Search

Background: School readiness is a measure of a child's cognitive, social, and emotional readiness to begin formal schooling. Children with low school readiness need additional support from schools for learning, developing required social and academic skills, and catching-up with their school-ready peers. This study aims to identify the most significant risk factors associated with low school readiness using linked routine data for children in Wales. Method: This was a longitudinal cohort study using linked data. The cohort comprises of children who completed the Foundation Phase assessment between 2012 and 2018. Individuals were identified by linking Welsh Demographic Service and Pre16 Education Attainment datasets. School readiness was assessed via the binary outcome of the Foundation Phase assessment (achieved/not achieved). This study used multivariable logistic regression model and a decision tree to identify and weight the most important risk factors associated with low school readiness. Results: In order of importance, logistic regression identified maternal learning difficulties (adjusted odds ratio 5.35(95% confidence interval 3.97–7.22)), childhood epilepsy (2.95(2.39–3.66)), very low birth weight (2.24(1.86–2.70), being a boy (2.11(2.04–2.19)), being on free school meals (1.85(1.78–1.93)), living in the most deprived areas (1.67(1.57–1.77)), maternal death (1.47(1.09–1.98)), and maternal diabetes (1.46(1.23–1.78)) as factors associated with low school readiness. Using a decision tree, eligibility for free school meals, being a boy, absence/low attendance at school, being born late in the academic year, being a low birthweight child, and not being breastfed were factors which were associated with low school readiness. Conclusion: This work suggests that public health interventions focusing on children who are: boys, living in deprived areas, have poor early years attendance, have parents with learning difficulties, have parents with an illness or have illnesses themselves, would make the most difference to school readiness in the population. [ABSTRACT FROM AUTHOR]

Background: Researchers have a responsibility to protect all participants, especially vulnerable participants, from harm. Vulnerability is increasingly understood to be context specific, yet limited guidance is available regarding the vulnerability and agency of research participants in different cultural settings. This study aims to explore research participants' daily vulnerability and agency, and how these interact with participants' research experiences in their own words. Researchers' views and responses were also explored. Methods: A qualitative study was conducted around two scrub typhus research studies in northern Thailand. A thematic analysis was carried out on 42 semi-structured interviews with research participants, their families, researchers and key informants. Results: The majority of the research participants belonged to a hill tribe ethnic minority group. Common challenges were related to Thai language barriers, travel difficulties, uncertain legal status, unstable employment, lack of education and healthcare. We did not identify new vulnerabilities but we found that the extent of these vulnerabilities might be underestimated or even hidden from researchers in some cases. Despite these challenges people demonstrated agency in their daily lives and were often motivated and supported in this by family members. The majority of perceived research benefits were related to healthcare and gaining knowledge, while attending follow-up visits could be a burden for some. Conclusions: Our approach to research in culturally and socioeconomically diverse settings should be more responsive to participants' specific vulnerabilities and abilities evidenced in their daily life, rather than attributing vulnerability on the basis of membership of pre-defined 'vulnerable groups'. Researchers need to be aware and responsive towards the challenges participants face locally in order to minimise the burdens of research participation whilst allowing participants to benefit from research. [ABSTRACT FROM AUTHOR]

Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways. [ABSTRACT FROM AUTHOR]

The Prenatal Assessment of Genome and Exomes (PAGE) project is a UK-wide study aiming to gain a better understanding of genetic variants causing developmental problems during pregnancy. A further aim of the study is to provide an evidence-base for the introduction of prenatal whole genome and exome sequencing (PWGES) into prenatal diagnostics provided by the NHS, which is expected in 2018. This paper presents the findings of a qualitative interview study undertaken with 20 health professionals and researchers involved in the PAGE project, and explores their implications for understandings of ‘good practice’ in the uses of prenatal genomics clinically. A number of critical issues are identified that will need to be addressed in the development of a model of good ethical practice for prenatal genomics: consent, management of expectations, return of results, and professional duties in the context of PWGES. The analysis presented identifies and illustrates a great deal of complexity and qualitative richness in these issues as they arise in the day-to-day work of genomics professionals. Inclusive, critical discussion of these findings, together with the findings from other empirical studies, normative analysis and scientific discoveries resulting from PAGE, will be required to inform the development of appropriate guidelines of good ethical practice that address the needs and concerns to be encountered in daily clinical practice. [ABSTRACT FROM AUTHOR]

