Probing the different chaperone activities of the bacterial HSP70-HSP40 system using a thermolabile luciferase substrate.

During mild heat-stress, a native thermolabile polypeptide may partially unfold and transiently expose water-avoiding hydrophobic segments that readily tend to associate into a stable misfolded species, rich in intra-molecular non-native beta-sheet structures. When the concentration of the heat-unfolded intermediates is elevated, the exposed hydrophobic segments tend to associate with other molecules into large stable insoluble complexes, also called "aggregates." In mammalian cells, stress- and mutation-induced protein misfolding and aggregation may cause degenerative diseases and aging. Young cells, however, effectively counteract toxic protein misfolding with a potent network of molecular chaperones that bind hydrophobic surfaces and actively unfold otherwise stable misfolded and aggregated polypeptides. Here, we followed the behavior of a purified, initially mostly native thermolabile luciferase mutant, in the presence or absence of the Escherichia coli DnaK-DnaJ-GrpE chaperones and/or of ATP, at 22 °C or under mild heat-stress. We concomitantly measured luciferase enzymatic activity, Thioflavin-T fluorescence, and light-scattering to assess the effects of temperature and chaperones on the formation, respectively, of native, unfolded, misfolded, and/or of aggregated species. During mild heat-denaturation, DnaK-DnaJ-GrpE+ATP best maintained, although transiently, high luciferase activity and best prevented heat-induced misfolding and aggregation. In contrast, the ATP-less DnaK and DnaJ did not maintain optimal luciferase activity and were less effective at preventing luciferase misfolding and aggregation. We present a model accounting for the experimental data, where native, unfolded, misfolded, and aggregated species spontaneously inter-convert, and in which DnaK-DnaJ-GrpE+ATP specifically convert stable misfolded species into unstable unfolded intermediates.

Stimulation of hematopoiesis in vivo by recombinant bacterial murine interleukin 3.

Mouse interleukin 3 (IL-3) cDNA was cloned into a plasmid construction, allowing the synthesis of very high quantities of IL-3 in Escherichia coli. The recombinant (r) IL-3, purified to homogeneity, was active in vitro on the proliferation and differentiation of various hematopoietic progenitor cells at 1 pM. To maintain detectable blood levels of IL-3, osmotic pumps containing rIL-3 or control solutions were placed under the skin of normal and irradiated C3H/HeJ and (BALB X B10) F1 mice. The effect of IL-3 on hematopoietic progenitor cell numbers in spleen and bone marrow was evaluated 3 and 7 days later by using an in vitro clonal assay. The results demonstrated the following: (i) Doses of IL-3 infused at the rate of 2.5-5 ng per g of body weight per hr were sufficient to increase the numbers of hematopoietic progenitors in normal mice by at least 2-fold within 3 days. (ii) In mice with progenitor cell levels depressed by sublethal irradiation, 7-day treatment with IL-3 resulted in a 10-fold increase to near normal levels. (iii) The erythroid and myeloid lineages appeared to be enhanced to the same extent. (iv) Enhancement of hematopoiesis occurred primarily in spleen, but hematopoietic foci were also evident in the liver; in contrast, total cell and progenitor cell numbers were decreased in the bone marrow.

Mycobacterium tuberculosis transmission in a country with low tuberculosis incidence: role of immigration and HIV infection.

Immigrants from high-burden countries and HIV-coinfected individuals are risk groups for tuberculosis (TB) in countries with low TB incidence. Therefore, we studied their role in transmission of Mycobacterium tuberculosis in Switzerland. We included all TB patients from the Swiss HIV Cohort and a sample of patients from the national TB registry. We identified molecular clusters by spoligotyping and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis and used weighted logistic regression adjusted for age and sex to identify risk factors for clustering, taking sampling proportions into account. In total, we analyzed 520 TB cases diagnosed between 2000 and 2008; 401 were foreign born, and 113 were HIV coinfected. The Euro-American M. tuberculosis lineage dominated throughout the study period (378 strains; 72.7%), with no evidence for another lineage, such as the Beijing genotype, emerging. We identified 35 molecular clusters with 90 patients, indicating recent transmission; 31 clusters involved foreign-born patients, and 15 involved HIV-infected patients. Birth origin was not associated with clustering (adjusted odds ratio [aOR], 1.58; 95% confidence interval [CI], 0.73 to 3.43; P = 0.25, comparing Swiss-born with foreign-born patients), but clustering was reduced in HIV-infected patients (aOR, 0.49; 95% CI, 0.26 to 0.93; P = 0.030). Cavitary disease, male sex, and younger age were all associated with molecular clustering. In conclusion, most TB patients in Switzerland were foreign born, but transmission of M. tuberculosis was not more common among immigrants and was reduced in HIV-infected patients followed up in the national HIV cohort study. Continued access to health services and clinical follow-up will be essential to control TB in this population.

