A user’s guide to golden gate cloning methods and standards

The continual demand for specialized molecular cloning techniques that suit a broad range of applications has driven the development of many different cloning strategies. One method that has gained significant traction is Golden Gate assembly, which achieves hierarchical assembly of DNA parts by utilizing Type IIS restriction enzymes to produce user-specified sticky ends on cut DNA fragments. This technique has been modularized and standardized, and includes different subfamilies of methods, the most widely adopted of which are the MoClo and Golden Braid standards. Moreover, specialized toolboxes tailored to specific applications or organisms are also available. Still, the quantity and range of assembly methods can constitute a barrier to adoption for new users, and even experienced scientists might find it difficult to discern which tools are best suited toward their goals. In this review, we provide a beginner-friendly guide to Golden Gate assembly, compare the different available standards, and detail the specific features and quirks of commonly used toolboxes. We also provide an update on the state-of-the-art in Golden Gate technology, discussing recent advances and challenges to inform existing users and promote standard practices

A colimit decomposition for homotopy algebras in Cat

Badzioch showed that in the category of simplicial sets each homotopy algebra of a Lawvere theory is weakly equivalent to a strict algebra. In seeking to extend this result to other contexts Rosicky observed a key point to be that each homotopy colimit in simplicial sets admits a decomposition into a homotopy sifted colimit of finite coproducts, and asked the author whether a similar decomposition holds in the 2-category of categories Cat. Our purpose in the present paper is to show that this is the case.Comment: Some notation changed; small amount of exposition added in intr

Process evaluation of the Bristol girls dance project

Background: The Bristol Girls Dance Project was a cluster randomised controlled trial that aimed to increase objectively measured moderate-to-vigorous physical activity (MVPA) levels of Year 7 (age 11–12) girls through a dance-based after-school intervention. The intervention was delivered in nine schools and consisted of up to forty after-school dance sessions. This paper reports on the main findings from the detailed process evaluation that was conducted. / Methods: Quantitative and qualitative data were collected from intervention schools. Dose and fidelity were reported by dance instructors at every session. Intervention dose was defined as attending two thirds of sessions and was measured by attendance registers. Fidelity to the intervention manual was reported by dance instructors. On four randomly-selected occasions, participants reported their perceived level of exertion and enjoyment. Reasons for non-attendance were self-reported at the end of the intervention. Semi-structured interviews were conducted with all dance instructors who delivered the intervention (n = 10) and school contacts (n = 9) in intervention schools. A focus group was conducted with girls who participated in each intervention school (n = 9). / Results: The study did not affect girls’ MVPA. An average of 31.7 girls participated in each school, with 9.1 per school receiving the intervention dose. Mean attendance and instructors’ fidelity to the intervention manual decreased over time. The decline in attendance was largely attributed to extraneous factors common to after-school activities. Qualitative data suggest that the training and intervention manual were helpful to most instructors. Participant ratings of session enjoyment were high but perceived exertion was low, however, girls found parts of the intervention challenging. / Conclusions: The intervention was enjoyed by participants. Attendance at the intervention sessions was low but typical of after-school activities. Participants reported that the intervention brought about numerous health and social benefits and improved their dance-based knowledge and skills. The intervention could be improved by reducing the number of girls allowed to participate in each school and providing longer and more in-depth training to those delivering the intervention. / Trial registration: ISRCTN52882523 Registered 25th April 2013

Using self-determination theory to promote adolescent girls' physical activity: Exploring the theoretical fidelity of the Bristol Girls Dance Project

OBJECTIVES: To report the theory-based process evaluation of the Bristol Girls' Dance Project, a cluster-randomised controlled trial to increase adolescent girls' physical activity. DESIGN: A mixed-method process evaluation of the intervention's self-determination theory components comprising lesson observations, post-intervention interviews and focus groups. METHOD: Four intervention dance lessons per dance instructor were observed, audio recorded and rated to estimate the use of need-supportive teaching strategies. Intervention participants (n = 281) reported their dance instructors' provision of autonomy-support. Semi-structured interviews with the dance instructors (n = 10) explored fidelity to the theory and focus groups were conducted with participants (n = 59) in each school to explore their receipt of the intervention and views on the dance instructors' motivating style. RESULTS: Although instructors accepted the theory-based approach, intervention fidelity was variable. Relatedness support was the most commonly observed need-supportive teaching behaviour, provision of structure was moderate and autonomy-support was comparatively low. The qualitative findings identified how instructors supported competence and developed trusting relationships with participants. Fidelity was challenged where autonomy provision was limited to option choices rather than input into the pace or direction of lessons and where controlling teaching styles were adopted, often to manage disruptive behaviour. CONCLUSION: The successes and challenges to achieving theoretical fidelity in the Bristol Girls' Dance Project may help explain the intervention effects and can more broadly inform the design of theory-based complex interventions aimed at increasing young people's physical activity in after-school settings

