Reference genome and comparative genome analysis for the WHO reference strain for Mycobacterium bovis BCG Danish, the present tuberculosis vaccine

Background: Mycobacterium bovis bacillus Calmette-Guerin (M. bovis BCG) is the only vaccine available against tuberculosis (TB). In an effort to standardize the vaccine production, three substrains, i.e. BCG Danish 1331, Tokyo 172-1 and Russia BCG-1 were established as the WHO reference strains. Both for BCG Tokyo 172-1 as Russia BCG-1, reference genomes exist, not for BCG Danish. In this study, we set out to determine the completely assembled genome sequence for BCG Danish and to establish a workflow for genome characterization of engineering-derived vaccine candidate strains.ResultsBy combining second (Illumina) and third (PacBio) generation sequencing in an integrated genome analysis workflow for BCG, we could construct the completely assembled genome sequence of BCG Danish 1331 (07/270) (and an engineered derivative that is studied as an improved vaccine candidate, a SapM KO), including the resolution of the analytically challenging long duplication regions. We report the presence of a DU1-like duplication in BCG Danish 1331, while this tandem duplication was previously thought to be exclusively restricted to BCG Pasteur. Furthermore, comparative genome analyses of publicly available data for BCG substrains showed the absence of a DU1 in certain BCG Pasteur substrains and the presence of a DU1-like duplication in some BCG China substrains. By integrating publicly available data, we provide an update to the genome features of the commonly used BCG strains. Conclusions: We demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of the BCG Danish vaccine strain. The BCG Danish WHO reference genome will serve as a reference for future engineered strains and the established workflow can be used to enhance BCG vaccine standardization

TH9 cells are required for tissue mast cell accumulation during allergic inflammation

BACKGROUND: IL-9 is important for the growth and survival of mast cells. IL-9 is produced by T cells, natural killer T cells, mast cells, eosinophils, and innate lymphoid cells, although the cells required for mast cell accumulation during allergic inflammation remain undefined. OBJECTIVE: We sought to elucidate the role of TH9 cells in promoting mast cell accumulation in models of allergic lung inflammation. METHODS: Adoptive transfer of ovalbumin-specific TH2 and TH9 cells was used to assess the ability of each subset to mediate mast cell accumulation in tissues. Mast cell accumulation was assessed in wild-type mice and mice with PU.1-deficient T cells subjected to acute and chronic models of allergic inflammation. RESULTS: Adoptive transfer experiments demonstrated that recipients of TH9 cells had significantly higher mast cell accumulation and expression of mast cell proteases compared with control or TH2 recipients. Mast cell accumulation was dependent on IL-9, but not IL-13, a cytokine required for many aspects of allergic inflammation. In models of acute and chronic allergic inflammation, decreased IL-9 levels in mice with PU.1-deficient T cells corresponded to diminished tissue mast cell numbers and expression of mast cell proteases. Mice with PU.1-deficient T cells have defects in IL-9 production from CD4(+) T cells, but not natural killer T cells or innate lymphoid cells, suggesting a TH cell-dependent phenotype. Rag1(-/-) mice subjected to a chronic model of allergic inflammation displayed reduced mast cell infiltration comparable with accumulation in mice with PU.1-deficient T cells, emphasizing the importance of IL-9 produced by T cells in mast cell recruitment. CONCLUSION: TH9 cells are a major source of IL-9 in models of allergic inflammation and play an important role in mast cell accumulation and activation

Development of a Recommender System for Dental Care Using Machine Learning

Resource mismanagement along with the underutilization of dental care has led to serious health and economic consequences. Artificial intelligence was applied to a national health database to develop recommendations for dental care. The data were obtained from the 2013–2014 National Health and Nutrition Examination Survey to perform machine learning. Feature selection was done using LASSO in R to determine the best regression model. Prediction models were developed using several supervised machine learning algorithms, including logistic regression, support vector machine, random forest, and classification and regression tree. Feature selection by LASSO along with the inclusion of additional clinically relevant variables identified 8 top features associated with recommendation for dental care. The top 3 features include gum health, number of prescription medications taken, and race. Gum health shows a significantly higher relative importance compared to other features. Demographics, healthcare access, and general health variables were identified as top features related to receiving additional dental care, consistent with prior research. Practicing dentists and other healthcare professionals can follow this model to enable precision dentistry through the incorporation of our algorithms into computerized screening tool or decision tree diagram to achieve more efficient and personalized preventive strategies and treatment protocols in dental care

IBEX:A versatile multiplex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues

The diverse composition of mammalian tissues poses challenges for understanding the cell–cell interactions required for organ homeostasis and how spatial relationships are perturbed during disease. Existing methods such as single-cell genomics, lacking a spatial context, and traditional immunofluorescence, capturing only two to six molecular features, cannot resolve these issues. Imaging technologies have been developed to address these problems, but each possesses limitations that constrain widespread use. Here we report a method that overcomes major impediments to highly multiplex tissue imaging. “Iterative bleaching extends multiplexity” (IBEX) uses an iterative staining and chemical bleaching method to enable high-resolution imaging of >65 parameters in the same tissue section without physical degradation. IBEX can be employed with various types of conventional microscopes and permits use of both commercially available and user-generated antibodies in an “open” system to allow easy adjustment of staining panels based on ongoing marker discovery efforts. We show how IBEX can also be used with amplified staining methods for imaging strongly fixed tissues with limited epitope retention and with oligonucleotide-based staining, allowing potential cross-referencing between flow cytometry, cellular indexing of transcriptomes and epitopes by sequencing, and IBEX analysis of the same tissue. To facilitate data processing, we provide an open-source platform for automated registration of iterative images. IBEX thus represents a technology that can be rapidly integrated into most current laboratory workflows to achieve high-content imaging to reveal the complex cellular landscape of diverse organs and tissues