Six novel susceptibility Loci for early-onset androgenetic alopecia and their unexpected association with common diseases.

Androgenetic alopecia (AGA) is a highly heritable condition and the most common form of hair loss in humans. Susceptibility loci have been described on the X chromosome and chromosome 20, but these loci explain a minority of its heritable variance. We conducted a large-scale meta-analysis of seven genome-wide association studies for early-onset AGA in 12,806 individuals of European ancestry. While replicating the two AGA loci on the X chromosome and chromosome 20, six novel susceptibility loci reached genome-wide significance (p = 2.62×10(-9)-1.01×10(-12)). Unexpectedly, we identified a risk allele at 17q21.31 that was recently associated with Parkinson's disease (PD) at a genome-wide significant level. We then tested the association between early-onset AGA and the risk of PD in a cross-sectional analysis of 568 PD cases and 7,664 controls. Early-onset AGA cases had significantly increased odds of subsequent PD (OR = 1.28, 95% confidence interval: 1.06-1.55, p = 8.9×10(-3)). Further, the AGA susceptibility alleles at the 17q21.31 locus are on the H1 haplotype, which is under negative selection in Europeans and has been linked to decreased fertility. Combining the risk alleles of six novel and two established susceptibility loci, we created a genotype risk score and tested its association with AGA in an additional sample. Individuals in the highest risk quartile of a genotype score had an approximately six-fold increased risk of early-onset AGA [odds ratio (OR) = 5.78, p = 1.4×10(-88)]. Our results highlight unexpected associations between early-onset AGA, Parkinson's disease, and decreased fertility, providing important insights into the pathophysiology of these conditions.

Within-population polymorphism of sex-determination systems in the common frog (Rana temporaria).

In sharp contrast with birds and mammals, the sex chromosomes of ectothermic vertebrates are often undifferentiated, for reasons that remain debated. A linkage map was recently published for Rana temporaria (Linnaeus, 1758) from Fennoscandia (Eastern European lineage), with a proposed sex-determining role for linkage group 2 (LG2). We analysed linkage patterns in lowland and highland populations from Switzerland (Western European lineage), with special focus on LG2. Sibship analyses showed large differences from the Fennoscandian map in terms of recombination rates and loci order, pointing to large-scale inversions or translocations. All linkage groups displayed extreme heterochiasmy (total map length was 12.2 cM in males, versus 869.8 cM in females). Sex determination was polymorphic within populations: a majority of families (with equal sex ratios) showed a strong correlation between offspring phenotypic sex and LG2 paternal haplotypes, whereas other families (some of which with female-biased sex ratios) did not show any correlation. The factors determining sex in the latter could not be identified. This coexistence of several sex-determination systems should induce frequent recombination of X and Y haplotypes, even in the absence of male recombination. Accordingly, we found no sex differences in allelic frequencies on LG2 markers among wild-caught male and female adults, except in one high-altitude population, where nonrecombinant Y haplotypes suggest sex to be entirely determined by LG2. Multifactorial sex determination certainly contributes to the lack of sex-chromosome differentiation in amphibians.

Developmental, metabolic and immunological costs of flea infestation in the common vole

Parasites use resources from their hosts, which can indirectly affect a number of host functions because of trade-offs in resource allocation. In order to get a comprehensive view of the costs imposed by blood sucking parasites to their hosts, it is important to monitor multiple components of the development and physiology of parasitized hosts over long time periods. The effect of infestation by fleas on body mass, body length growth, haematocrit, resistance to oxidative stress, resting metabolic rate and humoral immune response were experimentally evaluated. During a 3-month period, male common voles, Microtus arvalis, were either parasitized by rat fleas (Nosopsyllus fasciatus), which are naturally occurring generalist ectoparasites of voles, or reared without fleas. Then voles were challenged twice by injecting Keyhole Limpet Haemocyanin (KLH) to assess whether the presence of fleas affects the ability of voles to produce antibodies against a novel antigen. During the immune challenge we measured the evolution of body mass, haematocrit, resistance to oxidative stress and antibody production. Flea infestation negatively influenced the growth of voles. Moreover, parasitized voles had reduced haematocrit, higher resting metabolic rate and lower production of antibodies against the KLH. Resistance to oxidative stress was not influenced by the presence of fleas. During the immune challenge with KLH, body mass decreased in both groups, while the resistance to oxidative stress remained stable. In contrast, the haematocrit decreased only in parasitized voles. Our experiment shows that infestation by a haematophageous parasite negatively affects multiple traits like growth, energy consumption and immune response. Fleas may severely reduce the survival probability and reproductive success of their host in natural conditions.

