Mirna biogenesis pathway is differentially regulated during adipose derived stromal/stem cell differentiation.

Stromal/stem cell differentiation is controlled by a vast array of regulatory mechanisms. Included within these are methods of mRNA gene regulation that occur at the level of epigenetic, transcriptional, and/or posttranscriptional modifications. Current studies that evaluate the posttranscriptional regulation of mRNA demonstrate microRNAs (miRNAs) as key mediators of stem cell differentiation through the inhibition of mRNA translation. miRNA expression is enhanced during both adipogenic and osteogenic differentiation; however, the mechanism by which miRNA expression is altered during stem cell differentiation is less understood. Here we demonstrate for the first time that adipose-derived stromal/stem cells (ASCs) induced to an adipogenic or osteogenic lineage have differences in strand preference (-3p and -5p) for miRNAs originating from the same primary transcript. Furthermore, evaluation of miRNA expression in ASCs demonstrates alterations in both miRNA strand preference and 5'seed site heterogeneity. Additionally, we show that during stem cell differentiation there are alterations in expression of genes associated with the miRNA biogenesis pathway. Quantitative RT-PCR demonstrated changes in the Argonautes (AGO1-4), Drosha, and Dicer at intervals of ASC adipogenic and osteogenic differentiation compared to untreated ASCs. Specifically, we demonstrated altered expression of the AGOs occurring during both adipogenesis and osteogenesis, with osteogenesis increasing AGO1-4 expression and adipogenesis decreasing AGO1 gene and protein expression. These data demonstrate changes to components of the miRNA biogenesis pathway during stromal/stem cell differentiation. Identifying regulatory mechanisms for miRNA processing during ASC differentiation may lead to novel mechanisms for the manipulation of lineage differentiation of the ASC through the global regulation of miRNA as opposed to singular regulatory mechanisms.

Clinician response to Candida organisms in the urine of patients attending hospital.

The epidemiology of 54 episodes of candiduria with respect to clinical risk factors, species of Candida and physician response to the isolation of Candida in urine were studied in an observational survey over 3 months. Candida spp. were isolated from 4.7% of positive urine cultures. Common predisposing conditions included antibiotic use (74.1%), urinary drainage devices (57.4%), surgery (51.9%), intensive care unit (ICU) or high-dependency care unit (HDU) admission (42.6%) and urinary tract (UT) disease (18.5%). Upper UT infection was uncommon (n = 3). Of 65 Candida isolates, C. albicans predominated (85.2%), followed by C. glabrata (27.8%) and other Candida spp. (6.2%). All isolates were susceptible to fluconazole, itraconazole, voriconazole, amphotericin and caspofungin. Indwelling urinary catheters were removed in 76.2% of episodes. Antifungal therapy was initiated in 33.3% of cases independently of patient symptoms, underlying disease or Candida colony count. Patients in ICU/HDUs were significantly more likely to receive antifungal agents than those outside these units (p < 0.001). Fluconazole was the most common drug prescribed (77.8%). Clearance of candiduria occurred independently of antifungal therapy (p = 0.60). Physicians often did not follow up a positive urine result for Candida. Efforts to increase clinician awareness of current recommendations for managing candiduria and further study to elucidate specific risk factors in defined patient populations are warranted.

Comparison of use of phenotypic and genotypic characteristics for identification of species of the anamorph genus Candida and related teleomorph yeast species.

A total of 49 type and neotype isolates and 32 clinical isolates of the anamorph genus Candida and related teleomorph genera were obtained from different culture collections and clinical laboratories. Isolates were subjected to two phenotypic methods of identification, Vitek yeast biochemical card (YBC) and API ID 32C, both based on carbohydrate assimilation, and one genotypic method, PCR fingerprinting, based on the detection of DNA polymorphisms between minisatellite-specific sequences with the primer M13 (5' GAGGGTGGCGGTTCT 3'). The correct identification of a strain at the Centraalbureau voor Schimmelcultures was used as the gold standard for the identification of an isolate. When the study was restricted to species included in the respective biochemical databases, the Vitek YBC and API ID 32C systems performed adequately with positive identification rates of 87.3 and 76.8%, respectively. When uncommon species were added to the study, several of which are not included in the databases, the identification efficiencies were 76.5 and 77.5%, respectively. By comparison, all isolates were correctly identified by PCR fingerprinting, with 63 reference species profiles in the databank. Sufficient polymorphisms among the total set of banding patterns were observed, with adequate similarity in the major patterns obtained from a given species, to allow each isolate to be assigned unambiguously to a particular species. In addition, variations in minor bands allowed for differentiation to the strain level. PCR fingerprinting was found to be rapid, reproducible, and more cost-effective than either biochemical approach. Our results provide reference laboratories with an improved identification method for yeasts based on genotypic rather than phenotypic markers.