Boxed interim evaluation report March 2018

BoxED is an outreach and widening participation initiative designed, developed and delivered by members of the Faculty of Health and Applied Sciences at the University of the West of England (UWE), Bristol. The scheme aims to provide inspirational and engaging workshops to secondary school pupils in the local area, particularly targeting widening participation and attainment in order to encourage a wide range of young people into higher education. The following report documents the key findings of an evaluation undertaken in conjunction with the Centre for Research in Biosciences (CRIB) and the Science Communication Unit (SCU), UWE assessing the levels of satisfaction with the service and the breadth of science capital in school children aged 11-16 years in the Bristol area. The evaluation examined BoxED activities between October 2017 and March 2018. Post-activity questionnaires and semi-structured interviews were employed to gauge opinions from a total of 376 school pupils and 2 school teachers. This evaluation forms part of a wider series of BoxED evaluation activities including additional questionnaires, observations and teachers interviews which are currently ongoing

Development and validation of a high throughput, closed tube method for the determination of haemoglobin alpha gene (HBA1 and HBA2) numbers by gene ratio assay copy enumeration-PCR (GRACE-PCR)

© 2015 Turner et al. Background: Deletions of the aα-globin genes are the most common genetic abnormalities in the world. Currently multiplex Gap-PCRs are frequently used to identify specific sets of common deletions. However, these assays require significant post-amplification hands on time and cannot be used to identify novel or unexpected deletions. The aim of the current study was to develop a rapid screening test for the detection of all deletions of the aα-globin genes that can be integrated into a high volume clinical laboratory workflow. Methods: A gene ratio assay copy enumeration (GRACE) PCR method was developed by simultaneous amplification of targets in the aα-globin genes (HBA1 and HBA2) and the chloride channel voltage sensitive 7 (CLCN7) reference gene. A novel application of High Resolution Melting (HRM) analysis then allowed rapid determination of aα-globin gene copy numbers. The assay was validated using 105 samples with previously determined and 62 samples with unknown aα-globin genotypes. Results: The GRACE-PCR assay detected abnormal aα-globin gene copy numbers in 108 of the 167 samples evaluated. The results were consistent with those from a commercial reverse hybridization assay and no allele drop out was observed. Conclusions: We have successfully developed and validated a GRACE-PCR screening test for the detection of deletions and duplications of the aα-globin genes. The assay is based on copy number determination and has the ability to detect both known and novel deletions of the aα-globin genes. It is a closed tube technique; consequently the risk of amplicon contamination is negligible. Amplification, detection and analysis can be completed within one hour, making it faster, cheaper and simpler than other existing tests and thus well suited as a rapid first step in a clinical laboratory workflow

Dense core secretory vesicles revealed as a dynamic Ca2+ store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera

The role of dense core secretory vesicles in the control of cytosolic-free Ca2+ concentrations ([Ca2+]c) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2–synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet β-cells: (a) increases in [Ca2+]c cause a prompt increase in intravesicular-free Ca2+ concentration ([Ca2+]SV), which is mediated by a P-type Ca2+-ATPase distinct from the sarco(endo) plasmic reticulum Ca2+-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca2+ pumps; (b) steady state Ca2+ concentrations are 3–5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca2+; (c) inositol (1,4,5) trisphosphate has no impact on [Ca2+]SV in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca2+]SV. Thus, secretory vesicles represent a dynamic Ca2+ store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca2+-induced Ca2+ release from vesicles docked at the plasma membrane could participate in triggering exocytosis

Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR)

© 2016 The Author(s). Objectives: Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements. Methods: The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples. Results: A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %. Conclusion: We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts

Myosin Va Transports Dense Core Secretory Vesicles in Pancreatic MIN6 β-Cells

The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic β-cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative–acting globular tail domain of MyoVa decreased by ∼50% the number of vesicles docked at the plasma membrane and by 87% the number of depolarization-stimulated exocytotic events detected by total internal reflection fluorescence microscopy. We conclude that MyoVa-driven movements of vesicles along the cortical actin network are essential for the terminal stages of regulated exocytosis in β-cells

