Increased spread and replication efficiency of Listeria monocytogenes in organotypic brain-slices is related to multilocus variable number of tandem repeat analysis (MLVA) complex.

BACKGROUND Listeria (L.) monocytogenes causes fatal infections in many species including ruminants and humans. In ruminants, rhombencephalitis is the most prevalent form of listeriosis. Using multilocus variable number tandem repeat analysis (MLVA) we recently showed that L. monocytogenes isolates from ruminant rhombencephalitis cases are distributed over three genetic complexes (designated A, B and C). However, the majority of rhombencephalitis strains and virtually all those isolated from cattle cluster in MLVA complex A, indicating that strains of this complex may have increased neurotropism and neurovirulence. The aim of this study was to investigate whether ruminant rhombencephalitis strains have an increased ability to propagate in the bovine hippocampal brain-slice model and can be discriminated from strains of other sources. For this study, forty-seven strains were selected and assayed on brain-slice cultures, a bovine macrophage cell line (BoMac) and a human colorectal adenocarcinoma cell line (Caco-2). They were isolated from ruminant rhombencephalitis cases (n = 21) and other sources including the environment, food, human neurolisteriosis cases and ruminant/human non-encephalitic infection cases (n = 26). RESULTS All but one L. monocytogenes strain replicated in brain slices, irrespectively of the source of the isolate or MLVA complex. The replication of strains from MLVA complex A was increased in hippocampal brain-slice cultures compared to complex C. Immunofluorescence revealed that microglia are the main target cells for L. monocytogenes and that strains from MLVA complex A caused larger infection foci than strains from MLVA complex C. Additionally, they caused larger plaques in BoMac cells, but not CaCo-2 cells. CONCLUSIONS Our brain slice model data shows that all L. monocytogenes strains should be considered potentially neurovirulent. Secondly, encephalitis strains cannot be conclusively discriminated from non-encephalitis strains with the bovine organotypic brain slice model. The data indicates that MLVA complex A strains are particularly adept at establishing encephalitis possibly by virtue of their higher resistance to antibacterial defense mechanisms in microglia cells, the main target of L. monocytogenes.

Interaction between serotonin 5-HT2A receptor gene and dopamine transporter (DAT1) gene polymorphisms influences personality trait of persistence in Austrian Caucasians

We examined 89 normal volunteers using Cloninger's Temperament and Character Inventory (TCI). Genotyping the 102T/C polymorphism of the serotonin 5HT2A receptor gene and the ser9gly polymorphism in exon 1 of the dopamine D3 receptor (DRD3) gene was performed using PCR-RFLP, whereas the dopamine transporter (DAT1) gene variable number of tandem repeats (VNTR) polymorphism was investigated using PCR amplification followed by electrophoresis in an 8% acrylamide gel with a set of size markers. We found a nominally significant association between gender and harm avoidance (P=0.017; women showing higher scores). There was no association of either DAT1, DRD3 or 5HT2A alleles or genotypes with any dimension of the TCI applying Kruskal-Wallis rank-sum tests. Comparing homozygote and heterozygote DAT1 genotypes, we found higher novelty seeking scores in homozygotes (P=0.054). We further found a nominally significant interaction between DAT1 and 5HT2A homo-/heterozygous gene variants (P=0.0071; DAT1 and 5HT2A genotypes P value of 0.05), performing multivariate analysis of variance (MANOVA). Examining the temperamental TCI subscales, this interaction was associated with persistence (genotypes: P=0.004; homo-/heterozygous gene variants: P=0.0004). We conclude that an interaction between DAT1 and 5HT2A genes might influence the temperamental personality trait persistence.

A novel isolation method of Brucella species and molecular tracking of Brucella suis biovar 2 in domestic and wild animals

Brucella suis biovar 2 is the most common aetiological agent of porcine brucellosis in Europe. B. suis biovar 2 is considered to have low zoonotic potential, but is a causative agent of reproductive losses in pigs, and it is thus economically important. The multilocus variable-number of tandem repeats genotyping analysis of 16 loci (MLVA-16) has proven to be highly discriminatory and is the most suitable assay for simultaneously identifying B. suis and tracking infections. The aim of this study was to investigate the relatedness between isolates of B. suis biovar 2 obtained during a brucellosis outbreak in domestic pigs and isolates from wild boars and hares collected from proximal or remote geographical areas by MLVA-16. A cluster analysis of the MLVA-16 data revealed that most of the isolates obtained from Switzerland clustered together, with the exception of one isolate. The outbreak isolates constituted a unique subcluster (with a genetic similarity >93.8%) distinct from that of the isolates obtained from wild animals, suggesting that direct transmission of the bacterium from wild boars to domestic pigs did not occur in this outbreak. To obtain a representative number of isolates for MLVA-16, alternative methods of Brucella spp. isolation from tissue samples were compared with conventional direct cultivation on a Brucella-selective agar. We observed an enhanced sensitivity when mechanical homogenisation was followed by host cell lysis prior to cultivation on the Brucella-selective agar. This work demonstrates that MLVA-16 is an excellent tool for both monitoring brucellosis and investigating outbreaks. Additionally, we present efficient alternatives for the isolation of Brucella spp.