How do banks assess entrepreneurial competence? The role of voluntary information disclosure

This research adds to the literature on relationship lending in the small business context by discussing the roles of entrepreneurial competence and voluntarily disclosed information as determinants of credit access. More specifically, we propose that the loan manager’s evaluation of the information voluntarily disclosed by the entrepreneur is an important complement to publicly available financial data and soft information collected through observation and third parties in framing the loan manager’s perception of the entrepreneur’s competence. Further, we argue that banks charge lower interest rates if the loan manager perceives the entrepreneur to be competent. Econometric analysis based on 433 bank-firm relationships supports these hypothesised relationships. The results imply that entrepreneurs need to communicate their competence effectively to loan managers, and that banks should utilise their loan managers’ personal evaluations as inputs to lending decisions

Bristol Girls Dance Project: a cluster randomised controlled trial of an after-school dance programme to increase physical activity among 11- to 12-year-old girls

BACKGROUND: Many children do not meet UK physical activity (PA) guidelines. Girls are less active than boys, and the age-related decline in activity is steeper for girls. Dance is the favourite form of PA among adolescent girls in the UK. Participation in after-school dance classes could significantly contribute to girls’ PA. Therefore, after-school dance may be effective for increasing PA levels. OBJECTIVES: To determine the effectiveness and cost-effectiveness of a dance-based intervention to increase the objectively assessed mean weekday minutes of moderate- to vigorous-intensity physical activity (MVPA) of Year 7 girls (11- and 12-year olds) 1 year after baseline measurement. DESIGN:Two-arm cluster randomised controlled trial and economic evaluation. Year 7 girls in participant schools received a ‘taster’ session and were invited to participate. Up to 33 girls per school were able to participate. Schools were randomly assigned (equal numbers) to intervention or control arms. SETTING:A total of 18 mainstream secondary schools across greater Bristol. PARTICIPANTS: Year 7 girls in participating schools who could participate in physical education. INTERVENTION: Nine intervention schools received an after-school dance intervention (40 × 75-minute sessions) underpinned by self-determination theory, which attempts to improve intrinsic motivation for being active, and delivered by external dance instructors. Control schools continued as normal. MAIN OUTCOME MEASURES: The main outcome was accelerometer-assessed mean minutes of MVPA at T2. Measures were assessed at baseline (T0), the end of the intervention (T1) and at T0 + 52 weeks (T2). RESULTS:Baseline MVPA levels were high. A total of 508 girls were included in the primary analysis, which found no difference in weekday MVPA between trial arms. There was no effect on secondary accelerometer outcomes. Data were subjected to a per-protocol analysis and no effect was found. However, at T1, girls who attended dance classes had 4.61 minutes more of MVPA and 14.27 minutes more of light-intensity activity between 15.00 and 17.00 on the days on which they attended intervention sessions. The intervention was inexpensive at £73 per participant (£63 when excluding dance instructor travel) but was not cost-effective owing to the ineffectiveness of the intervention. The European Quality of Life-5 Dimensions Youth survey data were unresponsive to changes in the sample. The process evaluation reported that girls in attendance enjoyed the sessions, that exertion levels were low during sessions and that attendance was low and declined. Fidelity to the session-plan manual was low but theoretical fidelity (to self-determination theory) was good. Qualitative information provides information for improving future interventions. CONCLUSIONS: The intervention was enjoyed by participants. However, there was no difference in the MVPA levels (which were high at baseline) of girls allocated to receive dance compared with girls receiving the control. High baseline MVPA levels indicate that the study appealed to an already active cohort and, therefore, may not have targeted those most in need of an intervention. Dance is an enjoyable activity for adolescent girls and could be further trialled as a means by which to increase PA. Research might consider the impact of dividing the intervention period into smaller blocks. TRIAL REGISTRATION: Current Controlled Trials ISRCTN52882523. FUNDING: The National Institute for Health Research Public Health Research programme. The work was also undertaken with the support of the Centre for the Development and Evaluation of Complex Interventions for Public Health Improvement (DECIPHer), a UK Clinical Research Collaboration (UKCRC) PHR Centre of Excellence. Joint funding (MR/KO232331/1) from the British Heart Foundation, Cancer Research UK, Economic and Social Research Council, Medical Research Council, the Welsh Government and the Wellcome Trust, under the auspices of the UKCRC, is gratefully acknowledged. This study was designed and delivered in collaboration with the Bristol Randomised Trials Collaboration, a UKCRC Registered Clinical Trials Unit in receipt of NIHR Clinical Trials Unit support funding. All intervention costs were funded by the respective councils to which the participant schools were affiliated, namely North Somerset Council, Bristol City Council, and Bath and North East Somerset Council