Objective: To explore Cambodian community members’ understanding of and attitudes towards healthcare research. Design: This qualitative study generated data from semi-structured interviews and focus group discussions. This study was conducted at a non-governmental paediatric hospital and in nearby villages in Siem Reap province, Cambodia. A total of ten semi-structured interviews and four focus group discussions were conducted, involving 27 participants. Iterative data collection and analysis were performed concurrently. Data were analysed by thematic content analysis and the coding structure was developed using relevant literature. Results: Participants did not have a clear understanding of what activities related to research compared with those for routine healthcare. Key attitudes towards research were responsibility and trust: personal (trust of the researcher directly) and institutional (trust of the institution as a whole). Villagers believe the village headman holds responsibility for community activities, while the village headman believes that this responsibility should be shared across all levels of the government system. Conclusions: It is essential for researchers to understand the structure and relationship within the community they wish to work with in order to develop trust among community participants. This aids effective communication and understanding among all parties, enabling high quality ethical research to be conducted. [ABSTRACT FROM AUTHOR]

Photoproteins are responsible for light emission in a variety of marine ctenophores and coelenterates. The mechanism of light emission in both families occurs via the same reaction. However, the arrangement of amino acid residues surrounding the chromophore, and the catalytic mechanism of light emission is unknown for the ctenophore photoproteins. In this study, we used quantum mechanics/molecular mechanics (QM/MM) and site-directed mutagenesis studies to investigate the details of the catalytic mechanism in berovin, a member of the ctenophore family. In the absence of a crystal structure of the berovin-substrate complex, molecular docking was used to determine the binding mode of the protonated (2-hydroperoxy) and deprotonated (2-peroxy anion) forms of the substrate to berovin. A total of 13 mutants predicted to surround the binding site were targeted by site-directed mutagenesis which revealed their relative importance in substrate binding and catalysis. Molecular dynamics simulations and MM-PBSA (Molecular Mechanics Poisson-Boltzmann/surface area) calculations showed that electrostatic and polar solvation energy are +115.65 and -100.42 kcal/mol in the deprotonated form, respectively. QM/MM calculations and pKa analysis revealed the deprotonated form of substrate is unstable due to the generation of a dioxetane intermediate caused by nucleophilic attack of the substrate peroxy anion at its C3 position. This work also revealed that a hydrogen bonding network formed by a D158- R41-Y204 triad could be responsible for shuttling the proton from the 2- hydroperoxy group of the substrate to bulk solvent. [ABSTRACT FROM AUTHOR]

There has been a dramatic rise in the scale and scope of collaborative global health research. A number of structural and scientific factors explain this growth and there has been much discussion of these in the literature. Little, if any, attention has been paid, however, to the factors identified by scientists and other research actors as important to successful research collaboration. This is surprising given that their decisions are likely to play a key role in the sustainability and effectiveness of global health research initiatives. In this paper, we report on qualitative research with leading scientists involved in major international research collaborations about their views on good and bad collaborations and the factors that inform their decision-making about joining and participating actively in research networks. We identify and discuss eight factors that researchers see as essential in judging the merits of active participation in global health research collaborations: opportunities for active involvement in cutting-edge, interesting science; effective leadership; competence of potential partners in and commitment to good scientific practice; capacity building; respect for the needs, interests and agendas of partners; opportunities for discussion and disagreement; trust and confidence; and, justice and fairness in collaboration. Our findings suggest that the sustainability and effectiveness of global health research collaborations has an important ethical or moral dimension for the research actors involved. [ABSTRACT FROM AUTHOR]

Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions. [ABSTRACT FROM AUTHOR]

Background: There is increasing recognition of the importance of sharing research data within the international scientific community, but also of the ethical and social challenges this presents, particularly in the context of structural inequities and varied capacity in international research. Public involvement is essential to building locally responsive research policies, including on data sharing, but little research has involved stakeholders from low-to-middle income countries. Methods: Between January and June 2014, a qualitative study was conducted in Kenya involving sixty stakeholders with varying experiences of research in a deliberative process to explore views on benefits and challenges in research data sharing. In-depth interviews and extended small group discussions based on information sharing and facilitated debate were used to collect data. Data were analysed using Framework Analysis, and charting flow and dynamics in debates. Findings: The findings highlight both the opportunities and challenges of communicating about this complex and relatively novel topic for many stakeholders. For more and less research-experienced stakeholders, ethical research data sharing is likely to rest on the development and implementation of appropriate trust-building processes, linked to local perceptions of benefits and challenges. The central nature of trust is underpinned by uncertainties around who might request what data, for what purpose and when. Key benefits perceived in this consultation were concerned with the promotion of public health through science, with legitimate beneficiaries defined differently by different groups. Important challenges were risks to the interests of study participants, communities and originating researchers through stigmatisation, loss of privacy, impacting autonomy and unfair competition, including through forms of intentional and unintentional 'misuse' of data. Risks were also seen for science. Discussion: Given background structural inequities in much international research, building trust in this low-to-middle income setting includes ensuring that the interests of study participants, primary communities and originating researchers will be promoted as far as possible, as well as protected. Important ways of building trust in data sharing include involving the public in policy development and implementation, promoting scientific collaborations around data sharing and building close partnerships between researchers and government health authorities to provide checks and balances on data sharing, and promote near and long-term translational benefits. [ABSTRACT FROM AUTHOR]

