Phylogeny and Origin of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency Mutations in Indonesia

The aim of this study is to analyze the relationship between the types of G6PD mutations found in Indonesia and the relationships of mutations found in Indonesia to those found in other countries. We summarize the distribution of G6PDs in West Indonesia and East Indonesia. Moreover, we use bioinformatics methods to construct phylogenetic trees and compare the sequences containing the regions amplifi ed by the commonly used PCR primer pairs. Previous work has shown that Mediterranean G6PD and Chinese CoimbraG6PDare distributed in West Indonesia, whilst G6PD mutations in East Indonesia are Jammu/ViangchanG6PD and Chinese Gaohe G6PD. G6PD Jammu/Viangchan was mostly distributed in Flores Island, East Indonesia along with G6PDGaohe. We constructed phylogenetic trees using the G6PD sequences from various regions in Indonesia and other countries. It appears from phylogenetic trees and percentages of identity that FloresIndonesian G6PD defi ciency (Jammu/Viangchan G6PD, originating in India) is 92.5% identical to the G6PD defi ciency of Chinese origin (GaoheG6PD). It was interesting to note that the genetic region containing the Javanese Indonesian G6PD defi ciency (MediterraneanG6PD, fi rst found in Italy) located in the western parts of Indonesia is closely related (99% identity) to the Chinese G6PD defi ciency(Coimbra G6PD). We concludethat G6PD mutations in West Indonesia are closely related to G6PD mutations from China. G6PD mutations in East Indonesia are also closely related to G6PD mutations from India and China, but more distantly, and to different types to those in West Indonesia. A prediction of protein structure was carried out which allowed visualization of the locations of mutation on the three dimensional structure of G6PD. Key words: G6PD, phylogeny, origin, genetic mutation

The second gamma-H2AX assay inter-comparison exercise carried out in the framework of the European biodosimetry network (RENEB)

Purpose: Within the EU RENEB project, seven laboratories have taken part in training and harmonisation activities to strengthen triage gamma H2AX-based radiation exposure assessment. This has culminated in a second triage biodosimetry exercise. Materials and methods: Whole blood and separated lymphocyte samples were homogenously irradiated with 60Co gamma rays at 0.5, 2.5 (blind samples), 0 and 2 Gy (reference samples). Following post-exposure incubations of 4 and 24 h, 16 samples were shipped on ice packs to each partner. The samples were stained and scored for gamma-H2AX foci, using manual and/or automated fluorescence microscope scoring strategies. Dose estimates were obtained and used to assign triage categories to the samples. Results: Average dose estimates across all the laboratories correlated well with true doses. The most accurate assignment of triage category was achieved by manual scoring of the 4-h blood and lymphocyte samples. Only three samples out of a total of 46 were miscategorized in a way that could have adversely effected the clinical management of a radiation casualty. Conclusions: This inter-comparison exercise has demonstrated that following a recent acute radiation exposure, the gamma-H2AX assay could be a useful triage tool that can be successfully applied across a network of laboratories

The Effectiveness of the precision fluency shaping program controlling stuttering behaviour in adults

This study was done to test the effectiveness of the Precision Fluency Shaping Program in controlling stuttering behaviour in adults. Two sites were chosen, each using the Precision Fluency Shaping Program to treat stuttering. At each clinic, a Speech Patbologist made a random selection of the subjects' pre- and post-therapy video-taped interviews, totalling 20 in all. During the interviews, the clients were asked questions and re~d a short passage to determine the frequency of stuttering in natural conversation and in reading. Perceptions of Stuttering Inventory questionnaires vvere also filled in before and after therapy. Two judges were trained to identify stuttering behaviour, and were given an inter-rater reliability test at selected intervals throughout the study. Protocols",:m.a;d;6 of each interview tape, were scored for (a) stuttering behaviour and (b) words spoken or read. An Analysis of Variance Repeated Measures Test was used to compare before and after scores of conversations, readings, and Perceptions of Stuttering Inventory to determine whether the Precision Fluency Shaping Program controlled stuttering behaviour significantly. A Pearson R Correlation Test was also administered to determine if a relationship existed bet\veen Perceptions of Stuttering Inventory and (i) conversation and (ii) reading scores

Phylogeny and Origin of Glucose-6-Phosphate Dehydrogenase (G6PD) Defi ciency Mutations in Indonesia

