Matrix-assisted laser desorption ionization-time of flight mass spectrometry as an alternative to 16S rRNA gene sequencing for identification of difficult-to-identify bacterial strains.

Conventional methods are sometimes insufficient to identify human bacterial pathogens, and alternative techniques, often molecular, are required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified with a valid score 45.9% of 410 clinical isolates from 207 different difficult-to-identify species having required 16S rRNA gene sequencing. MALDI-TOF MS might represent an alternative to 16S rRNA gene sequencing.

Intergroup distinctiveness and discriminatory immigration attitudes: The role of national identification

We examined the moderating role of national identification in understanding when a focus on intergroup similarity versus difference on ingroup stereotypical traits-manipulated with scale anchors-leads to support for discriminatory immigration policies. In line with intergroup distinctiveness research, national identification moderated the similarity-difference manipulation effect. Low national identifiers supported discriminatory immigration policies more when intergroup difference rather than similarity was made salient, whereas the opposite pattern was found for high national identifiers: They trended toward being more discriminatory when similarity was made salient. The impact of assimilation expectations and national identity content on the findings is discussed.

Identification of HMX1 target genes: a predictive promoter model approach.

PURPOSE: A homozygous mutation in the H6 family homeobox 1 (HMX1) gene is responsible for a new oculoauricular defect leading to eye and auricular developmental abnormalities as well as early retinal degeneration (MIM 612109). However, the HMX1 pathway remains poorly understood, and in the first approach to better understand the pathway's function, we sought to identify the target genes. METHODS: We developed a predictive promoter model (PPM) approach using a comparative transcriptomic analysis in the retina at P15 of a mouse model lacking functional Hmx1 (dmbo mouse) and its respective wild-type. This PPM was based on the hypothesis that HMX1 binding site (HMX1-BS) clusters should be more represented in promoters of HMX1 target genes. The most differentially expressed genes in the microarray experiment that contained HMX1-BS clusters were used to generate the PPM, which was then statistically validated. Finally, we developed two genome-wide target prediction methods: one that focused on conserving PPM features in human and mouse and one that was based on the co-occurrence of HMX1-BS pairs fitting the PPM, in human or in mouse, independently. RESULTS: The PPM construction revealed that sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) (Sgcg), teashirt zinc finger homeobox 2 (Tshz2), and solute carrier family 6 (neurotransmitter transporter, glycine) (Slc6a9) genes represented Hmx1 targets in the mouse retina at P15. Moreover, the genome-wide target prediction revealed that mouse genes belonging to the retinal axon guidance pathway were targeted by Hmx1. Expression of these three genes was experimentally validated using a quantitative reverse transcription PCR approach. The inhibitory activity of Hmx1 on Sgcg, as well as protein tyrosine phosphatase, receptor type, O (Ptpro) and Sema3f, two targets identified by the PPM, were validated with luciferase assay. CONCLUSIONS: Gene expression analysis between wild-type and dmbo mice allowed us to develop a PPM that identified the first target genes of Hmx1.

Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers.

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.

Biochemical identification of three sympatric Apodemus species by protein electrophoresis of blood samples

The allelic pattern of seralbumine and general protein 1 of the three sympatric Apodemus species Apodemus sylvaticus, A. flavicollis, and A. alpicola, were studied using electrophoretic analysis of blood samples, This method appears to be a sensitive tool for distinguishing the three Apodemus species in the Alps, Their identification on the basis of external characteristics in the field is sometimes extremely difficult, even more so for juvenile specimens. Compared to previously described methods the electrophoretic analysis does not require killing animals and can be used on juveniles.