Consent processes have attracted significant research attention over the last decade, including in the global south. Although relevant studies suggest consent is a complex negotiated process involving multiple actors, most guidelines assume consent is a one-off encounter with a clear ‘yes’ or ‘no’ decision. In this paper we explore the concept of ‘silent refusals’, a situation where it is not clear whether potential participants want to join studies or those in studies want to withdraw from research, as they were not actively saying no. We draw on participant observation, in-depth interviews and group discussions conducted with a range of stakeholders in two large community based studies conducted by the KEMRI Wellcome Trust programme in coastal Kenya. We identified three broad inter-related rationales for silent refusals: 1) a strategy to avoid conflicts and safeguard relations within households, - for young women in particular—to appear to conform to the wishes of elders; 2) an approach to maintain friendly, appreciative and reciprocal relationships with fieldworkers, and the broader research programme; and 3) an effort to retain study benefits, either for individuals, whole households or wider communities. That refusals and underlying rationales were silent posed multiple dilemmas for fieldworkers, who are increasingly recognised to play a key interface role between researchers and communities in many settings. Silent refusals reflect and reinforce complex power relations embedded in decisions about research participation, with important implications for consent processes and broader research ethics practice. Fieldworkers need support to reflect upon and respond to the ethically charged environment they work in. [ABSTRACT FROM AUTHOR]

Mammalian HtrA (high temperature requirement factor A) proteases, comprising 4 multi-domain members HtrA1-4, play important roles in a number of normal cellular processes as well as pathological conditions such as cancer, arthritis, neurodegenerative diseases and pregnancy disorders. However, how HtrA activities are regulated is not well understood, and to date no inhibitors specific to individual HtrA proteins have been identified. Here we investigated five HtrA3 monoclonal antibodies (mAbs) that we have previously produced, and demonstrated that two of them regulated HtrA3 activity in an opposing fashion: one inhibited while the other stimulated. The inhibitory mAb also blocked HtrA3 activity in trophoblast cells and enhanced migration and invasion, confirming its potential in vivo utility. To understand how the binding of these mAbs modulated HtrA3 protease activity, their epitopes were visualized in relation to a 3-dimensional HtrA3 homology model. This model suggests that the inhibitory HtrA3 mAb blocks substrate access to the protease catalytic site, whereas the stimulatory mAb may bind to the PDZ domain alone or in combination with the N-terminal and protease domains. Since HtrA1, HtrA3 and HtrA4 share identical domain organization, our results establish important foundations for developing potential therapeutics to target these HtrA proteins specifically for the treatment of a number of diseases, including cancer and pregnancy disorders. [ABSTRACT FROM AUTHOR]

Uterine proprotein convertase (PC) 6 plays a critical role in embryo implantation and is pivotal for pregnancy establishment. Inhibition of PC6 may provide a novel approach for the development of non-hormonal and female-controlled contraceptives. We investigated a class of five synthetic non-peptidic small molecule compounds that were previously reported as potent inhibitors of furin, another PC member. We examined (i) the potency of these compounds in inhibiting PC6 activity in vitro; (ii) their binding modes in the PC6 active site in silico; (iii) their efficacy in inhibiting PC6-dependent cellular processes essential for embryo implantation using human cell-based models. All five compounds showed potent inhibition of PC6 activity in vitro, and in silico docking demonstrated that these inhibitors could adopt a similar binding mode in the PC6 active site. However, when these compounds were tested for their inhibition of decidualization of primary human endometrial stromal cells, a PC6-dependent cellular process critical for embryo implantation, only one (compound 1o) showed potent inhibition. The lack of activity in the cell-based assay may reflect the inability of the compounds to penetrate the cell membrane. Because compound's lipophilicity is linked to cell penetration, a measurement of lipophilicity (logP) was calculated for each compound. Compound 1o is unique as it appears the most lipophilic among the five compounds. Compound 1o also inhibited another crucial PC6-dependent process, the attachment of human trophoblast spheroids to endometrial epithelial cells (a model for human embryo attachment). We thus identified compound 1o as a potent small molecule PC6 inhibitor with pharmaceutical potential to inhibit embryo implantation. Our findings also highlight that human cell-based functional models are vital to complement the biochemical and in silico analyses in the selection of promising drug candidates. Further investigations for compound 1o are warranted in animal models to test its utility as an implantation-inhibiting contraceptive drug. [ABSTRACT FROM AUTHOR]

