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/

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

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

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

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

Diarrhea incidence in low- and middle-income countries in 1990 and 2010: a systematic review

<p>Abstract</p> <p>Background</p> <p>Diarrhea is recognized as a leading cause of morbidity and mortality among children under 5 years of age in low- and middle-income countries yet updated estimates of diarrhea incidence by age for these countries are greatly needed. We conducted a systematic literature review to identify cohort studies that sought to quantify diarrhea incidence among any age group of children 0-59 mo of age.</p> <p>Methods</p> <p>We used the Expectation-Maximization algorithm as a part of a two-stage regression model to handle diverse age data and overall incidence rate variation by study to generate country specific incidence rates for low- and middle-income countries for 1990 and 2010. We then calculated regional incidence rates and uncertainty ranges using the bootstrap method, and estimated the total number of episodes for children 0-59 mo of age in 1990 and 2010.</p> <p>Results</p> <p>We estimate that incidence has declined from 3.4 episodes/child year in 1990 to 2.9 episodes/child year in 2010. As was the case previously, incidence rates are highest among infants 6-11 mo of age; 4.5 episodes/child year in 2010. Among these 139 countries there were nearly 1.9 billion episodes of childhood diarrhea in 1990 and nearly 1.7 billion episodes in 2010.</p> <p>Conclusions</p> <p>Although our results indicate that diarrhea incidence rates may be declining slightly, the total burden on the health of each child due to multiple episodes per year is tremendous and additional funds are needed to improve both prevention and treatment practices in low- and middle-income countries.</p

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

Household overcrowding and risk of SARS-CoV-2: analysis of the Virus Watch prospective community cohort study in England and Wales

Background: Household overcrowding is associated with increased risk of infectious diseases across contexts and countries. Limited data exist linking household overcrowding and risk of COVID-19. We used data collected from the Virus Watch cohort to examine the association between overcrowded households and SARS-CoV-2. // Methods: The Virus Watch study is a household community cohort of acute respiratory infections in England and Wales. We calculated overcrowding using the measure of persons per room for each household. We considered two primary outcomes: PCR-confirmed positive SARS-CoV-2 antigen tests and laboratory-confirmed SARS-CoV-2 antibodies. We used mixed-effects logistic regression models that accounted for household structure to estimate the association between household overcrowding and SARS-CoV-2 infection. // Results: 26,367 participants were included in our analyses. The proportion of participants with a positive SARS-CoV-2 PCR result was highest in the overcrowded group (9.0%; 99/1,100) and lowest in the under-occupied group (4.2%; 980/23,196). In a mixed-effects logistic regression model, we found strong evidence of an increased odds of a positive PCR SARS-CoV-2 antigen result (odds ratio 2.45; 95% CI:1.43–4.19; p-value=0.001) and increased odds of a positive SARS-CoV-2 antibody result in individuals living in overcrowded houses (3.32; 95% CI:1.54–7.15; p-value<0.001) compared with people living in under-occupied houses. // Conclusion: Public health interventions to prevent and stop the spread of SARS-CoV-2 should consider the risk of infection for people living in overcrowded households and pay greater attention to reducing household transmission

Deprivation and exposure to public activities during the COVID-19 pandemic in England and Wales

BACKGROUND: Differential exposure to public activities may contribute to stark deprivation-related inequalities in SARS-CoV-2 infection and outcomes but has not been directly investigated. We set out to investigate whether participants in Virus Watch-a large community cohort study based in England and Wales-reported differential exposure to public activities and non-household contacts during the autumn-winter phase of the COVID-19 pandemic according to postcode-level socioeconomic deprivation. METHODS: Participants (n=20 120-25 228 across surveys) reported their daily activities during 3 weekly periods in late November 2020, late December 2020 and mid-February 2021. Deprivation was quantified based on participants' residential postcode using English or Welsh Index of Multiple Deprivation quintiles. We used Poisson mixed-effect models with robust standard errors to estimate the relationship between deprivation and risk of exposure to public activities during each survey period. RESULTS: Relative to participants in the least deprived areas, participants in the most deprived areas exhibited elevated risk of exposure to vehicle sharing (adjusted risk ratio (aRR) range across time points: 1.73-8.52), public transport (aRR: 3.13-5.73), work or education outside of the household (aRR: 1.09-1.21), essential shops (aRR: 1.09-1.13) and non-household contacts (aRR: 1.15-1.19) across multiple survey periods. CONCLUSION: Differential exposure to essential public activities-such as attending workplaces and visiting essential shops-is likely to contribute to inequalities in infection risk and outcomes. Public health interventions to reduce exposure during essential activities and financial and practical support to enable low-paid workers to stay at home during periods of intense transmission may reduce COVID-related inequalities