Rad51p and Rad54p,but not Rad52p,elevate gene repair in Saccharomyces cerevisiae directed by modified single-stranded oligonucleotide vectors

Synthetic single-stranded DNA vectors have been used to correct point and frameshift mutations in episomal or chromosomal targets in the yeast Saccharomyces cerevisiae. Certain parameters, such as the length of the vector and the genetic background of the organism, have a significant impact on the process of targeted gene repair, and point mutations are corrected at a higher frequency than frameshift mutations. Genetic analyses reveal that expression levels of the recombination/repair genes RAD51, RAD52 and RAD54 can affect the frequency of gene repair. Overexpression of RAD51 enhances the frequency 4-fold for correction of an episomal target and 5-fold for correction of a chromosomal target; overexpression of RAD54 is also effective in stimulating gene repair, to the same extent as RAD51 in the chromosomal target. In sharp contrast, RAD52 gene expression serves to reduce gene repair activity in rescue experiments and in experiments where RAD52 is overexpressed in a wild-type strain. This may suggest an antagonist role for Rad52p. Consistent with this notion, the highest level of targeted repair occurs when the RAD51 gene is overexpressed in a strain of yeast deficient in RAD52 gene function.     

A CART-based approach to discover emerging patterns in microarray data

MOTIVATION: Cancer diagnosis using gene expression profiles requires supervised learning and gene selection methods. Of the many suggested approaches, the method of emerging patterns (EPs) has the particular advantage of explicitly modeling interactions among genes, which improves classification accuracy. However, finding useful (i.e. short and statistically significant) EP is typically very hard. METHODS: Here we introduce a CART-based approach to discover EPs in microarray data. The method is based on growing decision trees from which the EPs are extracted. This approach combines pattern search with a statistical procedure based on Fisher's exact test to assess the significance of each EP. Subsequently, sample classification based on the inferred EPs is performed using maximum-likelihood linear discriminant analysis. RESULTS: Using simulated data as well as gene expression data from colon and leukemia cancer experiments we assessed the performance of our pattern search algorithm and classification procedure. In the simulations, our method recovers a large proportion of known EPs while for real data it is comparable in classification accuracy with three top-performing alternative classification algorithms. In addition, it assigns statistical significance to the inferred EPs and allows to rank the patterns while simultaneously avoiding overfit of the data. The new approach therefore provides a versatile and computationally fast tool for elucidating local gene interactions as well as for classification. AVAILABILITY: A computer program written in the statistical language R implementing the new approach is freely available from the web page http://www.stat.uni-muenchen.de/~socher/     

A versatile chondrogenic rat calvaria cell line R-tTA-24 that permits tetracycline-regulated gene expression

The clonal rat calvaria cell line RCJ3.1C5.18 (RCJ) undergoes chondrogenic differentiation after long-term culture post confluence. To allow flexible genetic manipulation, a tetracycline-regulated gene expression system was established in this cell line. Treatment with tetracycline in operational doses does not affect the differentiation of RCJ cells with respect to the markers tested. After stable transfection with pUHD15.1 containing the tetracycline transactivator (tTA) in the presence of pTK-hyg for hygromycin selection, 28 clones were isolated and characterized for alcian blue staining of cartilage-specific proteoglycans and for collagen type II expression. Clone R-tTA-24 was selected on the basis of phenotype and displayed tetracycline-dependent downregulation of luciferase activity (tet-OFF system) by two orders of magnitude (57–149-fold) after stable transfection with the reporter gene pBI-EGFP/luc. The novel, chondrogenic cell line R-tTA-24 may be stably transfected with various genes of interest for tetracycline- regulated gene expression using neomycin selection and may be a valuable tool to study the process of chondrogenic differentiation in vitro. Accepted: 30 November 1999     

Low Resistance to First and Second Line Anti-Tuberculosis Drugs among Treatment Naive Pulmonary Tuberculosis Patients in Southwestern Uganda

BackgroundThere are limited data on region-specific drug susceptibility of tuberculosis (TB) in Uganda. We performed resistance testing on specimens collected from treatment-naive patients with pulmonary TB in Southwestern Uganda for first and second line anti-TB drugs. We sought to provide data to guide regional recommendations for empiric TB therapy.MethodsArchived isolates, obtained from patients at Mbarara Regional Referral Hospital from February 2009 to February 2013, were tested for resistance to isoniazid and rifampicin using the MTBDRplus and Xpert MTB/RIF assays. A subset of randomly selected isolates was tested for second line agents, including fluoroquinolones (FQs), aminoglycosides, cyclic peptides, and ethambutol using the MTBDRsl assay. We performed confirmatory testing for FQ resistance using repeated MTBDRsl, the Mycobacteria growth indicator tube (MGIT) assay, and sequencing of the gyrA and gyrB genes.ResultsWe tested isolates from 190 patients. The cohort had a median age of 33 years (IQR 26-43), 69% (131/190) were male, and the HIV prevalence was 42% (80/190). No isolates (0/190) were rifampicin-resistant and only 1/190 (0.5%) was isoniazid-resistant. Among 92 isolates tested for second-line drug resistance, 71 (77%) had interpretable results, of which none were resistant to aminoglycosides, cyclic peptides or ethambutol. Although 7 (10%) initially tested as resistant to FQs by the MTBDRsl assay, they were confirmed as susceptible by repeat MTBDRsl testing as well as by MGIT and gyrase gene sequencingConclusionWe found no MDR-TB and no resistance to ethambutol, FQs, or injectable anti-TB drugs in treatment naïve patients with pulmonary TB in Southwestern Uganda. Standard treatment guidelines for susceptible TB should be adequate for most patients with TB in this population. Where possible, molecular susceptibility testing methods should be routinely validated by culture methods.     