We report the rational design and in vivo testing of mosaic proteins for a polyvalent pan-filoviral vaccine using a computational strategy designed for the Human Immunodeficiency Virus type 1 (HIV-1) but also appropriate for Hepatitis C virus (HCV) and potentially other diverse viruses. Mosaics are sets of artificial recombinant proteins that are based on natural proteins. The recombinants are computationally selected using a genetic algorithm to optimize the coverage of potential cytotoxic T lymphocyte (CTL) epitopes. Because evolutionary history differs markedly between HIV-1 and filoviruses, we devised an adapted computational technique that is effective for sparsely sampled taxa; our first significant result is that the mosaic technique is effective in creating high-quality mosaic filovirus proteins. The resulting coverage of potential epitopes across filovirus species is superior to coverage by any natural variants, including current vaccine strains with demonstrated cross-reactivity. The mosaic cocktails are also robust: mosaics substantially outperformed natural strains when computationally tested against poorly sampled species and more variable genes. Furthermore, in a computational comparison of cross-reactive potential a design constructed prior to the Bundibugyo outbreak performed nearly as well against all species as an updated design that included Bundibugyo. These points suggest that the mosaic designs would be more resilient than natural-variant vaccines against future Ebola outbreaks dominated by novel viral variants. We demonstrate in vivo immunogenicity and protection against a heterologous challenge in a mouse model. This design work delineates the likely requirements and limitations on broadly-protective filoviral CTL vaccines. [ABSTRACT FROM AUTHOR]

The TRIM family of proteins is distinguished by its tripartite motif (TRIM). Typically, TRIM proteins contain a RING finger domain, one or two B-box domains, a coiled-coil domain and the more variable C-terminal domains. TRIM16 does not have a RING domain but does harbour two B-box domains. Here we showed that TRIM16 homodimerized through its coiled-coil domain and heterodimerized with other TRIM family members; TRIM24, Promyelocytic leukaemia (PML) protein and Midline- 1 (MID1). Although, TRIM16 has no classic RING domain, three-dimensional modelling of TRIM16 suggested that its B-box domains adopts RING-like folds leading to the hypothesis that TRIM16 acts as an ubiquitin ligase. Consistent with this hypothesis, we demonstrated that TRIM16, devoid of a classical RING domain had auto-polyubiquitination activity and acted as an E3 ubiquitin ligase in vivo and in vitro assays. Thus via its unique structure, TRIM16 possesses both heterodimerization function with other TRIM proteins and also has E3 ubiquitin ligase activity. [ABSTRACT FROM AUTHOR]

Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a kcat = 22 s-1, KMPYR = 2.55 ± 0.05 mM and KMASA = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (TMapp) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (TMapp = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a KD4→2 = 1.7 nM, which is considerably tighter than its E. coli ortholog (KD4→2 = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å2) compared to its E. coli counterpart (500 Å2). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target. [ABSTRACT FROM AUTHOR]

Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a kcat = 22 s-1, KMPYR = 2.55 ± 0.05 mM and KMASA = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (TMapp) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (TMapp = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a KD4→2 = 1.7 nM, which is considerably tighter than its E. coli ortholog (KD4→2 = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å2) compared to its E. coli counterpart (500 Å2). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target. [ABSTRACT FROM AUTHOR]

Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a k(cat) = 22 s(-1), K(M)(PYR) = 2.55 ± 0.05 mM and K(M)(ASA) = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (T(M)(app)) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (T(M)(app) = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a K(D)(4→2) = 1.7 nM, which is considerably tighter than its E. coli ortholog (K(D)(4→2) = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å(2)) compared to its E. coli counterpart (500 Å(2)). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target.