Identification of dermatophyte species by 28S ribosomal DNA sequencing with a commercial kit

We have shown that dermatophyte species can be easily identified on the basis of a DNA sequence encoding a part of the large-subunit (LSU) rRNA (28S rRNA) by using the MicroSeq D2 LSU rRNA Fungal Sequencing Kit. Two taxa causing distinct dermatophytoses were clearly distinguished among isolates of the Trichophyton mentagrophytes species complex.     

A longitudinal and prospective study of Epstein-Barr virus load in AIDS-related non-Hodgkin lymphoma.

BACKGROUND: Epstein-Barr virus (EBV) may be causally associated with non-Hodgkin Lymphoma (NHL) in HIV-infected patients. OBJECTIVES: To compare EBV load in whole blood in AIDS-NHL patients, HIV non-AIDS patients and non-HIV-infected persons, and to prospectively measure EBV load in whole blood in AIDS-NHL patients. STUDY DESIGN: Longitudinal and prospective study. RESULTS: We observed no statistical difference in EBV load between AIDS-NHL (3.69log(10) copies/mL [interquartile range (IQR): 2.89-4.27]) and HIV non-AIDS patients (3.08log(10) copies/mL [IQR: 1.29-3.57]) but AIDS-NHL patients had significantly higher EBV loads than HIV-negative controls (1.19log(10) copies/mL [IQR: 0.00-3.29]). We noticed an inverse correlation between CD4+ lymphocytes count and EBV load in patients with AIDS-NHL (r(2)=0.41, P=0.01). In the longitudinal study, the mean EBV load three months after NHL diagnosis decreased significantly (mean difference=-1.69log(10) copies/mL [95% confidence interval: -0.32; -3.04]; P=0.03) under chemotherapy but was still elevated in patients with relapses or no response to chemotherapy. CONCLUSION: Although EBV load seems a suboptimal marker for the diagnosis of AIDS-NHL, we observed a significant decrease of EBV load in patients treated with chemotherapy and a strong association between NHL outcome and EBV load in whole blood.     

Interaction of enteropathogenic and Shiga toxin-producing Escherichia coli and porcine intestinal mucosa: role of intimin and Tir in adherence

The ileal in vitro organ culture (IVOC) model using tissues originating from colostrum-deprived newborn piglets has proven to be an effective way to study the attaching and effacing (A/E) phenotype of porcine enteropathogenic Escherichia coli (EPEC) ex vivo. The aim of this study was to investigate the role of intimin subtype and Tir in the adherence of EPEC and Shiga-toxin-producing E. coli (STEC), isolated from different animal species, to porcine intestinal IVOC. Moreover, the role of intimin in Tir-independent adherence of the human EPEC strain E2348/69 was investigated using intimin and Tir-deficient derivatives. Our results demonstrated that A/E E. coli strains (AEEC) from various animal species and humans induce the A/E phenotype in porcine ileal IVOC and that intimin subtype influences intestinal adherence and tropism of AEEC strains. We also showed that a tir mutant of EPEC strain E2348/69 demonstrates close adherence to the epithelial cells of porcine ileal IVOC segments, with microvillous effacement but with no evidence of actin polymerization or pedestal formation, and that intimin seems to be involved in this phenotype. Overall, this study provides further evidence for the existence of one or more host-cell-encoded intimin receptor(s) in the pig gut.     

Up-regulation of tRNA biosynthesis affects translational readthrough in maf1-delta mutant of Saccharomyces cerevisiae

Maf1p is a negative effector of RNA polymerase III in yeast. The maf1-delta mutation caused an increase in the level of cellular tRNAs, but a decrease of translational readthrough at nonsense codons. Using the lacZ- luc dual gene reporter system, we detected an almost twofold diminution of UAA and UAG readthrough in maf1-delta compared with the parental strain. The maf1-delta mutation did not affect the rate of protein biosynthesis and growth at standard conditions, but resulted in temperature-sensitive growth on non-fermentable carbon sources. We examined the correlation of the temperature sensitive and antisuppression phenotypes of maf1- Delta using a colour phenotype assay in the ade2-1 SUP11 strain. Antisuppression, but not the temperature-sensitive growth defect, was compensated either by increased dosage of SUP11or by [PSI(+)], the prion form of the translation termination factor Sup35p. Summarizing, the elevated tRNA levels in maf1- Delta increase translational fidelity and, independently, affect growth under special conditions.     

Levels of analysis in etiological research on schizophrenia

Etiological research spanning domains of inquiry as diverse as social psychology and molecular genetics has identified a number of potential factors that likely contribute to the development and clinical manifestation of schizophrenia. In this article, we first highlight the challenges inherent in developing cogent etiological models that represent both the diversity of suspected causal influences and their mechanisms of action. Then, using our own research program as a heuristic context, we present a general analytical framework for identifying and integrating multiple types of etiologic factors across different levels of analysis in the prediction of schizophrenia. In recognition of the myriad complexities of multifactorial causation, we argue that a multilevel causal perspective is required for the development and advancement of a fully nuanced theory of schizophrenia etiology and pathophysiology.     

