Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, which refutes the notion that they are statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cell's ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.

Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time.DOI: http://dx.doi.org/10.7554/eLife.02230.001

Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein–DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

Melatonin is a powerful antioxidant and a synchronizer of many physiological processes. Alteration in melatonin signaling has been reported in a broad range of diseases, but little is known about the genetic variability of this pathway in humans. Here, we sequenced all the genes of the melatonin pathway -AA-NAT, ASMT, MTNR1A, MTNR1B and GPR50 - in 321 individuals from Sweden including 101 patients with attention-deficit/hyperactivity disorder (ADHD) and 220 controls from the general population. We could find several damaging mutations in patients with ADHD, but no significant enrichment compared with the general population. Among these variations, we found a splice site mutation in ASMT (IVS5+2T>C) and one stop mutation in MTNR1A (Y170X) - detected exclusively in patients with ADHD - for which biochemical analyses indicated that they abolish the activity of ASMT and MTNR1A. These genetic and functional results represent the first comprehensive ascertainment of melatonin signaling deficiency in ADHD.

BackgroundGlobally, almost 40% of tuberculosis (TB) patients remain undiagnosed, and those that are diagnosed often experience prolonged delays before initiating correct treatment, leading to ongoing transmission. While there is a push for active case finding (ACF) to improve early detection and treatment of TB, there is extremely limited evidence about the relative cost-effectiveness of different ACF implementation models. Cambodia presents a unique opportunity for addressing this gap in evidence as ACF has been implemented using different models, but no comparisons have been conducted. The objective of our study is to contribute to knowledge and methodology on comparing cost-effectiveness of alternative ACF implementation models from the health service perspective, using programmatic data, in order to inform national policy and practice.MethodsWe retrospectively compared three distinct ACF implementation models - door to door symptom screening in urban slums, checking contacts of TB patients, and door to door symptom screening focusing on rural populations aged above 55 - in terms of the number of new bacteriologically-positive pulmonary TB cases diagnosed and the cost of implementation assuming activities are conducted by the national TB program of Cambodia. We calculated the cost per additional case detected using the alternative ACF models.ResultsOur analysis, which is the first of its kind for TB, revealed that the ACF model based on door to door screening in poor urban areas of Phnom Penh was the most cost-effective (249 USD per case detected, 737 cases diagnosed), followed by the model based on testing contacts of TB patients (308 USD per case detected, 807 cases diagnosed), and symptomatic screening of older rural populations (316 USD per case detected, 397 cases diagnosed).ConclusionsOur study provides new evidence on the relative effectiveness and economics of three implementation models for enhanced TB case finding, in line with calls for data from ‘routine conditions’ to be included in disease control program strategic planning. Such cost-effectiveness comparisons are essential to inform resource allocation decisions of national policy makers in resource constraint settings. We applied a novel, pragmatic methodological approach, which was designed to provide results that are directly relevant to policy makers, costing the interventions from Cambodia’s national TB program’s perspective and using case finding data from implementation activities, rather than experimental settings.

BackgroundChronic hepatitis B infection is a significant cause of morbidity and mortality worldwide; low- and middle-income countries (LMICs) are disproportionately affected. Economic evaluations are a useful decision tool to assess costs versus benefits of hepatitis B virus (HBV) screening. No published study reviewing economic evaluations of HBV screening in LMICs has been undertaken to date.MethodsThe following databases were searched from inception to 21 April 2017: MEDLINE, PubMed, EMBASE, CINAHL Plus, the Cochrane Library, Global Health and the Cost-effectiveness Analysis Registry. English-language studies were included if they assessed the costs against the benefits of HBV screening in LMICs. PROSPERO registration: CRD42015024391, 20 July 2015.ResultsNine studies fulfilled the eligibility criteria. One study from Thailand indicated that adding hepatitis B immunoglobulin (HBIG) to HBV vaccination for newborns following screening of pregnant women might be cost-effective for some LMICs, though inadequate total funding and health infrastructure were likely to limit feasibility. A similar study from China indicated a benefit to cost ratio of 2.7 from selective HBIG administration to newborns, if benefits were considered from a societal perspective. Of the two studies assessing screening amongst the general adult population, a single cost-benefit analysis from China found a benefit to cost ratio (BCR) of 1.73 with vaccination guided by HBV screening of adults aged 21–39, compared to 1.42 with vaccination with no screening, both from a societal perspective. Community-based screening of adults in The Gambia with linkage to treatment yielded an incremental cost per disability-adjusted life year averted of $566 (in 2017 USD), less than two-times gross domestic product per capita for that country.ConclusionsScreening with ‘catch-up’ vaccination for younger adults yielded benefits above costs, and screening linked with treatment has shown cost-effectiveness that may be affordable for some LMICs. However, interpretation needs to account for total cost implications and further research in LMICs is warranted as there were only nine included studies and evidence from high-income countries is not always directly applicable.Electronic supplementary materialThe online version of this article (10.1186/s12889-018-5261-8) contains supplementary material, which is available to authorized users.

