins, we need to discover the partners with which they carry out such novel functions. Although many technological and methodological tools have recently been reported for the detection of protein interactions, specific approaches to studying the interactions involving metalloproteins are not yet well developed. The task is even more challenging for metalloproteins, because they often form short-lived complexes that are difficult to detect. In this review, we gather the different proteomic techniques and biointeractomic tools reported in the literature. All of them have shown their applicability to the study of transient and weak protein-protein interactions, and are therefore suitable for metalloprotein interactions.     

Functional K(v)10.1 channels localize to the inner nuclear membrane

Ectopically expressed human K(V)10.1 channels are relevant players in tumor biology. However, their function as ion channels at the plasma membrane does not totally explain their crucial role in tumors. Both in native and heterologous systems, it has been observed that a majority of K(V)10.1 channels remain at intracellular locations. In this study we investigated the localization and possible roles of perinuclear K(V)10.1. We show that K(V)10.1 is expressed at the inner nuclear membrane in both human and rat models; it co-purifies with established inner nuclear membrane markers, shows resistance to detergent extraction and restricted mobility, all of them typical features of proteins at the inner nuclear membrane. K(V)10.1 channels at the inner nuclear membrane are not all transported directly from the ER but rather have been exposed to the extracellular milieu. Patch clamp experiments on nuclei devoid of external nuclear membrane reveal the existence of channel activity compatible with K(V)10.1. We hypothesize that K(V)10.1 channels at the nuclear envelope might participate in the homeostasis of nuclear K(+), or indirectly interact with heterochromatin, both factors known to affect gene expression.     

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients

Background

A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation.

Methodology/Findings

An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10⁻³ par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories.

Conclusion/Significance

This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.     

Detection of pathogenic mycobacteria based on functionalized quantum dots coupled with immunomagnetic separation

Mycobacteria have always proven difficult to identify due to their low growth rate and fastidious nature. Therefore molecular biology and more recently nanotechnology, have been exploited from early on for the detection of these pathogens. Here we present the first stage of development of an assay incorporating cadmium selenide quantum dots (QDs) for the detection of mycobacterial surface antigens. The principle of the assay is the separation of bacterial cells using magnetic beads coupled with genus-specific polyclonal antibodies and monoclonal antibodies for heparin-binding hemagglutinin. These complexes are then tagged with anti-mouse biotinylated antibody and finally streptavidin-conjugated QDs which leads to the detection of a fluorescent signal. For the evaluation of performance, the method under study was applied on Mycobacterium bovis BCG and Mycobacterium tuberculosis (positive controls), as well as E. coli and Salmonella spp. that constituted the negative controls. The direct observation of the latter category of samples did not reveal fluorescence as opposed to the mycobacteria mentioned above. The minimum detection limit of the assay was defined to 10(4) bacteria/ml, which could be further decreased by a 1 log when fluorescence was measured with a spectrofluorometer. The method described here can be easily adjusted for any other protein target of either the pathogen or the host, and once fully developed it will be directly applicable on clinical samples.     

Spatial distribution of the soil organic carbon pool in a Holm oak dehesa in Spain

Aims

Dehesas are agroforestry systems characterized by scattered trees among pastures, crops and/or fallows. A study at a Spanish dehesa has been carried out to estimate the spatial distribution of the soil organic carbon stock and to assess the influence of the tree cover.

Methods

The soil organic carbon stock was estimated from the five uppermost cm of the mineral soil with high spatial resolution at two plots with different grazing intensities. The Universal Kriging technique was used to assess the spatial distribution of the soil organic carbon stocks, using tree coverage within a buffering area as an auxiliary variable.

Results

A significant positive correlation between tree presence and soil organic carbon stocks up to distances of around 8 m from the trees was found. The tree crown cover within a buffer up to a distance similar to the crown radius around the point absorbed 30 % of the variance in the model for both grazing intensities, but residual variance showed stronger spatial autocorrelation under regular grazing conditions.

Conclusions

Tree cover increases soil organic carbon stocks, and can be satisfactorily estimated by means of crown parameters. However, other factors are involved in the spatial pattern of the soil organic carbon distribution. Livestock plays an interactive role together with tree presence in soil organic carbon distribution.