A Method for Serial Tissue Processing and Parallel Analysis of Aberrant Crypt Morphology, Mucin Depletion, and Beta-Catenin Staining in an Experimental Model of Colon Carcinogenesis

The use of architectural and morphological characteristics of cells for establishing prognostic indicators by which individual pathologies are assigned grade and stage is a well-accepted practice. Advances in automated micro- and macroscopic image acquisition and digital image analysis have created new opportunities in the field of prognostic assessment; but, one area in experimental pathology, animal models for colon cancer, has not taken advantage of these opportunities. This situation is primarily due to the methods available to evaluate the colon of the rodent for the presence of premalignant and malignant pathologies. We report a new method for the excision and processing of the entire colon of the rat and illustrate how this procedure permitted the quantitative assessment of aberrant crypt foci (ACF), a premalignant colon pathology, for characteristics consistent with progression to malignancy. ACF were detected by methylene blue staining and subjected to quantitative morphometric analysis. Colons were then restained with high iron diamine–alcian blue for assessment of mucin depletion using an image overlay to associate morphometric data with mucin depletion. The subsequent evaluation of ACF for beta-catenin staining is also demonstrated. The methods described are particularly relevant to the screening of compounds for cancer chemopreventive activity

Mapping poverty using mobile phone and satellite data

Poverty is one of the most important determinants of adverse health outcomes globally, a major cause of societal instability and one of the largest causes of lost human potential. Traditional approaches to measuring and targeting poverty rely heavily on census data, which in most low- and middle-income countries (LMICs) are unavailable or out-of-date. Alternate measures are needed to comp- lement and update estimates between censuses. This study demonstrates how public and private data sources that are commonly available for LMICs can be used to provide novel insight into the spatial distribution of poverty. We evalu- ate the relative value of modelling three traditional poverty measures using aggregate data from mobile operators and widely available geospatial data. Taken together, models combining these data sources providethebest predictive power (highest r 2 ¼ 0.78) and lowest error, but generally models employing mobile data only yield comparable results, offering the potential to measure poverty more frequently and at finer granularity. Stratifying models into urban and rural areas highlights the advantage of using mobile data in urban areas and different data in different contexts. The findings indicate the possibility to estimate and continually monitor poverty rates at high spatial resolution in countries with limited capacity to support traditional methods of datacollection

Inheritance of an Epigenetic Mark: The CpG DNA Methyltransferase 1 Is Required for De Novo Establishment of a Complex Pattern of Non-CpG Methylation

Site-specific methylation of cytosines is a key epigenetic mark of vertebrate DNA. While a majority of the methylated residues are in the symmetrical (meC)pG:Gp(meC) configuration, a smaller, but significant fraction is found in the CpA, CpT and CpC asymmetric (non-CpG) dinucleotides. CpG methylation is reproducibly maintained by the activity of the DNA methyltransferase 1 (Dnmt1) on the newly replicated hemimethylated substrates (meC)pG:GpC. On the other hand, establishment and hereditary maintenance of non-CpG methylation patterns have not been analyzed in detail. We previously reported the occurrence of site- and allele-specific methylation at both CpG and non-CpG sites. Here we characterize a hereditary complex of non-CpG methylation, with the transgenerational maintenance of three distinct profiles in a constant ratio, associated with extensive CpG methylation. These observations raised the question of the signal leading to the maintenance of the pattern of asymmetric methylation. The complete non-CpG pattern was reinstated at each generation in spite of the fact that the majority of the sperm genomes contained either none or only one methylated non-CpG site. This observation led us to the hypothesis that the stable CpG patterns might act as blueprints for the maintenance of non-CpG DNA methylation. As predicted, non-CpG DNA methylation profiles were abrogated in a mutant lacking Dnmt1, the enzymes responsible for CpG methylation, but not in mutants defective for either Dnmt3a or Dnmt2