Effectiveness of the human oral microbe identification microarray in identifying periodontal pathogens: A Systematic review

Objective: Investigating the effectiveness of the Human Oral Microbe Identification Microarray (HOMIM) in identifying and quantifying bacterial species of the oral microbiome in periodontal disease. Methods: Searches were conducted in CENTRAL, CINAHL, MEDLINE, EMBASE. Articles selected reported human observational studies in adults between 18 and 65 years, presenting specific predefined keywords. Articles were initially screened by title and abstract, then analysed for methodology quality using a detailed checklist. Data was extracted and reported using the PRISMA tool and risk of bias was evaluated using the Cochrane Risk of Bias tool. Results: Initial search retrieved 2931 articles of which 51 were selected by title and abstract. After full-text reading, 8 articles met the inclusion criteria, out of which 3 were classified as low, 3 as moderate and 2 as high methodological quality. The Human Oral Microbe Identification Microarray assay, compared to earlier techniques including Denaturing Gradient Gel Electrophoresis HOMIM represents a more effective approach for quantification due to its high sensitivity, highlighting major shifts between periodontal health and disease. Conclusions: The literature revealed moderate evidence that HOMIM is more effective in identifying and quantifying periodontal pathogens, compared to earlier molecular and non-molecular methods and culture-based approach with phenotypic tests

Additional file 1: of Development and validation of a high throughput, closed tube method for the determination of haemoglobin alpha gene (HBA1 and HBA2) numbers by gene ratio assay copy enumeration-PCR (GRACE-PCR)

Alternative GRACE-PCR primers. (DOCX 918 kb

3D scaffolds in the treatment of diabetic foot ulcers: New trends vs conventional approaches

Diabetic foot ulcer (DFU) is a serious complication of diabetes mellitus, affecting roughly 25% of diabetic patients and resulting in lower limb amputation in over 70% of known cases. In addition to the devastating physiological consequences of DFU and its impact on patient quality of life, DFU has significant clinical and economic implications. Various traditional therapies are implemented to effectively treat DFU. However, emerging technologies such as bioprinting and electrospinning, present an exciting opportunity to improve current treatment strategies through the development of 3D scaffolds, by overcoming the limitations of current wound healing strategies. This review provides a summary on (i) current prevention and treatment strategies available for DFU; (ii) methods of fabrication of 3D scaffolds relevant for this condition; (iii) suitable materials and commonly used molecules for the treatment of DFU; and (iv) future directions offered by emerging technologies

Involvement of conventional kinesin in glucose-stimulate secretory granule movements and exocytosis in clonal pancreatic β-cells

Recruitment of secretory vesicles to the cell surface is essential for the sustained secretion of insulin in response to glucose. At present, the molecular motors involved in this movement, and the mechanisms whereby they may be regulated, are undefined. To investigate the role of kinesin family members, we labelled densecore vesicles in clonal β-cells using an adenovirally expressed, vesicle-targeted green fluorescent protein (phogrin.EGFP), and employed immunoadsorption to obtain highly purified insulin-containing vesicles. Whereas several kinesin family members were expressed in this cell type, only conventional kinesin heavy chain (KHC) was detected in vesicle preparations. Expression of a dominant-negative KHC motor domain (KHCmut) blocked all vesicular movements with velocity >0.4 μm second-1, which demonstrates that kinesin activity was essential for vesicle motility in live β-cells. Moreover, expression of KHCmut strongly inhibited the sustained, but not acute, stimulation of secretion by glucose. Finally, vesicle movement was stimulated by ATP dose-dependently in permeabilized cells, which suggests that glucose-induced increases in cytosolic [ATP] mediate the effects of the sugar in vivo, by enhancing kinesin activity. These data therefore provide evidence for a novel mechanism whereby glucose may enhance insulin release