Designing and implementing an assay for the detection of rare and divergent NRPS and PKS clones in European, Antarctic and Cuban soils

The ever increasing microbial resistome means there is an urgent need for new antibiotics. Metagenomics is an underexploited tool in the field of drug discovery. In this study we aimed to produce a new updated assay for the discovery of biosynthetic gene clusters encoding bioactive secondary metabolites. PCR assays targeting the polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) were developed. A range of European soils were tested for their biosynthetic potential using clone libraries developed from metagenomic DNA. Results revealed a surprising number of NRPS and PKS clones with similarity to rare Actinomycetes. Many of the clones tested were phylogenetically divergent suggesting they were fragments from novel NRPS and PKS gene clusters. Soils did not appear to cluster by location but did represent NRPS and PKS clones of diverse taxonomic origin. Fosmid libraries were constructed from Cuban and Antarctic soil samples; 17 fosmids were positive for NRPS domains suggesting a hit rate of less than 1 in 10 genomes. NRPS hits had low similarities to both rare Actinobacteria and Proteobacteria; they also clustered with known antibiotic producers suggesting they may encode for pathways producing novel bioactive compounds. In conclusion we designed an assay capable of detecting divergent NRPS and PKS gene clusters from the rare biosphere; when tested on soil samples results suggest the majority of NRPS and PKS pathways and hence bioactive metabolites are yet to be discovere

Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens

<p>Abstract</p> <p>Background</p> <p>The influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean^®Fecal DNA Isolation Kit, M; QIAamp^®DNA Stool Mini Kit, Q; FastDNA^®SPIN Kit, FSp; FastDNA^®SPIN Kit for Soil, FSo) were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles.</p> <p>Method</p> <p>Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons.</p> <p>Results</p> <p>Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences.</p> <p>Conclusion</p> <p>We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial community fingerprints using PCR-DGGE technique. Subsequently, PCR-DGGE technique can be applied for studying variations in human intestinal microbial communities.</p

Preoperative medical treatment in Cushing&apos;s syndrome : frequency of use and its impact on postoperative assessment : data from ERCUSYN

Background: Surgery is the definitive treatment of Cushing's syndrome (CS) but medications may also be used as a first-line therapy. Whether preoperative medical treatment (PMT) affects postoperative outcome remains controversial. Objective: (1) Evaluate how frequently PMT is given to CS patients across Europe; (2) examine differences in preoperative characteristics of patients who receive PMT and those who undergo primary surgery and (3) determine if PMT influences postoperative outcome in pituitary-dependent CS (PIT-CS). Patients and methods: 1143 CS patients entered into the ERCUSYN database from 57 centers in 26 countries. Sixty-nine percent had PIT-CS, 25% adrenal-dependent CS (ADR-CS), 5% CS from an ectopic source (ECT-CS) and 1% were classified as having CS from other causes (OTH-CS). Results: Twenty per cent of patients took PMT. ECT-CS and PIT-CS were more likely to receive PMT compared to ADR-CS (P < 0.001). Most commonly used drugs were ketoconazole (62%), metyrapone (16%) and a combination of both (12%). Median (interquartile range) duration of PMT was 109 (98) days. PIT-CS patients treated with PMT had more severe clinical features at diagnosis and poorer quality of life compared to those undergoing primary surgery (SX) (P < 0.05). Within 7 days of surgery, PIT-CS patients treated with PMT were more likely to have normal cortisol (P < 0.01) and a lower remission rate (P < 0.01). Within 6 months of surgery, no differences in morbidity or remission rates were observed between SX and PMT groups. Conclusions: PMT may confound the interpretation of immediate postoperative outcome. Follow-up is recommended to definitely evaluate surgical results

Geometric and photometric image stabilization for detection of significant events in video from a low flying Unmanned Aerial Vehicles

Analysis and interpretation of the information present in a video stream from aerial surveys is demanding and time-consuming even for experts. On-line video sequences acquired by cameras on board of a small surveillance plane are very unstable. The brightness and contrast of the images are rapidly changing due to fast changes in illumination and the content of the scene. The movement of the small plane is non-uniform and it depends on the wind and other weather conditions

Oral health-related knowledge, attitudes and behaviours of elementary school teachers

Abstract: Background: elementary schoolteachers play a central role in shaping their students’ beliefs, attitudes, and behaviours related to health and oral hygiene. This study was designed to evaluate Turkish schoolteachers’ levels of oral health knowledge, attitudes, and behaviours. Methods: A crosssectional survey-based study was conducted among elementary schoolteachers in Istanbul using a validated self-administered questionnaire. The questionnaire was composed of 36 multiple-choice items categorised into six sections, and the participants were recruited using convenience sampling. (3) Results: A total of 385 elementary schoolteachers participated in this study. The majority were female (62.2%), qualified with a licensure degree (81.3%) and working in public schools (86.5%). Female gender and greater work experience were found to be promoters for oral health knowledge and positive attitudes. The correlation between their perceived knowledge and actual knowledge was very weak, thus suggesting that the teachers are inclined to overestimate their knowledge. Conclusions: The Turkish elementary schoolteachers showed satisfactory oral health knowledge and attitudes toward oral health education. The teachers’ knowledge about dental trauma management was inadequate, necessitating urgent educational interventions, especially for physical education teachers, who are at the greatest risk of encountering such events during their work. The oral hygiene behaviours were not associated with teachers’ oral health knowledge, attitudes, or practice, thus requiring further investigation

Pediatric multisystem inflammatory syndrome temporally associated with SARS-COV-2: Oral manifestations and implications

Dear Editor,In connection with the editorial of Mallineni et al12020on the coronavirus disease (COVID-19) characteristics inchildren, we aim to demonstrate the emerging pediatric multisystem inflammatory syndrome temporally associated withSARS-COV-2 (PMIS-TS) from oral health professionals perspective. The epidemiological burden of COVID-19 in children was unexplainably lower than adults; therefore, it waspredicted that the clinical course differs between children andadults, such hypothesis was confirmed by the surging casesof PMIS-TS.

Range Image Registration Driven By A Hierarchy Of Surface Differential Features

This work proposes a way how to register overlapping range images automatically. We explore the fact that the Euclidean transformation is determined by three pairs of corresponding points only. The main idea of the proposed approach is to reduce the number of points by finding intrinsic (significant) ones first. For that, differential structures of the surface as curves of zero-mean curvature which are invariant to Euclidean transformation are used. The differential structures on a surface provide us with a hierarchy of intrinsic features, i.e. in a top down manner: surface ! curves ! points. The first estimate of the Euclidean transform is done using points then it is refined on curves and finally improved on surfaces. The performance of the approach is satisfactory for complicated surfaces which have rich differential structure

Studies of microbial community structure and function below ground in a managed upland grassland site at Sourhope Research Station

An upland grassland site was subjected to a range of techniques focused on the study of microbial community structure and function below ground. An overriding aim was to achieve an improved understanding of trophic interactions in the field, and apply innovative carbon tracking techniques to determine routes of carbon flow between different microbial groups. Stable isotopes proved to be a valuable experimental tool allowing the fate of plant photosynthate to be traced through the food web below ground. Established techniques for analysis of DNA and RNA were used to interrogate labelled biomass providing a unique in depth study of active microbial fractions. A key finding was the extensive role of mycorrhizal fungi in facilitating the flow of carbon into the soil microbial community. A number of novel techniques were developed which will assist in future studies of soil microbiology. (c) 2006 Elsevier B.V. All rights reserved