MicroRNA expression profiling in the prefrontal cortex: putative mechanisms for the cognitive effects of adolescent high fat feeding

The medial prefrontal cortex (mPFC), master regulator of higher-order cognitive functions, is the only brain region that matures until late adolescence. During this period, the mPFC is sensitive to stressful events or suboptimal nutrition. For instance, high-fat diet (HFD) feeding during adolescence markedly impairs prefrontal-dependent cognition. It also provokes multiple changes at the cellular and synaptic scales within the mPFC, suggesting that major transcriptional events are elicited by HFD during this maturational period. The nature of this transcriptional reprogramming remains unknown, but may include epigenetic processes, in particular microRNAs, known to directly regulate synaptic functions. We used high–throughput screening in the adolescent mouse mPFC and identified 38 microRNAs differentially regulated by HFD, in particular mir-30e-5p. We used a luciferase assay to confirm the functional effect of mir-30e-5p on a chosen target: Ephrin-A3. Using global pathway analyses of predicted microRNA targets, we identified biological pathways putatively affected by HFD. Axon guidance was the top-1 pathway, validated by identifying gene expression changes of axon guidance molecules following HFD. Our findings delineate major microRNA transcriptional reprogramming within the mPFC induced by adolescent HFD. These results will help understanding the contribution of microRNAs in the emergence of cognitive deficits following early-life environmental events

Use of the Bruker MALDI Biotyper for identification of molds in the clinical mycology laboratory

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is increasingly used for the identification of bacteria and fungi in the diagnostic laboratory. We evaluated the mold database of Bruker Daltonik (Bremen, Germany), the Filamentous Fungi Library 1.0. First, we studied 83 phenotypically and molecularly well-characterized, nondermatophyte, nondematiaceous molds from a clinical strain collection. Using the manufacturer-recommended interpretation criteria, genus and species identification rates were 78.3% and 54.2%, respectively. Reducing the species cutoff from 2.0 to 1.7 significantly increased species identification to 71.1% without increasing misidentifications. In a subsequent prospective study, 200 consecutive clinical mold isolates were identified by the MALDI Biotyper and our conventional identification algorithm. Discrepancies were resolved by ribosomal DNA (rDNA) internal transcribed spacer region sequence analysis. For the MALDI Biotyper, genus and species identification rates were 83.5% and 79.0%, respectively, when using a species cutoff of 1.7. Not identified were 16.5% of the isolates. Concordant genus and species assignments of MALDI-TOF MS and the conventional identification algorithm were observed for 98.2% and 64.2% of the isolates, respectively. Four erroneous species assignments were observed using the MALDI Biotyper. The MALDI Biotyper seems highly reliable for the identification of molds when using the Filamentous Fungi Library 1.0 and a species cutoff of 1.7. However, expansion of the database is required to reduce the number of nonidentified isolates

Evaluation of the Bruker MALDI Biotyper for the identification of fastidious Gram-negative rods

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has entered clinical laboratories facilitating identification of bacteria. We here evaluated the MALDI Biotyper (Bruker Daltonics) for the identification of fastidious Gram-negative rods (GNR). Three sample preparation methods were analyzed for 151 clinical isolates: direct colony transfer, direct transfer plus on-target formic acid preparation, and ethanol-formic acid extraction. Direct colony transfer applied with the manufacturer's interpretation criteria resulted in overall species and genus identification rates of 43.0% and 32.5%, respectively. 23.2% of the isolates were not identified, and two misidentifications (1.3%) were observed. Species identification rates increased to 46.4% and 53.7% for direct transfer plus formic acid preparation and ethanol-formic acid extraction, respectively. In addition, we evaluated score value cutoff alterations. Identification rates were hardly increased by reducing the genus cutoff, while reducing the 2.0 species cutoff to 1.9 and to 1.8 increased identification rates to up to 66.2% without increasing the rate of misidentifications. This study shows that fastidious GNR can reliably be identified using the MALDI Biotyper. However, identification rates do not reach those of non-fastidious GNR such as the Enterobacteriaceae. In addition, two approaches optimizing the identification of fastidious GNR by the MALDI Biotyper were demonstrated: (i) formic acid based on-target sample treatment, and (ii) reduction of cutoff scores to increase species identification rates

Performance of copan WASP for routine urine microbiology

This study compared manual workup of urine clinical samples with fully automated WASPLab processing. As a first step two different inocula (1 and 10 μl) and different streaking patterns were compared using WASP and InoqulA BT™ instrumentation. A 10 μl inoculum produced significantly more single colonies than a 1 μl inoculum and automated streaking yielded significantly more single colonies as compared to manual streaking on whole plates (p<0.001). In a second step, 379 clinical urine samples were evaluated using WASP and manual workup. Average numbers of detected morphologies, recovered species, and CFU/ml of all 379 urine samples showed excellent agreement of WASPLab and manual workup. The percentage of clinical categorization of urine samples as "positive" or "negative" did not differ between automated and manual work-flow but within the positive samples automated processing by WASPLab resulted in the detection of more potential pathogens. In summary, the present study demonstrates that i) the streaking pattern, i.e. primarily the number of zigzags/length of streaking lines, is critical for optimizing the number of single colonies yielded from primary cultures of urine samples, ii) automated streaking by the WASP instrument was superior to manual streaking regarding the number of single colonies yielded, (for 32.2%) iii) automated streaking leads to higher numbers of detected morphologies (for 47.5%), species (for 17.4%) and pathogens (for 3.4%). The results of this study point to an improved quality of microbiological analyses and laboratory reports when using automated sample processing by WASP and WASPLab

Frequent detection of Streptococcus tigurinus in the human oral microbial flora by a specific 16S rRNA gene real-time TaqMan PCR

Background: Many bacteria causing systemic invasive infections originate from the oral cavity by entering the bloodstream. Recently, a novel pathogenic bacterium, Streptococcus tigurinus, was identified as causative agent of infective endocarditis, spondylodiscitis and meningitis. In this study, we sought to determine the prevalence of S. tigurinus in the human oral microbial flora and analyzed its association with periodontal disease or health. Results: We developed a diagnostic highly sensitive and specific real-time TaqMan PCR assay for detection of S. tigurinus in clinical samples, based on the 16S rRNA gene. We analyzed saliva samples and subgingival plaque samples of a periodontally healthy control group (n = 26) and a periodontitis group (n = 25). Overall, S. tigurinus was detected in 27 (53%) out of 51 patients. There is no significant difference of the frequency of S. tigurinus detection by RT-PCR in the saliva and dental plaque samples in the two groups: in the control group, 14 (54%) out of 26 individuals had S. tigurinus either in the saliva samples and/or in the plaque samples; and in the periodontitis group, 13 (52%) out of 25 patients had S. tigurinus in the mouth samples, respectively (P = 0.895). The consumption of nicotine was no determining factor. Conclusion: Although S. tigurinus was a frequently detected species of the human oral microbial flora, it was not associated with periodontal disease. Further investigations are required to determine whether S. tigurinus is a commensal or an opportunistic oral pathogen with a potential for development of invasive infections