Luminal Plasma Treatment for Small Diameter Polyvinyl Alcohol Tubular Scaffolds

Plasma-based surface modification is recognized as an effective way to activate biomaterial surfaces, and modulate their interactions with cells, extracellular matrix proteins, and other materials. However, treatment of a luminal surface of a tubular scaffold remains non-trivial to perform in small diameter tubes. Polyvinyl alcohol (PVA) hydrogel, which has been widely used for medical applications, lacks functional groups to mediate cell attachment. This poses an issue for vascular applications, as endothelialization in a vascular graft lumen is crucial to maintain long term graft patency. In this study, a Radio Frequency Glow Discharges (RFGD) treatment in the presence of NH3 was used to modify the luminal surface of 3-mm diameter dehydrated PVA vascular grafts. The grafted nitrogen containing functional groups demonstrated stability, and in vitro endothelialization was successfully maintained for at least 30 days. The plasma-modified PVA displayed a higher percentage of carbonyl groups over the untreated PVA control. Plasma treatment on PVA patterned with microtopographies was also studied, with only the concave microlenses topography demonstrating a significant increase in platelet adhesion. Thus, the study has shown the possibility of modifying a small diameter hydrogel tubular scaffold with the RFGD plasma treatment technique and demonstrated stability in ambient storage conditions for up to 30 days

A comparison between data requirements and availability for calibrating predictive ecological models for lowland UK woodlands: learning new tricks from old trees

© 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.Woodlands provide valuable ecosystem services, and it is important to understand their dynamics. To predict the way in which these might change, we need process-based predictive ecological models, but these are necessarily very data intensive. We tested the ability of existing datasets to provide the parameters necessary to instantiate a well-used forest model (SORTIE) for a well-studied woodland (Wytham Woods). Only five of SORTIE's 16 equations describing different aspects of the life history and behavior of individual trees could be parameterized without additional data collection. One age class – seedlings – was completely missed as they are shorter than the height at which Diameter at Breast Height (DBH) is measured. The mensuration of trees has changed little in the last 400 years (focussing almost exclusively on DBH) despite major changes in the nature of the source of value obtained from trees over this time. This results in there being insufficient data to parameterize process-based models in order to meet the societal demand for ecological prediction. We do not advocate ceasing the measurement of DBH, but we do recommend that those concerned with tree mensuration consider whether additional measures of trees could be added to their data collection protocols. We also see advantages in integrating techniques such as ground-based LIDAR or remote sensing techniques with long-term datasets to both preserve continuity with what has been performed in the past and to expand the range of measurements made.published_or_final_versio

High Expression Levels of Total IGF-1R and Sensitivity of NSCLC Cells In Vitro to an Anti-IGF-1R Antibody (R1507)

© 2009 Gong et al

Ombitasvir, Paritaprevir, Ritonavir, and Dasabuvir Mini-Tabs Plus Ribavirin for Children Aged 3–11 Years with Hepatitis C Genotype 1a

Introduction To assess the safety, efficacy, and pharmacokinetics of mini-tablet formulations of ombitasvir (OBV), paritaprevir (PTV), ritonavir, and dasabuvir (DSV) with or without ribavirin for 12 weeks in children infected with chronic hepatitis C virus (HCV) genotype (GT) 1. Methods This is an ongoing, open-label, Phase 2/3 study in children 3–11 years old infected with HCV GT1 who were HCV treatment-naïve and non-cirrhotic. Pediatric mini-tablet formulations of OBV, PTV, ritonavir, and DSV plus ribavirin oral solution were administered for 12 weeks based on body weight. Endpoints included SVR12, adverse events (AEs), and pharmacokinetic parameters. Results Overall, 26 children received OBV, PTV, ritonavir, and DSV plus ribavirin; 14 were 3–8 years old and 12 were 9–11 years old; 35% were male; and all had chronic HCV GT1a infection. The SVR12 rate was 96% (25/26; 95% CI 81.1–99.3), with 1 child failing to achieve SVR12 due to non-adherence and treatment discontinuation. Treatment-emergent AEs of Grade ≥ 3 occurred in 3 children; 2 events in 1 child were considered serious; and none were considered treatment-related. No AEs led to discontinuation of study treatment. The most common AEs were headache (27%), fatigue (23%), pyrexia (19%), and vomiting (19%). Pharmacokinetic results showed mini-tablet formulations of OBV, PTV, DSV, and ritonavir drug exposures were comparable to the adult formulation. Conclusion The mini-tablet combination of OBV, PTV, ritonavir, and DSV plus ribavirin to treat HCV GT1a infection for 12 weeks was highly effective and suitable in children 3–11 years of age