Plasma RNA viral load in human immunodeficiency virus type 2 subtype A and subtype B infections

Human immunodeficiency virus type 2 (HIV-2) is much less pathogenic than HIV-1, and HIV-2 infection is associated with plasma viral loads significantly lower than those found in HIV-1 infection. We have developed a real-time quantitative PCR method for measuring the HIV-2 RNA load that covers the range of genetic diversity of HIV-2 isolates and that detects extremely low viral loads. Samples from 49 patients were studied. Proviral DNA was first detected and quantified. The strains that were detected were then genotyped: 21 patients were infected with HIV-2 subtype A and 15 patients were infected with HIV-2 subtype B; 1 patient was infected with a highly divergent strain. Env PCR failed for the remaining 12 patients, so subtypes could not be determined. For viral RNA quantification, a stock of HIV-2 strain NIHZ, which was counted by electron microscopy, was used as the standard. Several primer sets targeting the highly conserved gag region were evaluated. Various primer combinations failed to amplify subtype B strains. With the final primer pair selected, which detected both subtype A and subtype B strains, the sensitivity of the assay was 100% at a viral load of 250 copies/ml and 66% at a viral load of 125 copies/ml. We found a correlation between the CD4(+)-cell count, the clinical stage, and the plasma HIV-2 RNA level. The median plasma HIV-2 RNA value for the 33 asymptomatic patients was 2.14 log(10), whereas it was 3.1 log(10) for the 16 patients with AIDS (P < 0.01). Proviral DNA was detectable in 18 symptom-free patients with high CD4(+)-cell counts, in whom viral RNA was undetectable.     

Formalin-fixed and paraffin-embedded nodal non-Hodgkin's lymphomas demonstrate the same chromosome changes as those found in frozen samples: a comparative study using interphase fluorescence in situ hybridization.

Cytogenetic studies in lymphomas classically require fresh or frozen tissue, whereas in many instances only paraffin-embedded biopsies are available. We applied an interphase FISH assay on nuclei extracted from thick paraffin sections to determine accuracy of molecular cytogenetics in such samples. Twenty-three lymphoma samples and 4 reactive lymph nodes were tested with various commercially available DNA probes, and hybridization patterns were compared with those obtained on frozen nuclei counterparts. Successful hybridization with all probes tested was observed for 23/27 (85%) paraffin-embedded tissues and for all (100%) frozen samples, and cut-off levels defining positivity were superimposable for both situations. Chromosome changes were detected in the same way, without any false-positive or false-negative cases. Hybridization signals observed on dewaxed samples were either those classically expected to define the relevant chromosome change or were atypical: all atypical changes could be demonstrated also into nuclei from the frozen counterpart. Moreover, all typical and atypical chromosome changes observed on frozen nuclei were also detected in paraffin-embedded tissues. Our study shows that our interphase FISH assay performed on paraffin-embedded samples is a valuable alternate to conventional methods to ascertain diagnosis of lymphomas as to include patients into therapeutic trials.     

Refined genetic mapping of autosomal recessive chronic distal spinal muscular atrophy to chromosome 11q13.3 and evidence of linkage disequilibrium in European families

Chronic distal spinal muscular atrophy (Chronic DSMA, MIM (*)607088) is a rare autosomal recessive disorder characterized by a progressive motor weakness and muscular atrophy, predominating in the distal parts of the limbs. A form of Chronic DSMA gene has been previously mapped to chromosome 11q13 in the 10.3 cM interval defined by loci D11S1889 and D11S1321. By linkage analysis in 12 European Chronic DSMA families, we showed that a disease gene maps to chromosome 11q13.3 (Z(max)=6.66 at theta=0.00 at the DSM4 locus) and suggested that this condition is genetically homogeneous. Recombination events allowed us to reduce the genetic interval to a 2.6 cM region, telomeric to the IGHMBP2 gene, excluding this gene as the disease causing gene in Chronic DSMA. Moreover, partial linkage disequilibrium was found between three rare alleles at loci D11S1369, DSM4 and D11S4184 and the mutant chromosome in European patients. Analysis of the markers at these loci strongly suggests that most Chronic DSMA chromosomes are derived from a single ancestor. Refinement of the Chronic DSMA locus will hopefully allow to test candidate genes and lead to identification of the disease-causing mutations.     

Chronic pneumonia despite adaptive immune response to Mycobacterium bovis BCG in MyD88-deficient mice

To assess the role of Toll-like receptor (TLR) signalling in host response to mycobacterial infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) were infected with the vaccine strain Mycobacterium bovis (BCG), and the immune response and bacterial burden were investigated. Macrophages and dendritic cells from MyD88-deficient mice stimulated in vitro with BCG mycobacterial antigens produced very low levels of proinflammatory cytokines, while the expression of costimulatory molecules such as CD40 and CD86 was preserved. Upon systemic infection with BCG (2 x 10(6) CFU i.v.) MyD88-deficient mice developed confluent chronic pneumonia with two log higher CFU than wild-type mice. Interestingly, the infection was controlled in liver and spleen and there was efficient systemic T-cell priming with high IFNgamma production by CD4+ splenic T cells in MyD88-deficient mice. Lung infiltrating cells showed IFNgamma production by pulmonary CD4+ T cells upon specific restimulation, and a reduced capacity to produce nitric oxide and IL-10. In summary, despite the dramatic reduction of the innate immune response, MyD88-deficient mice were able to mount an efficient T-cell response to mycobacterial antigens, which was however insufficient to control infection in the lung, resulting in chronic pneumonia in MyD88-deficient mice.     

Estimating the relative frequency of leukodystrophies and recommendations for carrier screening in the era of next‐generation sequencing

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A‐related leukodystrophy, Peroxisomal biogenesis disorders, POLR3‐related Leukodystrophy, Vanishing White Matter, and Pelizaeus‐Merzbacher Disease. Despite the relative frequency of these conditions, carrier‐screening laboratories regularly test only 20 of the 55 leukodystrophy‐related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening.