Identification of direction in gene networks from expression and methylation

Background: Reverse-engineering gene regulatory networks from expression data is difficult, especially without temporal measurements or interventional experiments. In particular, the causal direction of an edge is generally not statistically identifiable, i.e., cannot be inferred as a statistical parameter, even from an unlimited amount of non-time series observational mRNA expression data. Some additional evidence is required and high-throughput methylation data can viewed as a natural multifactorial gene perturbation experiment. Results: We introduce IDEM (Identifying Direction from Expression and Methylation), a method for identifying the causal direction of edges by combining DNA methylation and mRNA transcription data. We describe the circumstances under which edge directions become identifiable and experiments with both real and synthetic data demonstrate that the accuracy of IDEM for inferring both edge placement and edge direction in gene regulatory networks is significantly improved relative to other methods. Conclusion: Reverse-engineering directed gene regulatory networks from static observational data becomes feasible by exploiting the context provided by high-throughput DNA methylation data. An implementation of the algorithm described is available at http://code.google.com/p/idem/

Opportunities to reduce antibiotic prescribing for patients with COPD in primary care: a cohort study using electronic health records from the Clinical Practice Research Datalink (CPRD)

Background In primary care there is uncertainty about which patients with acute exacerbations of COPD (AECOPD) benefit from antibiotics. Objectives To identify which types of COPD patients get the most antibiotics in primary care to support targeted antibiotic stewardship. Methods Observational study of COPD patients using a large English primary care database with 12 month follow-up. We estimated the incidence of and risk factors for antibiotic prescribing relative to the number of AECOPD during follow-up, considering COPD severity, smoking, obesity and comorbidity. Results From 157 practices, 19594 patients were diagnosed with COPD, representing 2.6% of patients and 11.5% of all prescribed antibiotics. Eight hundred and thirty-three (4.5%) patients with severe COPD and frequent AECOPD were prescribed six to nine prescriptions per year and accounted for 13.0% of antibiotics. Individuals with mild to moderate COPD and zero or one AECOPD received one to three prescriptions per year but accounted for 42.5% of all prescriptions. In addition to COPD severity, asthma, chronic heart disease, diabetes, heart failure and influenza vaccination were independently associated with increased antibiotic use. Conclusions Patients with severe COPD have the highest rates of antibiotic prescribing but most antibiotics are prescribed for patients with mild to moderate COPD. Antibiotic stewardship should focus on the dual goals of safely reducing the volume of prescribing in patients with mild to moderate COPD, and optimizing prescribing in patients with severe disease who are at significant risk of drug resistance

Personalized Epigenomic Signatures That Are Stable Over Time and Covary with Body Mass Index

International audienc

Altering, Improving, And Defining The Specificities Of Crispr-Cas Nucleases

CRISPR-Cas9 nucleases have been widely adopted for genome editing applications to knockout genes or to introduce desired changes. While these nucleases have shown immense promise, two notable limitations of the wild-type form of the broadly used Streptococcus pyogenes Cas9 (SpCas9) are the restriction of targeting range to sites that contain an NGG protospacer adjacent motif (PAM), and the undesirable ability of the enzyme to cleave off-target sites that resemble the on-target site. Scarcity of PAM motifs can limit implementations that require precise targeting, whereas off-target effects can confound research applications and are important considerations for therapeutics. To improve the targeting range of SpCas9 and an orthogonal Cas9 from Staphylococcus aureus (called SaCas9), we optimized a heterologous genetic selection system that enabled us to perform directed evolution of PAM specificity. With SpCas9, we evolved two separate variants that can target NGA and NGCG PAMs1, and with SaCas9 relaxed the PAM from NNGRRT to NNNRRT2, increasing the targetability of these enzyme 2- to 4-fold. The genome-wide specificity profiles of SpCas9 and SaCas9 variants, determine by GUIDE-seq3, indicate that they are at least as, if not more, specific than the wild-type enzyme1,2. Together, these results demonstrate that the inherent PAM specificity of multiple different Cas9 orthologues can be purposefully modified to improve the accuracy of targeting. Existing strategies for improving the genome-wide specificity of SpCas9 have thus far proven to be incompletely effective and/or have other limitations that constrain their use. To address the off-target potential of SpCas9, we engineered a high-fidelity variant of SpCas9 (called SpCas9-HF1), that contains alterations designed to reduce non-specific contacts to the target strand DNA backbone. In comparison to wild-type SpCas9, SpCas9-HF1 rendered all or nearly all off-target events imperceptible by GUIDE-seq and targeted deep-sequencing methods with standard non-repetitive target sites in human cells4. Even for atypical, repetitive target sites, the vast majority of off-targets induced by SpCas9-HF1 and optimized derivatives were not detected4. With its exceptional precision, SpCas9-HF1 provides an important and easily employed alternative to wild-type SpCas9 that can eliminate off-target effects when using CRISPR-Cas9 for research and therapeutic applications. Finally, on-target activity and genome-wide specificity are two important properties of engineered nucleases that should be characterized prior to adoption of such technologies for research or therapeutic applications. CRISPR-Cas Cpf1 nucleases have recently been described as an alternative genome-editing platform5, yet their activities and genome-wide specificities remain largely undefined. Based on assessment of on-target activity across more than 40 target sites, we demonstrate that two Cpf1 orthologues function robustly in human cells with efficiencies comparable to those of the widely used Streptococcus pyogenes Cas9. We also demonstrate that four to six bases at the 3’ end of the short CRISPR RNA (crRNA) used to program Cpf1 are insensitive to single base mismatches, but that many of the other bases within the crRNA targeting region are highly sensitive to single or double substitutions6. Consistent with these results, GUIDE-seq performed in multiple cell types and targeted deep sequencing analyses of two Cpf1 nucleases revealed no detectable off-target cleavage for over half of 20 different crRNAs we examined. Our results suggest that the two Cpf1 nucleases we characterized generally possess robust on-target activity and high specificities in human cells, findings that should encourage broader use of these genome editing enzymes. 1. Kleinstiver, BP, et al. (2015) Nature, 523(7561):481-5 2. Kleinstiver, BP, et al. (2015) Nature Biotechnology, 33(12):1293-98 3. Tsai, SQ et al. (2015) Nature Biotechnology, 33(2):187-97 4. Kleinstiver, BP and Pattanayak, V, et al. (2016), Nature, 529(7587):490-5 5. Zetsche, B, et al. (2015) Cell, 163(3):759-71 6. Kleinstiver, BP and Tsai, SQ, et al. (2016), Nature Biotechnology, 34(8):869-7

The role of mentorship in protege performance

The role of mentorship on protege performance is a matter of importance to academic, business, and governmental organizations. While the benefits of mentorship for proteges, mentors and their organizations are apparent, the extent to which proteges mimic their mentors' career choices and acquire their mentorship skills is unclear. Here, we investigate one aspect of mentor emulation by studying mentorship fecundity---the number of proteges a mentor trains---with data from the Mathematics Genealogy Project, which tracks the mentorship record of thousands of mathematicians over several centuries. We demonstrate that fecundity among academic mathematicians is correlated with other measures of academic success. We also find that the average fecundity of mentors remains stable over 60 years of recorded mentorship. We further uncover three significant correlations in mentorship fecundity. First, mentors with small mentorship fecundity train proteges that go on to have a 37% larger than expected mentorship fecundity. Second, in the first third of their career, mentors with large fecundity train proteges that go on to have a 29% larger than expected fecundity. Finally, in the last third of their career, mentors with large fecundity train proteges that go on to have a 31% smaller than expected fecundity.Comment: 23 pages double-spaced, 4 figure

Smart-RRBS for single-cell methylome and transcriptome analysis

The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications. We recently reported a new method for profiling the DNA methylome and transcriptome from the same single cells in a cancer research study. Here, we present details of the protocol and provide guidance on its utility. Our Smart-RRBS (reduced representation bisulfite sequencing) protocol combines Smart-seq2 and RRBS and entails physically separating mRNA from the genomic DNA. It generates paired epigenetic promoter and RNA-expression measurements for ~24% of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding flow sorting of cells and sequencing, takes ~3 d to process up to 192 samples manually. It requires basic molecular biology expertise and laboratory equipment, including a PCR workstation with UV sterilization, a DNA fluorometer and a microfluidic electrophoresis system

Improving nutrition through biofortification–A systematic review

Nutritious foods are essential for human health and development. However, malnutrition and hidden hunger continue to be a challenge globally. In most developing countries, access to adequate and nutritious food continues to be a challenge. Although hidden hunger is less prevalent in developed countries compared to developing countries where iron (Fe) and zinc (Zn) deficiencies are common. The United Nations (UN) 2nd Sustainable Development Goal was set to eradicate malnutrition and hidden hunger. Hidden hunger has led to numerous cases of infant and maternal mortalities, and has greatly impacted growth, development, cognitive ability, and physical working capacity. This has influenced several countries to develop interventions that could help combat malnutrition and hidden hunger. Interventions such as dietary diversification and food supplementation are being adopted. However, fortification but mainly biofortification has been projected to be the most sustainable solution to malnutrition and hidden hunger. Plant-based foods (PBFs) form a greater proportion of diets in certain populations; hence, fortification of PBFs is relevant in combating malnutrition and hidden hunger. Agronomic biofortification, plant breeding, and transgenic approaches are some currently used strategies in food crops. Crops such as cereals, legumes, oilseeds, vegetables, and fruits have been biofortified through all these three strategies. The transgenic approach is sustainable, efficient, and rapid, making it suitable for biofortification programs. Omics technology has also been introduced to improve the efficiency of the transgenic approach

Surveillance of bacterial pathogens of diarrhoea in two selected sub metros within the Accra metropolis

Background: In recent years, many localities within the Greater Accra Region (GAR) have witnessed several episodes of cholera outbreaks, with some deaths.Compared to previous epidemics, which usually followed heavy rains, recent outbreaks show no seasonality.Objectives: To investigate infective bacterial diseases in selected sub metros within the GAR.Methods: We used existing disease surveillance systems in Ghana, and investigated all reported cases of diarrhoea that met our case-definition. A three-daytraining workshop was done prior to the start of study, to sensitize prescribers at the Korle-Bu Polyclinic and Maamobi General hospital. A case-based investigationform was completed per patient, and two rectal swabs were taken for culture at the National Public Health and Reference Laboratory. Serotyping and antibiogramprofiles of identified bacteria were determined. Potential risk factors were also assessed using a questionnaire.Results: Between January and June 2012, a total of 361 diarrhoeal cases with 5 deaths were recorded. Out of a total of 218 rectal swabs cultured, 71 (32.6%) Vibrio cholerae O1 Ogawa serotypes, and 1 (0.5%) Salmonella (O group B) were laboratory confirmed. No Shigella was isolated. The Vibrio cholerae isolates were susceptible to ciprofloxacin and tetracycline. Greater than 80% of patients reported having drank sachet water 24 h prior to diarrhoea onset, and many (144/361) young adults (20-29 years) reported with diarrhoea.Conclusion: Enhanced surveillance of diarrhoeal diseases (enteric pathogens) within cholera endemic regions, will serve as an early warning signal, and reduce fatalities associated with infective diarrhoea.Keywords: Diarrhoeal disease surveillance, enteric pathogens, Vibrio cholerae, Salmonell

Reported exposure to SARS-CoV-2 and relative perceived importance of different settings for SARS-CoV-2 acquisition in England and Wales: Analysis of the Virus Watch Community Cohort [version 1; peer review: awaiting peer review]

We aimed to assess the relative importance of different settings for SARS-CoV-2 transmission in a large community cohort based on perceived location of infection for self-reported confirmed SARS-COV-2 cases. We demonstrate the importance of home, work and education as perceived venues for transmission. In children, education was most important and in older adults essential shopping was of high importance. Our findings support public health messaging about infection control at home, advice on working from home and restrictions in different venues

Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD) followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Results Examining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes) and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes) hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines. Conclusions Comparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease.Published versio