The ancillary protein 1 of Streptococcus pyogenes FCT-1 pili mediates cell adhesion and biofilm formation through heterophilic as well as homophilic interactions

Gram‐positive pili are known to play a role in bacterial adhesion to epithelial cells and in the formation of biofilm microbial communities. In the present study we undertook the functional characterization of the pilus ancillary protein 1 (AP1_M6) from Streptococcus pyogenes isolates expressing the FCT‐1 pilus variant, known to be strong biofilm formers. Cell binding and biofilm formation assays using S. pyogenes in‐frame deletion mutants, Lactococcus expressing heterologous FCT‐1 pili and purified recombinant AP1_M6, indicated that this pilin is a strong cell adhesin that is also involved in bacterial biofilm formation. Moreover, we show that AP1_M6 establishes homophilic interactions that mediate inter‐bacterial contact, possibly promoting bacterial colonization of target epithelial cells in the form of three‐dimensional microcolonies. Finally, AP1_M6 knockout mutants were less virulent in mice, indicating that this protein is also implicated in GAS systemic infection.     

Excitability of the Motor Cortex in De Novo Patients with Celiac Disease

Introduction

Celiac disease (CD) may initially present as a neurological disorder or may be complicated by neurological changes. To date, neurophysiological studies aiming to an objective evaluation of the potential central nervous system involvement in CD are lacking.

Objective

To assess the profile of cortical excitability to Transcranial Magnetic Stimulation (TMS) in a group of de novo CD patients.

Materials and methods

Twenty CD patients underwent a screening for cognitive and neuropsychiatric symptoms by means of the Mini Mental State Examination and the Structured Clinical Interview for DSM-IV Axis I Disorders, respectively. Instrumental exams, including electroencephalography and brain computed tomography, were also performed. Cortico-spinal excitability was assessed by means of single and paired-pulse TMS using the first dorsal interosseus muscle of the dominant hand. TMS measures consisted of resting motor threshold, motor evoked potentials, cortical silent period (CSP), intracortical inhibition (ICI) and facilitation (ICF). None of the CD was on gluten-free diet. A group of 20 age-matched healthy controls was used for comparisons.

Results

CD showed a significantly shorter CSP (78.0 vs 125.0 ms, p<0.025), a reduced ICI (0.3 vs 0.2, p<0.045) and an enhanced ICF (1.1 vs 0.7, p<0.042) compared to controls. A dysthymic disorder was identified in five patients. The effect size between dysthymic and non-dysthymic CD patients indicated a low probability of interference with the CSP (Cohen''s d -0.414), ICI (-0.278) and ICF (-0.292) measurements.

Conclusion

A pattern of cortical excitability characterized by “disinhibition” and “hyperfacilitation” was found in CD patients. Immune system dysregulation might play a central role in triggering changes of the motor cortex excitability.     

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

Background

Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results

We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion

We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.     

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X<Subscript>7</Subscript>-KO mice

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X₇-invalidated (KO) mice was tested. Low concentrations (1–100 μM) of ATP transiently increased the intracellular concentration of calcium ([Ca²⁺]_i) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X₇ receptors. One millimolar ATP provoked a sustained increase in the [Ca²⁺]_i only in WT mice. The response to 10 μM ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K⁺]_i) and stimulated the secretion of interleukin-1β (IL-1β) only in cells from WT mice. Ten micromolar ATP in combination with 3 μM ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1β was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150 μM BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10 μM ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3 μM ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X₄ receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K⁺]_i triggered the secretion of IL-1β. Pore formation was also triggered by activation of P2X₄ receptors. Higher concentrations of ATP elicited similar responses after binding to P2X₇ receptors. The expression of the P2X₇ receptors was also coupled to a better response to P2Y receptors.     

Aquaporin-6 is expressed along the rat gastrointestinal tract and upregulated by feeding in the small intestine

Background  

Several aquaporins (a family of integral membrane proteins) have been recently identified in the mammalian gastrointestinal tract, and their involvement in the movement of fluid and small solutes has been suggested. In this direction we investigated, in some regions of the rat gastrointestinal tract, the presence and localization of aquaporin-6, given its peculiar function as an ion selective channel.     

Cell Survival and Polarity of Drosophila Follicle Cells Require the Activity of Ecdysone Receptor B1 Isoform

Proper assembly and maintenance of epithelia are critical for normal development and homeostasis. Here, using the Drosophila ovary as a model, we identify a role for the B1 isoform of the ecdysone receptor (EcR-B1) in this process. We performed a reverse genetic analysis of EcR-B1 function during oogenesis and demonstrate that silencing of this receptor isoform causes loss of integrity and multilayering of the follicular epithelium. We show that multilayered follicle cells lack proper cell polarity with altered distribution of apical and basolateral cell polarity markers including atypical-protein kinase C (aPKC), Discs-large (Dlg), and Scribble (Scrib) and aberrant accumulation of adherens junctions and F-actin cytoskeleton. We find that the EcR-B1 isoform is required for proper follicle cell polarity both during early stages of oogenesis, when follicle cells undergo the mitotic cell cycle, and at midoogenesis when these cells stop dividing and undergo several endocycles. In addition, we show that the EcR-B1 isoform is required during early oogenesis for follicle cell survival and that disruption of its function causes apoptotic cell death induced by caspase.     

A proteomic study of Xanthomonas oryzae pv. oryzae in rice xylem sap

Xanthomonas oryzae pv. oryzae (Xoo) is the second most important rice pathogen, causing a disease called bacterial leaf blight. Xoo colonizes and infects the vascular tissue resulting in tissue necrosis and wilting causing significant yield losses worldwide. In this study Xoo infected vascular fluid (xylem sap) was recovered and analyzed for secreted Xoo proteins. Three independent experiments resulted in the identification of 324 different proteins, 64 proteins were found in all three samples which included many of the known virulence-associated factors. In addition, 10 genes encoding for the identified proteins were inactivated and one mutant displayed statistically a significant loss in virulence when compared to the wild type Xoo, suggesting that a new virulence-associated factor has been revealed. The usefulness of this approach in understanding the lifestyle and unraveling the virulence-associated factors of phytopathogenic vascular bacteria is discussed.     

Imprints of multiple glacial refugia in the Pyrenees revealed by phylogeography and palaeodistribution modelling of an endemic spider

Mediterranean mountain ranges harbour highly endemic biota in islandlike habitats. Their topographic diversity offered the opportunity for mountain species to persist in refugial areas during episodes of major climatic change. We investigate the role of Quaternary climatic oscillations in shaping the demographic history and distribution ranges in the spider Harpactocrates ravastellus, endemic to the Pyrenees. Gene trees and multispecies coalescent analyses on mitochondrial and nuclear DNA sequences unveiled two distinct lineages with a hybrid zone around the northwestern area of the Catalan Pyrenees. The lineages were further supported by morphological differences. Climatic niche‐based species distribution models (SDMs) identified two lowland refugia at the western and eastern extremes of the mountain range, which would suggest secondary contact following postglacial expansion of populations from both refugia. Neutrality test and approximate Bayesian computation (ABC) analyses indicated that several local populations underwent severe bottlenecks followed by population expansions, which in combination with the deep population differentiation provided evidence for population survival during glacial periods in microrefugia across the mountain range, in addition to the main Atlantic and Mediterranean (western and eastern) refugia. This study sheds light on the complexities of Quaternary climatic oscillations in building up genetic diversity and local endemicity in the southern Europe mountain ranges.