A database to aid the identification of chemicals potentially posing a health risk through percutaneous exposure

In the context of recent attempts to redefine the 'skin notation' concept, a position paper summarizing an international workshop on the topic stated that the skin notation should be a hazard indicator related to the degree of toxicity and the potential for transdermal exposure of a chemical. Within the framework of developing a web-based tool integrating this concept, we constructed a database of 7101 agents for which a percutaneous permeation constant can be estimated (using molecular weight and octanol-water partition constant), and for which at least one of the following toxicity indices could be retrieved: Inhalation occupational exposure limit (n=644), Oral lethal dose 50 (LD50, n=6708), cutaneous LD50 (n=1801), Oral no observed adverse effect level (NOAEL, n=1600), and cutaneous NOAEL (n=187). Data sources included the Registry of toxic effects of chemical substances (RTECS, MDL information systems, Inc.), PHYSPROP (Syracuse Research Corp.) and safety cards from the International Programme on Chemical Safety (IPCS). A hazard index, which corresponds to the product of exposure duration and skin surface exposed that would yield an internal dose equal to a toxic reference dose was calculated. This presentation provides a descriptive summary of the database, correlations between toxicity indices, and an example of how the web tool will help industrial hygienist decide on the possibility of a dermal risk using the hazard index.

Clinical follow-up of women infected with human papillomavirus-16, either alone or with other human papillomavirus types : identification of different risk groups

Rapport de synthèse Objectifs : Évaluer l'impact clinique de femmes infectées par de multiples papillomavirus human (HPV) à haut risque dont le HPV 16 en comparaison de l'évolution de femmes infectées par du HPV 16 seul. Méthode : 169 femmes ont été classifiées en trois groupes, dépendant de leur profile HPV: HPV-16 seul, HPV-16 et un HPV de type bas risque, HPV-16 et un autre HPV à haut risque. Le HPV-DNA des frottis cervicaux a été analysé par polymerase chain reaction (PCR) et reverse line blot hybridization (RLBH). Toutes les femmes ont été suivies à la consultation de colposcopie pour une durée de 24 mois ou plus. La prise en charge s'est faite selon les recommandations de Bethesda. Résultats : Les femmes infectées par du HPV 16 et un autre HPV à haut risque n'ont présenté aucun changement voire une progression de leur dysplasie en comparaison des femmes des autres groupes (RR: 1.39; 95%CI: 1.07 à 1.82; p value: 0.02 à 6 mois; RR: 2.10; 95%CI: 1.46 à 3.02; p value: <0.001 à 12 mois; RR: 1.82; 95%CI: 1.21 à 2.72; p value: 0.004 à 24 mois). Conclusions : Les femmes présentant une co-infection par du HPV 16 ainsi qu'un autre HPV de haut risque voient leur risque d'évolution défavorable augmenter.

Identification of the minimal promoter region of the mouse NKX5-3, a transcription factor implicated in eye development.

Early ocular development is controlled by a complex network of transcription factors, cell cycle regulators, and diffusible signalling molecules. Together, these molecules regulate cell proliferation and apoptosis, and specify retinal fate. NKX5-3 is a homeobox transcription factor implicated in eye development. The analysis of the 5'-flanking region of the mouse Nkx5-3 gene revealed a predicted TATA-less promoter sequence between -416 and -166 of the translation start site. To functionally characterise Nkx5-3 promoter activity, serial deletions of the promoter sequence were introduced in pGL-3 basic vector and promoter activity of these 5'- and 3'-deleted constructions was tested in HeLa and CHO cells. Transactivation assays identified a region between -350 and -296 exhibiting promoter-like activity. Combined analysis by deletions and point mutations showed that this sequence, containing multiple Sp1 binding sites was necessary to promote transcriptional activity. Binding of Sp1 to this region was confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation, using an antibody specific for Sp1. Altogether, these results demonstrated that the immediate upstream region of Nkx5-3 gene possessed a strong intrinsic promoter activity in vitro, suggesting a potential role in Nkx5-3 transcription in vivo.