Spatio-temporal Genetic Structuring of Leishmania major in Tunisia by Microsatellite Analysis

In Tunisia, cases of zoonotic cutaneous leishmaniasis caused by Leishmania major are increasing and spreading from the south-west to new areas in the center. To improve the current knowledge on L. major evolution and population dynamics, we performed multi-locus microsatellite typing of human isolates from Tunisian governorates where the disease is endemic (Gafsa, Kairouan and Sidi Bouzid governorates) and collected during two periods: 1991–1992 and 2008–2012. Analysis (F-statistics and Bayesian model-based approach) of the genotyping results of isolates collected in Sidi Bouzid in 1991–1992 and 2008–2012 shows that, over two decades, in the same area, Leishmania parasites evolved by generating genetically differentiated populations. The genetic patterns of 2008–2012 isolates from the three governorates indicate that L. major populations did not spread gradually from the south to the center of Tunisia, according to a geographical gradient, suggesting that human activities might be the source of the disease expansion. The genotype analysis also suggests previous (Bayesian model-based approach) and current (F-statistics) flows of genotypes between governorates and districts. Human activities as well as reservoir dynamics and the effects of environmental changes could explain how the disease progresses. This study provides new insights into the evolution and spread of L. major in Tunisia that might improve our understanding of the parasite flow between geographically and temporally distinct populations.     

Systemic and Cardiac Depletion of M2 Macrophage through CSF-1R Signaling Inhibition Alters Cardiac Function Post Myocardial Infarction

The heart hosts tissue resident macrophages which are capable of modulating cardiac inflammation and function by multiple mechanisms. At present, the consequences of phenotypic diversity in macrophages in the heart are incompletely understood. The contribution of cardiac M2-polarized macrophages to the resolution of inflammation and repair response following myocardial infarction remains to be fully defined. In this study, the role of M2 macrophages was investigated utilising a specific CSF-1 receptor signalling inhibition strategy to achieve their depletion. In mice, oral administration of GW2580, a CSF-1R kinase inhibitor, induced significant decreases in Gr1^lo and F4/80^hi monocyte populations in the circulation and the spleen. GW2580 administration also induced a significant depletion of M2 macrophages in the heart after 1 week treatment as well as a reduction of cardiac arginase1 and CD206 gene expression indicative of M2 macrophage activity. In a murine myocardial infarction model, reduced M2 macrophage content was associated with increased M1-related gene expression (IL-6 and IL-1β), and decreased M2-related gene expression (Arginase1 and CD206) in the heart of GW2580-treated animals versus vehicle-treated controls. M2 depletion was also associated with a loss in left ventricular contractile function, infarct enlargement, decreased collagen staining and increased inflammatory cell infiltration into the infarct zone, specifically neutrophils and M1 macrophages. Taken together, these data indicate that CSF-1R signalling is critical for maintaining cardiac tissue resident M2-polarized macrophage population, which is required for the resolution of inflammation post myocardial infarction and, in turn, for preservation of ventricular function.     

Evidence that yeast frataxin is not an iron storage protein in vivo

Yeast cells deficient in the yeast frataxin homolog (Yfh1p) accumulate iron in their mitochondria. Whether this iron is toxic, however, remains unclear. We showed that large excesses of iron in the growth medium did not inhibit growth and did not decrease cell viability. Increasing the ratio of mitochondrial iron-to-Yfh1p by decreasing the steady-state level of Yfh1p to less than 100 molecules per cell had very few deleterious effects on cell physiology, even though the mitochondrial iron concentration greatly exceeded the iron-binding capacity of Yfh1p in these conditions. Mössbauer spectroscopy and FPLC analyses of whole mitochondria or of isolated mitochondrial matrices showed that the chemical and biochemical forms of the accumulated iron in mitochondria of mutant yeast strains (Δyfh1, Δggc1 and Δssq1) displayed a nearly identical distribution. This was also the case for Δggc1 cells, in which Yfh1p was overproduced. In these mitochondria, most of the iron was insoluble, and the ratio of soluble-to-insoluble iron did not change when the amount of Yfh1p was increased up to 4500 molecules per cell. Our results do not privilege the hypothesis of Yfh1p being an iron storage protein in vivo.