The list of the chromosome races of the common shrew Sorex araneus (updated 2002)

In 1996 the International Sorex araneus Cytogenetics Committee (ISACC) published a comprehensive list of 50 chromosome races of the common shrew Sorex araneus (lima et al. 1996). Since that time twenty one new races have been described and three races have been removed from the list. The present list summarises the data about races described since the 1996 publication. The rules introduced by Searle et al. (1991) and Hausser et al. (1994) were followed in the compilation of the list. It can be considered a reference for further studies of evolutionary relationships between the chromosome races of Sorex araneus. A summary table of all the 68 known races, arranged alphabetically according to their names, is given.

Illuminating the detection chain of bacterial bioreporters.

Engineering bacteria for measuring chemicals of environmental or toxicological concern (bioreporter bacteria) has grown slowly into a mature research area. Despite many potential advantages, current bioreporters do not perform well enough to comply with environmental detection standards. Basically, the reasons for this are the lack of engineering principles in the detection chain in the bioreporters. Here, we dissect critical steps in the detection chain and illustrate how bioreporter design could be improved by mutagenizing specificity and selectivity of the sensing and regulatory proteins, by newer expression strategies and application of different signalling networks. Furthermore, we describe how redesigning bioreporter assays with respect to pollutant transport into the cells and application of other detection devices can decrease detection limits and increase the speed of detection.

Exome sequencing identifies DYNC2H1 mutations as a common cause of asphyxiating thoracic dystrophy (Jeune syndrome) without major polydactyly, renal or retinal involvement.

BACKGROUND: Jeune asphyxiating thoracic dystrophy (JATD) is a rare, often lethal, recessively inherited chondrodysplasia characterised by shortened ribs and long bones, sometimes accompanied by polydactyly, and renal, liver and retinal disease. Mutations in intraflagellar transport (IFT) genes cause JATD, including the IFT dynein-2 motor subunit gene DYNC2H1. Genetic heterogeneity and the large DYNC2H1 gene size have hindered JATD genetic diagnosis. AIMS AND METHODS: To determine the contribution to JATD we screened DYNC2H1 in 71 JATD patients JATD patients combining SNP mapping, Sanger sequencing and exome sequencing. RESULTS AND CONCLUSIONS: We detected 34 DYNC2H1 mutations in 29/71 (41%) patients from 19/57 families (33%), showing it as a major cause of JATD especially in Northern European patients. This included 13 early protein termination mutations (nonsense/frameshift, deletion, splice site) but no patients carried these in combination, suggesting the human phenotype is at least partly hypomorphic. In addition, 21 missense mutations were distributed across DYNC2H1 and these showed some clustering to functional domains, especially the ATP motor domain. DYNC2H1 patients largely lacked significant extra-skeletal involvement, demonstrating an important genotype-phenotype correlation in JATD. Significant variability exists in the course and severity of the thoracic phenotype, both between affected siblings with identical DYNC2H1 alleles and among individuals with different alleles, which suggests the DYNC2H1 phenotype might be subject to modifier alleles, non-genetic or epigenetic factors. Assessment of fibroblasts from patients showed accumulation of anterograde IFT proteins in the ciliary tips, confirming defects similar to patients with other retrograde IFT machinery mutations, which may be of undervalued potential for diagnostic purposes.

Small bowel bacterial overgrowth is found in 71% of IBS patients and associated symptoms respond well to Rifaximin in a phase IV trial

Background a nd A ims: The prevalence of small intestinal bowel bacterial o vergrowth (SIBO) i n patients w ith irritable bowel syndrome (IBS) ranges from 43% to 78% as determined by t he lactulose hydrogen breath (LHBT) t est. Although rifaximine, a non-absorbable antibiotic, h as b een able to decrease I BS s ymptoms i n placebo-controlled r andomized trials, these results were not repeated in phase IV studies. We aimed to assess the prevalence of SIBO in an IBS cohort and to evaluate the response to rifaximin. Methods: I BS p atients f ulfilled Rome III criteria, had an absence of alarm symptoms, n ormal f ecal c alproectin, and normal e ndoscopic workup. They underwent lactulose hydrogen breath t esting (LHBT) for SIBO diagnosis. P atients with SIBO were t reated w ith rifaximine tablets f or 14 d ays. Symptoms were a ssessed by q uestionnaires before rifaximin treatment and at week 6. Results: Hundred-fifty IBS patients were enrolled (76% female, mean age 44 ± 16 years), of whom 106 (71%) were diagnosed with SIBO and consequently treated with rifaximine. Rifaximine treatment s ignificantly reduced the following symptoms as assessed by t he s ymptom q uestionnaire: bloating (5.5 ± 2.6 before vs. 3 .6 ± 2.7 after treatment, p <0.001), flatulence (5 ± 2.7 vs. 4 ± 2.7, p = 0.015), diarrhea (2.9 ± 2.4 vs. 2 ± 2.4, p = 0.005), abdominal pain (4.8 ± 2.7 vs. 3.3 ± 2.5, p <0.001) and resulted in improved overall well-being (3.9 ± 2.4 vs. 2.7 ± 2.3, p <0.001). The LHBT was repeated 2-4 weeks after rifaximine treatment in 6 5/93 (70%) patients. Eradication of SIBO was documented in 85% of all patients (55/65). Conclusions: The results o f our phase IV trial i ndicate that a high proportion of IBS p atients t ested positive f or SIBO. I BS symptoms w ere significantly diminished following a 2-week treatment with rifaximine.

