Optimization of DNA extraction from human urinary samples for mycobiome community profiling.

IntroductionRecent data suggest the urinary tract hosts a microbial community of varying composition, even in the absence of infection. Culture-independent methodologies, such as next-generation sequencing of conserved ribosomal DNA sequences, provide an expansive look at these communities, identifying both common commensals and fastidious organisms. A fundamental challenge has been the isolation of DNA representative of the entire resident microbial community, including fungi.Materials and methodsWe evaluated multiple modifications of commonly-used DNA extraction procedures using standardized male and female urine samples, comparing resulting overall, fungal and bacterial DNA yields by quantitative PCR. After identifying protocol modifications that increased DNA yields (lyticase/lysozyme digestion, bead beating, boil/freeze cycles, proteinase K treatment, and carrier DNA use), all modifications were combined for systematic confirmation of optimal protocol conditions. This optimized protocol was tested against commercially available methodologies to compare overall and microbial DNA yields, community representation and diversity by next-generation sequencing (NGS).ResultsOverall and fungal-specific DNA yields from standardized urine samples demonstrated that microbial abundances differed significantly among the eight methods used. Methodologies that included multiple disruption steps, including enzymatic, mechanical, and thermal disruption and proteinase digestion, particularly in combination with small volume processing and pooling steps, provided more comprehensive representation of the range of bacterial and fungal species. Concentration of larger volume urine specimens at low speed centrifugation proved highly effective, increasing resulting DNA levels and providing greater microbial representation and diversity.ConclusionsAlterations in the methodology of urine storage, preparation, and DNA processing improve microbial community profiling using culture-independent sequencing methods. Our optimized protocol for DNA extraction from urine samples provided improved fungal community representation. Use of this technique resulted in equivalent representation of the bacterial populations as well, making this a useful technique for the concurrent evaluation of bacterial and fungal populations by NGS

Retrospective Analysis of Factors Leading to Pediatric Tracheostomy Decannulation Failure. A Single-Institution Experience

RATIONALE: There is a lack of evidence regarding factors associated with failure of tracheostomy decannulation. OBJECTIVES: We aimed to identify characteristics of pediatric patients who fail a tracheostomy decannulation challenge Methods: A retrospective review was performed on all patients who had a decannulation challenge at a tertiary care center from June 2006 to October 2013. Tracheostomy decannulation failure was defined as reinsertion of the tracheostomy tube within 6 months of the challenge. Data on demographics, indications for tracheostomy, home mechanical ventilation, and comorbidities were collected. Data were also collected on specific airway endoscopic findings during the predecannulation bronchoscopy and airway surgical procedures before decannulation. We attempted to predict the decannulation outcome by analyzing associations. MEASUREMENTS AND MAIN RESULTS: 147 of 189 (77.8%) patients were successfully decannulated on the first attempt. Tracheostomy performed due to chronic respiratory failure decreased odds for decannulation failure (odds ratio = 0.34, 95% confidence interval = 0.15-0.77). Genetic abnormalities (45%) and feeding dysfunction (93%) were increased in the population of patients failing their first attempt. The presence of one comorbidity increased the odds of failure by 68% (odds ratio = 1.68, 95% confidence interval = 1.23-2.29). Decannulation pursuit based on parental expectation of success, rather than medically determined readiness, was associated with a higher chance of failure (P = 0.01). CONCLUSIONS: Our study highlights the role of genetic abnormalities, feeding dysfunction, and multiple comorbidities in patients who fail decannulation. Our findings also demonstrate that the outcome of decannulation may be predicted by the indication for tracheostomy. Patients who had tracheostomy placed for chronic respiratory support had a higher likelihood of success. Absence of a surgically treatable airway obstruction abnormality on the predecannulation bronchoscopy increased the chances of success

Cloning and Sequence Analysis of the Structural Gene for the bc 1 - Type Rieske Iron-Sulfur Protein from Thermus thermophilus HB8

The structural gene encoding the Rieske iron-sulfur protein from Thermus thermophilus HB8 has been cloned and sequenced. The gene encodes a protein of 209 amino acids that begins with a hydrophilic N-terminus followed by a stretch of 21 hydrophobic amino acids that could serve as a transmembrane helix. The remainder of the protein has a hydrophobicity pattern typical of a water-soluble protein. A phylogenetic analysis of 26 Rieske proteins that are part of bc 1 or b 6 f complexes shows that they fall into three major groups: eubacterial and mitochondrial, cyanobacterial and plastid, and five highly divergent outliers, including that of Thermus . Although the overall homology with other Rieske proteins is very low, the C-terminal half of the Thermus protein contains the signature sequence CTHLGC-(13X)-CPCH that most likely provides the ligands of the [2Fe-2S] cluster. It is proposed that this region of the protein represents a small domain that folds independently and that the encoding DNA sequence may have been transferred during evolution to several unrelated genes to provide the cluster attachment site to proteins of different origin. The role of individual residues in this domain of the Thermus protein is discussed vis-a-vis the three-dimensional structure of the bovine protein (Iwata et al ., 1996 Structure 4 , 567–579).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44801/1/10863_2004_Article_409077.pd

Discharge characteristics at bioswale installation sites along I-294 in northern Cook County, Illinois

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Using bioswales to improve the quality of roadway runoff from I-294 in northern Cook County, Illinois

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The effects of bioswale construction on groundwater adjacent to I-294 in northern Cook County, Illinois

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Reductions in turbidity and specific conductivity in runoff treated by bioswales along I-294 in northern Cook County, Illinois

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From Providers to PHOs: an institutional analysis of nonprofit primary health care governance in New Zealand

Policy reforms to primary health care delivery in New Zealand required government-funded firms overseeing care delivery to be constituted as nonprofit entities with governance shared between consumer and producers. This paper examines the consumer and producer interests in the allocation of ownership and control of New Zealand firms delivering primary health care utilising theories of competition in the markets for ownership and control of firms. Consistent with pre-reform patterns of ownership and control provider interests appear to have exerted effective control over the formation and governance of the new entities in all but a few cases where community (consumer) control was already established. Their ability to do so is implied from the absence of a defined ownership stake via which the balance of governance control could shift as a consequence of changes to incentives facing the different stakeholding groups. It appears that the pre-existing patterns will prevail and further intervention will be required if policymakers are to achieve their underlying aims