The aim of this study is to analyze the relationship between the types of G6PD mutations found in Indonesia and the relationships of mutations found in Indonesia to those found in other countries. We summarize the distribution of G6PDs in West Indonesia and East Indonesia. Moreover, we use bioinformatics methods to construct phylogenetic trees and compare the sequences containing the regions amplifi ed by the commonly used PCR primer pairs. Previous work has shown that Mediterranean G6PD and Chinese CoimbraG6PDare distributed in West Indonesia, whilst G6PD mutations in East Indonesia are Jammu/ViangchanG6PD and Chinese Gaohe G6PD. G6PD Jammu/Viangchan was mostly distributed in Flores Island, East Indonesia along with G6PDGaohe. We constructed phylogenetic trees using the G6PD sequences from various regions in Indonesia and other countries. It appears from phylogenetic trees and percentages of identity that FloresIndonesian G6PD defi ciency (Jammu/Viangchan G6PD, originating in India) is 92.5% identical to the G6PD defi ciency of Chinese origin (GaoheG6PD). It was interesting to note that the genetic region containing the Javanese Indonesian G6PD defi ciency (MediterraneanG6PD, fi rst found in Italy) located in the western parts of Indonesia is closely related (99% identity) to the Chinese G6PD defi ciency(Coimbra G6PD). We concludethat G6PD mutations in West Indonesia are closely related to G6PD mutations from China. G6PD mutations in East Indonesia are also closely related to G6PD mutations from India and China, but more distantly, and to different types to those in West Indonesia. A prediction of protein structure was carried out which allowed visualization of the locations of mutation on the three dimensional structure of G6PD.Key words: G6PD, phylogeny, origin, genetic mutations</div

iPSC-Derived Vascular Cell Spheroids as Building Blocks for Scaffold-Free Biofabrication

Recently a protocol is established to obtain large quantities of human induced pluripotent stem cells (iPSC)-derived endothelial progenitors, called endothelial colony forming cells (ECFC), and of candidate smooth-muscle forming cells (SMFC). Here, the suitability for assembling in spheroids, and in larger 3D cell constructs is tested. iPSC-derived ECFC and SMFC are labeled with tdTomato and eGFP, respectively. Spheroids are formed in ultra-low adhesive wells, and their dynamic proprieties are studied by time-lapse microscopy, or by confocal microscopy. Spheroids are also tested for fusion ability either in the wells, or assembled on the Regenova 3D bioprinter which laces them in stainless steel micro-needles (the “Kenzan” method). It is found that both ECFC and SMFC formed spheroids in about 24 h. Fluorescence monitoring indicated a continuous compaction of ECFC spheroids, but stabilization in those prepared from SMFC. In mixed spheroids, the cell distribution changed continuously, with ECFC relocating to the core, and showing pre-vascular organization. All spheroids have the ability of in-well fusion, but only those containing SMFC are robust enough to sustain assembling in tubular structures. In these constructs a layered distribution of alpha smooth muscle actin-positive cells and extracellular matrix deposition is found. In conclusion, iPSC-derived vascular cell spheroids represent a promising new cellular material for scaffold-free biofabrication

Enhancement of Adeno-Associated Virus-Mediated Gene Therapy Using Hydroxychloroquine in Murine and Human Tissues.

The therapeutic effects of gene therapy using adeno-associated virus (AAV) vectors are dependent on the efficacy of viral transduction. Currently, we have reached the safe limits of AAV vector dose, beyond which damaging inflammatory responses are seen. To improve the efficacy of AAV transduction, we treated mouse embryonic fibroblasts, primate retinal pigment epithelial cells, and human retinal explants with hydroxychloroquine (HCQ) 1 h prior to transduction with an AAV2 vector encoding GFP driven by a ubiquitous CAG promoter. This led to a consistent increase in GFP expression, up to 3-fold, compared with vector alone. Comparing subretinal injections of AAV2.CAG.GFP vector alone versus co-injection with 18.75 μM HCQ in paired eyes in mice, mean GFP expression was 4.6-fold higher in retinae co-treated with HCQ without retinal toxicity. A comparative 5.9-fold effect was seen with an AAV8(Y733F).GRK1.GFP vector containing the photoreceptor-specific rhodopsin kinase promoter. While the mechanism of action remains to be fully elucidated, our data suggest that a single pulse of adjunctive HCQ could safely improve AAV transduction in vivo, thus providing a novel strategy for enhancing the clinical effects of gene therapy

Molecular characterization of Mycobacterium bovis infection in cattle and buffalo in Amazon Region, Brazil

