Optimizing DNA Extraction Methods for Nanopore Sequencing of Neisseria gonorrhoeae Directly from Urine Samples

Empirical gonorrhea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance. We investigated if Nanopore sequencing can detect sufficient Neisseria gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae-spiked urine samples and samples from gonorrhea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced while minimizing contaminating host DNA. In simulated infections, the Qiagen UCP pathogen mini kit provided the highest ratio of N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections but decreased yields in clinical samples. In 10 urine samples from men with symptomatic urethral gonorrhea, ≥92.8% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥93.8% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections, if ≥104 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR medium tubes and from urethral swabs and in the presence of simulated Chlamydia coinfection. Using Nanopore sequencing of urine samples from men with urethral gonorrhea, sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture

What factors are associated with adolescents\u27 school break time physical activity and sedentary time?

Purpose Adolescents\u27 physical activity levels during school break time are low and understanding correlates of physical activity and sedentary time in this context is important. This study investigated cross-sectional and longitudinal associations between a range of individual, behavioural, social and policy/organisational correlates and objectively measured school break time physical activity and sedentary time.Methods In 2006, 146 adolescents (50% males; mean age = 14.1&plusmn;0.6 years) completed a questionnaire and wore an accelerometer for &ge;3 school days. Time spent engaged in sedentary, light (LPA) and moderate-to-vigorous physical activity (MVPA) during school break times (recess and lunchtime) were calculated using existing cut-points. Measures were repeated in 2008 among 111 adolescents. Multilevel models examined cross-sectional and longitudinal associations.Results Bringing in equipment was cross-sectionally associated with 3.2% more MVPA during break times. Females engaged in 5.1% more sedentary time than males, whilst older adolescents engaged in less MVPA than younger adolescents. Few longitudinal associations were observed. Adolescents who brought sports equipment to school engaged in 7.2% less LPA during break times two years later compared to those who did not bring equipment to school.Conclusion These data suggest that providing equipment and reducing restrictions on bringing in sports equipment to school may promote physical activity during school recess. Strategies targeting females\u27 and older adolescents\u27, in particular, are warranted.<br /

‘Do i care?’ young adults' recalled experiences of early adolescent overweight and obesity: a qualitative study

&lt;p&gt;Objective: Individual behaviour change to reduce obesity requires awareness of, and concern about, weight. This paper therefore describes how young adults, known to have been overweight or obese during early adolescence, recalled early adolescent weight-related awareness and concerns. Associations between recalled concerns and weight-, health- and peer-related survey responses collected during adolescence are also examined.&lt;/p&gt; &lt;p&gt;Design: Qualitative semi-structured interviews with young adults; data compared with responses to self-report questionnaires obtained in adolescence.&lt;/p&gt; &lt;p&gt;Participants: A total of 35 participants, purposively sub-sampled at age 24 from a longitudinal study of a school year cohort, previously surveyed at ages 11, 13 and 15. Physical measures during previous surveys allowed identification of participants with a body mass index (BMI) indicative of overweight or obesity (based on British 1990 growth reference) during early adolescence. Overall, 26 had been obese, of whom 11 had BMI99.6th centile, whereas 9 had been overweight (BMI=95th–97.9th centile).&lt;/p&gt; &lt;p&gt;Measures: Qualitative interview responses describing teenage life, with prompts for school-, social- and health-related concerns. Early adolescent self-report questionnaire data on weight-worries, self-esteem, friends and victimisation (closed questions).&lt;/p&gt; &lt;p&gt;Results: Most, but not all recalled having been aware of their overweight. None referred to themselves as having been obese. None recalled weight-related health worries. Recollection of early adolescent obesity varied from major concerns impacting on much of an individual's life to almost no concern, with little relation to actual severity of overweight. Recalled concerns were not clearly patterned by gender, but young adult males recalling concerns had previously reported more worries about weight, lower self-esteem, fewer friends and more victimisation in early adolescence; no such pattern was seen among females. Conclusion: The popular image of the unhappy overweight teenager was not borne out. Many obese adolescents, although well aware of their overweight recalled neither major dissatisfaction nor concern. Weight-reduction behaviours are unlikely in such circumstances.&lt;/p&gt

Associations between cardiorespiratory fitness, physical activity and clustered cardiometabolic risk in children and adolescents: the HAPPY study

Clustering of cardiometabolic risk factors can occur during childhood and predisposes individuals to cardiometabolic disease. This study calculated clustered cardiometabolic risk in 100 children and adolescents aged 10-14 years (59 girls) and explored differences according to cardiorespiratory fitness (CRF) levels and time spent at different physical activity (PA) intensities. CRF was determined using a maximal cycle ergometer test, and PA was assessed using accelerometry. A cardiometabolic risk score was computed as the sum of the standardised scores for waist circumference, blood pressure, total cholesterol/high-density lipoprotein ratio, triglycerides and glucose. Differences in clustered cardiometabolic risk between fit and unfit participants, according to previously proposed health-related threshold values, and between tertiles for PA subcomponents were assessed using ANCOVA. Clustered risk was significantly lower (p < 0.001) in the fit group (mean 1.21 ± 3.42) compared to the unfit group (mean -0.74 ± 2.22), while no differences existed between tertiles for any subcomponent of PA. Conclusion These findings suggest that CRF may have an important cardioprotective role in children and adolescents and highlights the importance of promoting CRF in youth