Molecular epidemiology of the nasal colonization by methicillin-susceptible Staphylococcus aureus in Swiss children

Nasal carriage of Staphylococcus aureus contributes to an increased risk of developing an infection with the same bacterial strain. Genetic regulatory elements and toxin-expressing genes are virulence factors associated with the pathogenic potential of S. aureus. We undertook an extensive molecular characterization of methicillin-susceptible S. aureus (MSSA) carried by children. MSSA were recovered from the nostrils of children. The presence of Panton-Valentine leukocidin (PVL), exfoliatins A and B (exfoA and exfoB), and the toxic-shock staphylococcal toxin (TSST-1) and agr group typing were determined by quantitative PCR. A multiple-locus variable-number of tandem repeat analysis (MLVA) assay was also performed for genotyping. Five hundred and seventy-two strains of MSSA were analysed. Overall, 30% were positive for toxin-expressing genes: 29% contained one toxin and 1.6% two toxins. The most commonly detected toxin gene was tst, which was present in 145 (25%) strains. The TSST-1 gene was significantly associated with the agr group 3 (OR 56.8, 95% CI 32.0-100.8). MLVA analysis revealed a large diversity of genetic content and no clonal relationship was demonstrated among the analysed MSSA strains. Multilocus sequence typing confirmed this observation of diversity and identified ST45 as a frequent colonizer. This broad diversity in MSSA carriage strains suggests a limited selection pressure in our geographical area.

Splitting of a prevalent Mycobacterium bovis spoligotype by variable-number tandem-repeat typing reveals high heterogeneity in an evolving clonal group

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

Ruminant rhombencephalitis-associated Listeria monocytogenes alleles linked to a multilocus variable-number tandem-repeat analysis complex

Listeria monocytogenes is among the most important food-borne pathogens and is well adapted to persist in the environment. To gain insight into the genetic relatedness and potential virulence of L. monocytogenes strains causing central nervous system (CNS) infections, we used multilocus variable-number tandem-repeat analysis (MLVA) to subtype 183 L. monocytogenes isolates, most from ruminant rhombencephalitis and some from human patients, food, and the environment. Allelic-profile-based comparisons grouped L. monocytogenes strains mainly into three clonal complexes and linked single-locus variants (SLVs). Clonal complex A essentially consisted of isolates from human and ruminant brain samples. All but one rhombencephalitis isolate from cattle were located in clonal complex A. In contrast, food and environmental isolates mainly clustered into clonal complex C, and none was classified as clonal complex A. Isolates of the two main clonal complexes (A and C) obtained by MLVA were analyzed by PCR for the presence of 11 virulence-associated genes (prfA, actA, inlA, inlB, inlC, inlD, inlE, inlF, inlG, inlJ, and inlC2H). Virulence gene analysis revealed significant differences in the actA, inlF, inlG, and inlJ allelic profiles between clinical isolates (complex A) and nonclinical isolates (complex C). The association of particular alleles of actA, inlF, and newly described alleles of inlJ with isolates from CNS infections (particularly rhombencephalitis) suggests that these virulence genes participate in neurovirulence of L. monocytogenes. The overall absence of inlG in clinical complex A and its presence in complex C isolates suggests that the InlG protein is more relevant for the survival of L. monocytogenes in the environment.

Testing an alternative search algorithm for compound identification with the 'Wiley Registry of Tandem Mass Spectral Data. MSforID'

A tandem mass spectral database system consists of a library of reference spectra and a search program. State-of-the-art search programs show a high tolerance for variability in compound-specific fragmentation patterns produced by collision-induced decomposition and enable sensitive and specific 'identity search'. In this communication, performance characteristics of two search algorithms combined with the 'Wiley Registry of Tandem Mass Spectral Data, MSforID' (Wiley Registry MSMS, John Wiley and Sons, Hoboken, NJ, USA) were evaluated. The search algorithms tested were the MSMS search algorithm implemented in the NIST MS Search program 2.0g (NIST, Gaithersburg, MD, USA) and the MSforID algorithm (John Wiley and Sons, Hoboken, NJ, USA). Sample spectra were acquired on different instruments and, thus, covered a broad range of possible experimental conditions or were generated in silico. For each algorithm, more than 30,000 matches were performed. Statistical evaluation of the library search results revealed that principally both search algorithms can be combined with the Wiley Registry MSMS to create a reliable identification tool. It appears, however, that a higher degree of spectral similarity is necessary to obtain a correct match with the NIST MS Search program. This characteristic of the NIST MS Search program has a positive effect on specificity as it helps to avoid false positive matches (type I errors), but reduces sensitivity. Thus, particularly with sample spectra acquired on instruments differing in their Setup from tandem-in-space type fragmentation, a comparably higher number of false negative matches (type II errors) were observed by searching the Wiley Registry MSMS.

Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci

We undertook a meta-analysis of six Crohn's disease genome-wide association studies (GWAS) comprising 6,333 affected individuals (cases) and 15,056 controls and followed up the top association signals in 15,694 cases, 14,026 controls and 414 parent-offspring trios. We identified 30 new susceptibility loci meeting genome-wide significance (P < 5 × 10 ? ? ). A series of in silico analyses highlighted particular genes within these loci and, together with manual curation, implicated functionally interesting candidate genes including SMAD3, ERAP2, IL10, IL2RA, TYK2, FUT2, DNMT3A, DENND1B, BACH2 and TAGAP. Combined with previously confirmed loci, these results identify 71 distinct loci with genome-wide significant evidence for association with Crohn's disease.

Quality evaluation of tandem mass spectral libraries

Tandem mass spectral libraries are gaining more and more importance for the identification of unknowns in different fields of research, including metabolomics, forensics, toxicology, and environmental analysis. Particularly, the recent invention of reliable, robust, and transferable libraries has increased the general acceptance of these tools. Herein, we report on results obtained from thorough evaluation of the match reliabilities of two tandem mass spectral libraries: the MSforID library established by the Oberacher group in Innsbruck and the Weinmann library established by the Weinmann group in Freiburg. Three different experiments were performed: (1) Spectra of the libraries were searched against their corresponding library after excluding either this single compound-specific spectrum or all compound-specific spectra prior to searching; (2) the libraries were searched against each other using either library as reference set or sample set; (3) spectra acquired on different mass spectrometric instruments were matched to both libraries. Almost 13,000 tandem mass spectra were included in this study. The MSforID search algorithm was used for spectral matching. Statistical evaluation of the library search results revealed that principally both libraries enable the sensitive and specific identification of compounds. Due to higher mass accuracy of the QqTOF compared with the QTrap instrument, matches to the MSforID library were more reliable when comparing spectra with both libraries. Furthermore, only the MSforID library was shown to be efficiently transferable to different kinds of tandem mass spectrometers, including "tandem-in-time" instruments; this is due to the coverage of a large range of different collision energy settings-including the very low range-which is an outstanding characteristics of the MSforID library.

Meta-analysis identifies 29 additional ulcerative colitis risk loci, increasing the number of confirmed associations to 47

Genome-wide association studies and candidate gene studies in ulcerative colitis have identified 18 susceptibility loci. We conducted a meta-analysis of six ulcerative colitis genome-wide association study datasets, comprising 6,687 cases and 19,718 controls, and followed up the top association signals in 9,628 cases and 12,917 controls. We identified 29 additional risk loci (P < 5 × 10(-8)), increasing the number of ulcerative colitis-associated loci to 47. After annotating associated regions using GRAIL, expression quantitative trait loci data and correlations with non-synonymous SNPs, we identified many candidate genes that provide potentially important insights into disease pathogenesis, including IL1R2, IL8RA-IL8RB, IL7R, IL12B, DAP, PRDM1, JAK2, IRF5, GNA12 and LSP1. The total number of confirmed inflammatory bowel disease risk loci is now 99, including a minimum of 28 shared association signals between Crohn's disease and ulcerative colitis.

Evaluating the Impact of the Number of Access Points in Mobile Robots Localization Using Artificial Neural Networks

Localization is information of fundamental importance to carry out various tasks in the mobile robotic area. The exact degree of precision required in the localization depends on the nature of the task. The GPS provides global position estimation but is restricted to outdoor environments and has an inherent imprecision of a few meters. In indoor spaces, other sensors like lasers and cameras are commonly used for position estimation, but these require landmarks (or maps) in the environment and a fair amount of computation to process complex algorithms. These sensors also have a limited field of vision. Currently, Wireless Networks (WN) are widely available in indoor environments and can allow efficient global localization that requires relatively low computing resources. However, the inherent instability in the wireless signal prevents it from being used for very accurate position estimation. The growth in the number of Access Points (AP) increases the overlap signals areas and this could be a useful means of improving the precision of the localization. In this paper we evaluate the impact of the number of Access Points in mobile nodes localization using Artificial Neural Networks (ANN). We use three to eight APs as a source signal and show how the ANNs learn and generalize the data. Added to this, we evaluate the robustness of the ANNs and evaluate a heuristic to try to decrease the error in the localization. In order to validate our approach several ANNs topologies have been evaluated in experimental tests that were conducted with a mobile node in an indoor space.

What is the optimal number of implants for fixed reconstructions: a systematic review

To assess the 5-year and 10-year survival and complication rates of implant-supported fixed reconstructions in partially and totally edentulous patients with regard to the optimal number and distribution of dental implants.

What is the optimal number of implants for removable reconstructions? A systematic review on implant-supported overdentures

The aim of this systematic review was to assess the optimal number of implants for removable reconstructions.