Identification of novel arsenic resistance genes in yeast.

Arsenic is a toxic metalloid that affects human health by causing numerous diseases and by being used in the treatment of acute promyelocytic leukemia. Saccharomyces cerevisiae (budding yeast) has been extensively utilized to elucidate the molecular mechanisms underlying arsenic toxicity and resistance in eukaryotes. In this study, we applied a genomic DNA overexpression strategy to identify yeast genes that provide arsenic resistance in wild-type and arsenic-sensitive S. cerevisiae cells. In addition to known arsenic-related genes, our genetic screen revealed novel genes, including PHO86, VBA3, UGP1, and TUL1, whose overexpression conferred resistance. To gain insights into possible resistance mechanisms, we addressed the contribution of these genes to cell growth, intracellular arsenic, and protein aggregation during arsenate exposure. Overexpression of PHO86 resulted in higher cellular arsenic levels but no additional effect on protein aggregation, indicating that these cells efficiently protect their intracellular environment. VBA3 overexpression caused resistance despite higher intracellular arsenic and protein aggregation levels. Overexpression of UGP1 led to lower intracellular arsenic and protein aggregation levels while TUL1 overexpression had no impact on intracellular arsenic or protein aggregation levels. Thus, the identified genes appear to confer arsenic resistance through distinct mechanisms but the molecular details remain to be elucidated.

Genomewide Elucidation of Drug Resistance Mechanisms for Systemically Used Antifungal Drugs Amphotericin B, Caspofungin, and Voriconazole in the Budding Yeast.

There are only a few antifungal drugs used systemically in treatment, and invasive fungal infections that are resistant to these drugs are an emerging problem in health care. In this study, we performed a high-copy-number genomic DNA (gDNA) library screening to find and characterize genes that reduce susceptibility to amphotericin B, caspofungin, and voriconazole in Saccharomyces cerevisiae We identified the PDR16 and PMP3 genes for amphotericin B, the RMD9 and SWH1 genes for caspofungin, and the MRS3 and TRI1 genes for voriconazole. The deletion mutants for PDR16 and PMP3 were drug susceptible, but the other mutants had no apparent susceptibility. Quantitative-PCR analyses suggested that the corresponding drugs upregulated expression of the PDR16, PMP3, SWH1, and MRS3 genes. To further characterize these genes, we also profiled the global expression patterns of the cells after treatment with the antifungals and determined the genes and paths that were up- or downregulated. We also cloned Candida albicans homologs of the PDR16, PMP3, MRS3, and TRI1 genes and expressed them in S. cerevisiae Heterologous expression of Candida homologs also provided reduced drug susceptibility to the budding yeast cells. Our analyses suggest the involvement of new genes in antifungal drug resistance.

Comparison of multiplex real-time polymerase chain reaction with serological tests and culture for diagnosing human brucellosis.

OBJECTIVE: Brucellosis is a zoonotic disease with various clinical presentations and early diagnosis is crucial to avoid severe complications. Due to limitations of conventional diagnostic methods, polymerase chain reaction (PCR) based approaches have gained importance in diagnosis.We aimed to evaluate diagnostic value of multiplex real time-PCR (mRT-PCR) in serum samples collected from brucellosis suspected patients by comparision sensitivity of mRT-PCR with those of conventional diagnostic methods. MATERIAL AND METHODS: A total of 249 serum samples collected from the suspected brucellosis patients admitted to the hospitals in three different provinces were analyzed by serological tests, culture and mRT-PCR. In laboratories of the participating hospital, serum samples were tested for the Brucella specific antibody by commercial serological kits including standart tube agglutination test (STAT), Coombs' test, and immunocapture test (ICT). Blood culture was performed for 153 of the patients in the participating hospital. All serum samples were analyzed for the presence of Brucella DNA by mRT-PCR. RESULTS: According to laboratory test results, 215 of the 249 suspected cases having comparible clinical data were identified as brucellosis cases. Of the 215 brucellosis cases, 36 were diagnosed as definitive cases, the remaning 179 patients were presumptive cases. Sensitivity of mRT-PCR in the samples that were positive by ICT, STAT, Coombs' test, and blood culture was 70.2%, 77.3%, 83%, and 97.2%, respectively. By using mRT-PCR, additional 17 suspected patients were diagnosed as presumptive cases. Among the mRT-PCR positive serum samples, Brucella abortus was detected in 3 samples (1.9%), the remaining 156 samples (98.1%) had B. melitensis DNA. CONCLUSION: Our results indicate that mRT-PCR can be considered a useful diagnostic tool in patients who have negative serologic test results, and in detection of Brucella species.

Effects of boiling dairy products on human brucellosis.

OBJECTIVE: In this study, we determined the seroprevalence of Brucella in the blood of bovine and ovine animals and in the blood of the people who raise these animals to produce cheese in two rural counties that use two different methods of cheese production in Erzurum Province in Turkey. MATERIALS AND METHODS: Samples are taken from 100 bovine animals, 100 ovine animals, 100 young people between the ages of 10-20 years and 100 adults between the ages of 20-60 years. The samples were tested with the Rose Bengal Plate Test (RBPT), the Serum Agglutination Test (SAT), the Coombs' Test and a micro-ELISA. RESULTS: We found the following rates of Brucella in the province that makes cheese with raw milk: bovine (3.00%), ovine (5.00%), people between 10-20 years of age (2.00%) and people between 20-60 years of age (10.00%). However, the corresponding rates in the region that makes cheese with boiling milk were 2%, 4%, 1% and 5%, respectively. CONCLUSION: The results were analyzed descriptively and in comparison to the results from the other region. There was a significant difference found between the two regions among the Hinis and Oltu individuals aged 10-20 and 20-60 (z=0.6<1.96 with a 95% confidence interval).