Mycobacterium tuberculosis ClpP Proteases Are Co-transcribed but Exhibit Different Substrate Specificities

PMCID: PMC3613350This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Identification of elderly fallers by muscle strength measures

For efficient prevention of falls among older adults, individuals at a high risk of falling need to be identified. In this study, we searched for muscle strength measures that best identified those individuals who would fall after a gait perturbation and those who recovered their balance. Seventeen healthy older adults performed a range of muscle strength tests. We measured maximum and rate of development of ankle plantar flexion moment, knee extension moment and whole leg push-off force, as well as maximum jump height and hand grip strength. Subsequently, their capacity to regain balance after tripping over an obstacle was determined experimentally. Seven of the participants were classified as fallers based on the tripping outcome. Maximum isometric push-off force in a leg press apparatus was the best measure to identify the fallers, as cross-validation of a discriminant model with this variable resulted in the best classification (86% sensitivity and 90% specificity). Jump height and hand grip strength were strongly correlated to leg press force (r = 0.82 and 0.59, respectively) and can also be used to identify fallers, although with slightly lower specificity. These results indicate that whole leg extension strength is associated with the ability to prevent a fall after a gait perturbation and might be used to identify the elderly at risk of falling

Worldwide Genetic Variability of the Duffy Binding Protein: Insights into Plasmodium vivax Vaccine Development

The dependence of Plasmodium vivax on invasion mediated by Duffy binding protein (DBP) makes this protein a prime candidate for development of a vaccine. However, the development of a DBP-based vaccine might be hampered by the high variability of the protein ligand (DBPII), known to bias the immune response toward a specific DBP variant. Here, the hypothesis being investigated is that the analysis of the worldwide DBPII sequences will allow us to determine the minimum number of haplotypes (MNH) to be included in a DBP-based vaccine of broad coverage. For that, all DBPII sequences available were compiled and MNH was based on the most frequent nonsynonymous single nucleotide polymorphisms, the majority mapped on B and T cell epitopes. A preliminary analysis of DBPII genetic diversity from eight malaria-endemic countries estimated that a number between two to six DBP haplotypes (17 in total) would target at least 50% of parasite population circulating in each endemic region. Aiming to avoid region-specific haplotypes, we next analyzed the MNH that broadly cover worldwide parasite population. The results demonstrated that seven haplotypes would be required to cover around 60% of DBPII sequences available. Trying to validate these selected haplotypes per country, we found that five out of the eight countries will be covered by the MNH (67% of parasite populations, range 48–84%). In addition, to identify related subgroups of DBPII sequences we used a Bayesian clustering algorithm. The algorithm grouped all DBPII sequences in six populations that were independent of geographic origin, with ancestral populations present in different proportions in each country. In conclusion, in this first attempt to undertake a global analysis about DBPII variability, the results suggest that the development of DBP-based vaccine should consider multi-haplotype strategies; otherwise a putative P. vivax vaccine may not target some parasite populations

The influence of minimum sitting period of the ActivPAL™ on the measurement of breaks in sitting in young children

Sitting time and breaks in sitting influence cardio-metabolic health. New monitors (e.g. activPAL™) may be more accurate for measurement of sitting time and breaks in sitting although how to optimize measurement accuracy is not yet clear. One important issue is the minimum sitting/upright period (MSUP) to define a new posture. Using the activPAL™, we investigated the effect of variations in MSUP on total sitting time and breaks in sitting, and also determined the criterion validity of different activPAL™ settings for both construct

Investigating the metabolic capabilities of Mycobacterium tuberculosis H37Rv using the in silico strain iNJ661 and proposing alternative drug targets

<p>Abstract</p> <p>Background:</p> <p><it>Mycobacterium tuberculosis </it>continues to be a major pathogen in the third world, killing almost 2 million people a year by the most recent estimates. Even in industrialized countries, the emergence of multi-drug resistant (MDR) strains of tuberculosis hails the need to develop additional medications for treatment. Many of the drugs used for treatment of tuberculosis target metabolic enzymes. Genome-scale models can be used for analysis, discovery, and as hypothesis generating tools, which will hopefully assist the rational drug development process. These models need to be able to assimilate data from large datasets and analyze them.</p> <p>Results:</p> <p>We completed a bottom up reconstruction of the metabolic network of <it>Mycobacterium tuberculosis </it>H37Rv. This functional <it>in silico </it>bacterium, <it>iNJ</it>661, contains 661 genes and 939 reactions and can produce many of the complex compounds characteristic to tuberculosis, such as mycolic acids and mycocerosates. We grew this bacterium <it>in silico </it>on various media, analyzed the model in the context of multiple high-throughput data sets, and finally we analyzed the network in an 'unbiased' manner by calculating the Hard Coupled Reaction (HCR) sets, groups of reactions that are forced to operate in unison due to mass conservation and connectivity constraints.</p> <p>Conclusion:</p> <p>Although we observed growth rates comparable to experimental observations (doubling times ranging from about 12 to 24 hours) in different media, comparisons of gene essentiality with experimental data were less encouraging (generally about 55%). The reasons for the often conflicting results were multi-fold, including gene expression variability under different conditions and lack of complete biological knowledge. Some of the inconsistencies between <it>in vitro </it>and <it>in silico </it>or <it>in vivo </it>and <it>in silico </it>results highlight specific loci that are worth further experimental investigations. Finally, by considering the HCR sets in the context of known drug targets for tuberculosis treatment we proposed new alternative, but equivalent drug targets.</p