Background Family planning (FP) has the potential to improve maternal and child health outcomes and to reduce poverty in sub-Saharan Africa. However, substantial unmet need for modern contraceptive methods (MCMs) persists in this region. Current literature highlights multi-level barriers, including socio-cultural norms that discourage the use of MCMs. This paper explores women’s choices and decision-making around MCM use and examines whether integrating FP services with childhood immunisations influenced women’s perceptions of, and decision to use, an MCM. Methods 94 semi-structured interviews and 21 focus group discussions with women, health providers, and community members (N = 253) were conducted in health facilities and outreach clinics where an intervention was delivering integrated FP and childhood immunisation services in Benin, Ethiopia, Kenya, Malawi and Uganda. Data were coded using Nvivo software and an analytical framework was developed to support interpretative and thematic analyses on women’s decision-making about MCM use. Results Most women shared the reproductive desire to space or limit births because of the perceived benefits of improved health and welfare for themselves and for their children, including the economic advantages. For some, choices about MCM use were restricted because of wider societal influences. Women’s decision to use MCMs was driven by their reproductive desires, but for some that was stymied by fears of side effects, community stigma, and disapproving husbands, which led to clandestine MCM use. Health providers acknowledged that women understood the benefits of using MCMs, but highlighted that the wider socio-cultural norms of their community often contributed to a reluctance to use them. Integration of FP and childhood immunisation services provided repeat opportunities for health providers to counter misinformation and it improved access to MCMs, including for women who needed to use them covertly. Conclusions Some women chose to use MCMs without the approval of their husbands, and/or despite cultural norms, because of the perceived health and economic benefits for themselves and for their families, and because they lived with the consequences of short birth intervals and large families. Integrated FP and childhood immunisation services expanded women’s choices about MCM use and created opportunities for women to make decisions autonomously.

With funding for tuberculosis (TB) research decreasing, and the high global disease burden persisting, there are calls for increased investment in TB research. However, justification of such investments is questionable, when translation of research outputs into policy and health care improvements remains a challenge for TB and other diseases. Using TB in Cambodia as a case study, we investigate how evidence needs of national policy makers are addressed by topics covered in research publications. We first conducted a systematic review to compile all studies on TB in Cambodia published since 2000. We then identified priority areas in which evidence for policy and programme planning are required from the perspective of key national TB control stakeholders. Finally, results from the literature review were analysed in relation to the priority research areas for national policy makers to assess overlap and highlight gaps in evidence. Priority research areas were: TB-HIV co-infection; childhood TB; multidrug resistant TB (MDR-TB); and universal and equitable access to quality diagnosis and treatment. On screening 1687 unique papers retrieved from our literature search, 253 were eligible publications focusing on TB in Cambodia. Of these, only 73 (29%) addressed one of the four priority research areas. Overall, 30 (11%), five (2%), seven (2%) and 37 (14%) studies reported findings relevant to TB-HIV, childhood TB, MDR-TB and access to quality diagnosis and treatment respectively. Our analysis shows that a small proportion of the research outputs in Cambodia address priority areas for informing policy and programme planning. This case study illustrates that there is substantial room for improvement in alignment between research outputs and evidence gaps that national policy makers would like to see addressed; better coordination between researchers, funders and policy makers' on identifying priority research topics may increase the relevance of research findings to health policies and programmes.