Association Study of Common Genetic Variants and HIV-1 Acquisition in 6,300 Infected Cases and 7,200 Controls.

Multiple genome-wide association studies (GWAS) have been performed in HIV-1 infected individuals, identifying common genetic influences on viral control and disease course. Similarly, common genetic correlates of acquisition of HIV-1 after exposure have been interrogated using GWAS, although in generally small samples. Under the auspices of the International Collaboration for the Genomics of HIV, we have combined the genome-wide single nucleotide polymorphism (SNP) data collected by 25 cohorts, studies, or institutions on HIV-1 infected individuals and compared them to carefully matched population-level data sets (a list of all collaborators appears in Note S1 in Text S1). After imputation using the 1,000 Genomes Project reference panel, we tested approximately 8 million common DNA variants (SNPs and indels) for association with HIV-1 acquisition in 6,334 infected patients and 7,247 population samples of European ancestry. Initial association testing identified the SNP rs4418214, the C allele of which is known to tag the HLA-B*57:01 and B*27:05 alleles, as genome-wide significant (p = 3.6×10(-11)). However, restricting analysis to individuals with a known date of seroconversion suggested that this association was due to the frailty bias in studies of lethal diseases. Further analyses including testing recessive genetic models, testing for bulk effects of non-genome-wide significant variants, stratifying by sexual or parenteral transmission risk and testing previously reported associations showed no evidence for genetic influence on HIV-1 acquisition (with the exception of CCR5Δ32 homozygosity). Thus, these data suggest that genetic influences on HIV acquisition are either rare or have smaller effects than can be detected by this sample size.

Expression of neuroectodermal antigens common to melanomas, gliomas, and neuroblastomas. I. Identification by monoclonal anti-melanoma and anti-glioma antibodies.

The reactivity spectrum of five different monoclonal anti-melanoma antibodies cross-reacting with gliomas and neuroblastomas and one monoclonal anti-glioma antibody cross-reacting with melanomas and neuroblastomas was investigated. Comparison of the binding activity of these monoclonal antibodies for 11 melanoma, seven glioma, and three neuroblastoma cell lines showed that each of these clones had a different pattern of cross-reactivity. The results indicated that the antigenic determinants detected by these antibodies were not associated with the same antigen and thus suggested the existence of at least six different antigens common to melanomas, gliomas, and neuroblastomas. Since all these tumors are known to derive from cells originating embryologically from the neural crest, it can be assumed that the antigens recognized by our monoclonal antibodies are neuroectodermal differentiation antigens. However, absorption with fetal brain homogenates abolished only the binding of monoclonal anti-glioma antibody, but did not modify the binding of monoclonal anti-melanoma antibodies.

Comparison of antibiotic drops placed in the conjunctival cul-de-sac to antibiotic ointment applied to the lid margin in reduction of bacterial colonization on the lid margin.

PURPOSE: To compare the efficacy of antibiotic drops placed in the conjunctival cul-de-sac to antibiotic ointment applied to the lid margin in reduction of bacterial colonization on the lid margin. METHODS: A randomized, prospective, single-masked study was conducted on 19 patients with culture-proven colonization of bacteria on the lid margins. Ophthalmic eligibility criteria included the presence of > or =50 colony-forming units/mL (CFU/mL) of bacteria on both right and left lids. Each patient received one drop of ofloxacin in one eye every night for one week, followed by one drop once a week for one month. In the same manner, each patient received bacitracin ointment (erythromycin or gentamicin ointment if lid margin bacteria were resistant to bacitracin) to the lid margin of the fellow eye. Quantitative lid cultures were taken at initial visit, one week, one month, and two months. Fifteen volunteers (30 lids) served as controls. Lid cultures were taken at initial visit, one week, and one month. RESULTS: Both antibiotic drop and ointment reduced average bacterial CFU/mL at one week and one month. Average bacterial CFU/mL reestablished to baseline values at two months. There was no statistically significant difference between antibiotic drop and ointment in reducing bacterial colonization on the lid margin. CONCLUSION: Antibiotic drops placed in the conjunctival cul-de-sac appear to be as effective as ointment applied to the lid margins in reducing bacterial colonization in patients with > or =50 CFU/mL of bacteria on the lid margins.