Identification des dermatophytes causant des teignes de la peau glabre (Tinea glabra) et du cuir chevelu (Tinea capitis) par PCR

Contexte: Le nombre de teignes du cuir chevelu et de la peau glabre étant en nette augmentation, l'identification du pathogène qui est indispensable pour un traitement ciblé, a, par conséquence, un grand intérêt pour la santé publique. Dans divers cas, un animal de compagnie peut être identifié en tant que source du pathogène. La fréquence de cultures restant stériles est particulièrement élevée en cas de prétraitement antifongique. Objectif: Le but de ce travail est de mettre au point une méthode rapide d'identification du dermatophyte pathogène in situ par PCR/séquençage dans les cas de teignes du cuir chevelu et/ou de la peau glabre. Matériel et méthodes : De l'ADN a été extrait de squames (N=5) et cheveux (N=21) dont l'examen direct démontrait une infection fongique (N=26) ou se révèlait négatif (N=1). Ensuite, une première PCR du segment 28s de l'ADN ribosomale fongique a été effectuée, suivie par une PCR nichée intérieure à ce segment. L'amplicon a été séquencé et le champignon est identifié par alignement. Résultats : Seule la PCR enchainée a permis d'obtenir une quantité suffisante d'amplicon pour permettre le séquençage. Dans 4 cas sur 5 de tinea pedis, 10 sur 12 de tinea glabra, respectivement 4 sur 4 de tinea capitis, dans lesquels un dermato- phyte a été identifié en culture, le même dermatophyte a été identifié par PCR/séquençage. Une fois sur 27 prélèvements, un autre dermatophyte a été identifié par PCR/séquençage. Ce résultat pourrait être dû à une fausse identification du champignon en culture. Dans un cas de tinea pedis et un cas de tinea corporis, la culture est restée stérile, mais un dermatophyte a pu être identifié par PCR et séquençage. Conclusions : La méthode décrite est à la fois rapide (24 h au lieu de deux semaines pour la culture), sensible et très spécifique. Elle est particulièrement utile dans les cas de teigne du cuir chevelu, dans lesquels le traitement est différent selon l'espèce de dermatophyte et où il s'agit d'un traitement systémique lourd, souvent chez l'enfant.

Mutation screening of the urocortin gene: identification of new single nucleotide polymorphisms and association studies with obesity in French Caucasians.

A linkage between obesity-related phenotypes and the 2p21-23 locus has been reported previously. The urocortin (UCN) gene resides at this interval, and its protein decreases appetite behavior, suggesting that UCN may be a candidate gene for susceptibility to obesity. We localized the UCN gene by radiation hybrid mapping, and the surrounding markers were genotyped in a collection of French families. Evidence for linkage was shown between the marker D2S165 and leptin levels (LOD score, 1.34; P = 0.006) and between D2S2247 and the z-score of body mass index (LOD score, 1.829; P = 0.0019). The gene was screened for SNPs in 96 obese patients. Four new variants were established. Two single nucleotide polymorphisms were located in the promoter (-535 A-->G, -286 G-->A), one in intron 1 (+31 C-->G), and one in the 3'-untranslated region (+34 C-->T). Association studies in cohorts of 722 unrelated obese and 381 control subjects and transmission disequilibrium tests, performed for the two frequent promoter polymorphisms, in 120 families (894 individuals) showed that no association was present between these variants and obesity, obesity-related phenotypes, and diabetes. Thus, our analyses of the genetic variations of the UCN gene suggest that, at least in French Caucasians, they do not represent a major cause of obesity.

Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL infiltrating metastatic melanoma.

The tumor Ag SSX-2 (HOM-MEL-40) was found by serological identification of Ags by recombinant expression cloning and was shown to be a cancer/testis Ag expressed in a wide variety of tumors. It may therefore represent a source of CD8(+) T cell epitopes useful for specific immunotherapy of cancer. To identify potential SSX-2-derived epitopes that can be recognized by CD8(+) T cells, we used an approach that combined: 1) the in vitro proteasomal digestion of precursor peptides overlapping the complete SSX-2 sequence; 2) the prediction of SSX-2-derived peptides with an appropriate HLA-A2 binding score; and 3) the analysis of a tumor-infiltrated lymph node cell population from an HLA-A2(+) melanoma patient with detectable anti-SSX-2 serum Abs. This strategy allowed us to identify peptide SSX-2(41-49) as an HLA-A2-restricted epitope. SSX2(41-49)-specific CD8(+) T cells were readily detectable in the tumor-infiltrated lymph node population by multimer staining, and CTL clones isolated by multimer-guided cell sorting were able to lyse HLA-A2(+) tumor cells expressing SSX-2.