The aim of this study was to characterize Mycobacterium bovis from cattle and buffalo tissue samples, from two Brazilian states, and to analyse their genetic diversity by spoligotyping. Tissue samples from tuberculosis suspect animals, 57 in Amazonas State (12 cattle and 45 buffaloes) and six from Pará State (5 cattle and one buffalo) from slaughterhouses under State Veterinary Inspection, were isolated in culture medium Stonebrink. The positive cultures were confirmed by PCR and analysed by the spoligotyping technique and the patterns (spoligotypes) were identified and compared at the Mycobacterium bovis Spoligotype Database (http://www.mbovis.org/). There was bacterial growth in 44 (69.8%) of the tissues of the 63 animals, of which PCR for region of differentiation 4 identified 35/44 (79.5%) as Mycobacterium bovis. Six different spoligotypes were identified among the 35 Mycobacterium bovis isolates, of which SB0295, SB1869, SB0121 and SB1800 had already been described in Brazil, and SB0822 and SB1608 had not been described. The most frequent spoligotype in this study (SB0822) had already been described in buffaloes in Colombia, a neighbouring country of Amazonas state. The other identified spoligotypes were also described in other South American countries, such as Argentina and Venezuela, and described in the Brazilian states of Rio Grande do Sul, Santa Catarina, São Paulo, Minas Gerais, Mato Grosso do Sul, Mato Grosso and Goiás, indicating an active movement of Mycobacterium bovis strains within Brazil.Instituto de BiotecnologíaFil: Carneiro, Paulo A. M. Michigan State University. Center for Comparative Epidemiology; Estados UnidosFil: Pasquatti, Taynara N. Dom Bosco Catholic University; BrasilFil: Takatani, Haruo. Agencia de Defesa Agropecuaria do Amazonas; BrasilFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Marfil, Maria Jimena. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Barnard, Christian. Agencia de Defesa Agropecuaria do Amazonas; BrasilFil: Fitzgerald, Scott D. Michigan State University. Veterinary Diagnostic Laboratory; Estados UnidosFil: Abramovitch, Robert B. Michigan State University. Department of Microbiology and Molecular Genetics; Estados UnidosFil: Araujo, Flabio Ribeiro de. Centro Nacional de Pesquisa de Gado de Corte; BrasilFil: Kaneene, John B. Michigan State University. Center for Comparative Epidemiology; Estados Unido

Design, formulation and sensory evaluation of a polyphenol-rich food placebo: an example of aronia juice for food intervention studies

Products suitable for use as controls in food interventions designed to demonstrate the role of minor components are largely lacking. In the present study, we aimed to develop a formulation to be used as a placebo in a clinical trial designed to assess the effects of aronia juice polyphenols on platelet function. Three formulations with the same nutrient composition as aronia juice were prepared by mixing various nutrients, artificial colours and flavours with water. The similarity of formulations to aronia juice in terms of taste, colour, smell and texture was assessed by six food panellists. The final placebo was tested for its impact on platelet function, biochemical and anthropometric parameters in a 4-week long study. No significant changes in platelet function, or in several cardiovascular and safety markers were recorded. Formulation suitable for use as a placebo for dietary intervention studies using aronia juice has been developed and demonstrated to be well tolerated in humans

Developing an implementation strategy for a digital health intervention: an example in routine healthcare.

BACKGROUND: Evidence on how to implement new interventions into complex healthcare environments is often poorly reported and indexed, reducing its potential to inform initiatives to improve healthcare services. Using the implementation of a digital intervention within routine National Health Service (NHS) practice, we provide an example of how to develop a theoretically based implementation plan and how to report it transparently. In doing so we also highlight some of the challenges to implementation in routine healthcare. METHODS: The implemented intervention was HeLP-Diabetes, a digital self-management programme for people with Type 2 Diabetes, which was effective in improving diabetes control. The target setting for the implementation was an inner city London Clinical Commissioning Group in the NHS comprised of 34 general practices. HeLP-Diabetes was designed to be offered to patients as part of routine diabetes care across England. Evidence synthesis, engagement of local stakeholders, a theory of implementation (Normalization Process Theory), feedback, qualitative interviews and usage data were used to develop an implementation plan. RESULTS: A new implementation plan was developed to implement HeLP-Diabetes within routine practice. Individual component strategies were selected and developed informed by Normalization Process Theory. These strategies included: engagement of local opinion leaders, provision of educational materials, educational visits, educational meetings, audit and feedback and reminders. Additional strategies were introduced iteratively to address barriers that arose during the implementation. Barriers largely related to difficulties in allocating resources to implement the intervention within routine care. CONCLUSION: This paper provides a worked example of implementing a digital health intervention. The learning from this work can inform others undertaking the work of planning and executing implementation activities in routine healthcare. Of particular importance is: the selection of appropriate theory to guide the implementation process and selection of strategies; ensuring that enough attention is paid to planning implementation; and a flexible approach that allows response to emerging barriers