The design and statistical analysis of single-cell RNA-sequencing experiments

Next-generation DNA- and RNA-sequencing (RNA-seq) technologies have expanded rapidly in both throughput and accuracy within the last decade. The momentum continues as emerging techniques become increasingly capable of profiling molecular content at the level of individual cells. One goal of this research is to put forward best practices in the design of single-cell RNA-sequencing (scRNA-seq) experiments, specifically as it relates to choices regarding the trade-off between sequencing depth and sample size. In addition to general guidelines, an interactive tool is presented to aid researchers in making experiment-specific decisions that are informed by real data and practical constraints. Further, a new approach to the modeling and testing of differential gene expression in scRNA-seq data is proposed, which notably incorporates salient features (e.g. highly zero-inflated expression values) of single-cell transcription that are otherwise obscured at the tissue level. As single-cell technologies offer an unprecedented window into cell-to-cell heterogeneity and its biological consequences, it is essential that suitable approaches are adopted for both the design and analysis of these experiments

Well-Being and Social Capital on Planet Earth: Cross-National Evidence from 142 Countries

High levels of social trust and social support are associated with life satisfaction around the world. However, it is not known whether this association extends to other indicators of social capital and of subjective well-being globally. We examine associations between three measures of social capital and three indicators of subjective well-being in 142 low-, middle- and high-income countries. Furthermore, we explore whether positive and negative feelings mirror each other or if they are separate constructs that behave differently in relation to social capital. Data comes from the Gallup World Poll, an international cross-sectional comparable survey conducted yearly from 2005 to 2009 for those 15 years of age and over. The poll represents 95% of the world's population. Social capital was measured with self-reports of access to support from relatives and friends, of volunteering to an organization in the past month, and of trusting others. Subjective well-being was measured with self-reports of life satisfaction, positive affect, and negative affect. We first estimate random coefficient (multi-level) models and then use multivariate (individual-level) Ordinary Least Square (OLS) regression to model subjective well-being as a function of social support, volunteering and social trust, controlling for age, gender, education, marital status, household income and religiosity. We found that having somebody to count on in case of need and reporting high levels of social trust are associated with better life evaluations and more positive feelings and an absence of negative feelings in most countries around the world. Associations, however, are stronger for high- and middle-income countries. Volunteering is also associated with better life evaluations and a higher frequency of positive emotions. There is not an association, however, between volunteering and experiencing negative feelings, except for low-income countries. Finally, we present evidence that the two affective components of subjective well-being behave differently in relation to different indicators of social capital and social support across countries

Influence of trans-Pacific pollution transport on acyl peroxy nitrate abundances and speciation at Mount Bachelor Observatory during INTEX-B

International audienceWe present month-long observations of speciated acyl peroxy nitrates (APNs), including PAN, PPN, MPAN, APAN, and the sum of PiBN and PnBN, measured at the Mount Bachelor Observatory (MBO) as part of the INTEX-B collaborative field campaign during spring 2006. APN abundances, measured by thermal dissociation-chemical ionization mass spectrometry (TD-CIMS), are discussed in terms of differing contributions from the boundary layer and the free troposphere and in the context of previous APN measurements in the NE Pacific region. PAN mixing ratios range from 11 to 3955 pptv, with a mean value of 334 pptv for the full measurement period. PPN is linearly correlated with PAN (r2=0.96), with an average abundance of 6.5% relative to PAN; other APNs are generally <1% of PAN. Diurnal cycles and relationships of APNs with ozone reveal a gradient in hydrocarbon chemistry between the boundary layer and the free troposphere. On average, the highest levels of APNs, ozone and PPN/PAN are found in free tropospheric air masses, suggesting that this site is strongly influenced by distant pollution sources. To estimate the impact of long-range transport of Asian pollution on atmospheric composition at MBO, we perform a detailed analysis utilizing HYSPLIT back trajectories. This analysis suggests that trans-Pacific transport of Asian pollution leads to substantial increases in APN and ozone mixing ratios at MBO, especially when transport occurs via the free troposphere. The ensemble of trajectories indicate that Asian-influenced free tropospheric air was sampled in ~16% of our data and contained a median PAN mixing ratio double that of the full dataset

Cholesterol Sulfotransferase SULT2B1b Modulates Sensitivity to Death Receptor Ligand TNFα in Castration-Resistant Prostate Cancer

Cholesterol sulfotransferase, SULT2B1b, has been demonstrated to modulate both androgen receptor activity and cell growth properties. However, the mechanism(s) by which SULT2B1b alters these properties within prostate cancer cells has not been described. Furthermore, specific advantages of SULT2B1b expression in prostate cancer cells is not understood. In these studies, single-cell mRNA sequencing (scRNA-seq) was conducted to compare the transcriptomes of SULT2B1b knockdown (KD) versus Control KD LNCaP cells. Over 2,000 differentially expressed (DE) genes were identified along with alterations in numerous canonical pathways, including the death receptor signaling pathway. The studies herein demonstrate that SULT2B1b KD increases tumor necrosis factor alpha (TNF) expression in prostate cancer cells and results in NF-κB activation in a TNF-dependent manner. More importantly, SULT2B1b KD significantly enhances TNF-mediated apoptosis in both TNF-sensitive LNCaP cells and TNF-resistant C4–2 cells. Overexpression of SULT2B1b in LNCaP cells also decreases sensitivity to TNF-mediated cell death, suggesting that SULT2B1b modulates pathways dictating the TNF sensitivity capacity of prostate cancer cells. Probing human prostate cancer patient datasets further support this work by providing evidence that SULT2B1b expression is inversely correlated with TNF-related genes, including TNF, CD40LG, FADD, and NFKB1. Together, these data provide evidence that SULT2B1b expression in prostate cancer cells enhances resistance to TNF and may provide a growth advantage. In addition, targeting SULT2B1b may induce an enhanced therapeutic response to TNF treatment in advanced prostate cancer

Next-generation transcriptome sequencing of the premenopausal breast epithelium using specimens from a normal human breast tissue bank

Introduction Our efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. In order to provide the specimens that will facilitate such an understanding, The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center (KTB) was established. The KTB is, to our knowledge, the only biorepository in the world prospectively established to collect normal, healthy breast tissue from volunteer donors. As a first initiative toward a molecular understanding of the biology and developmental genetics of the normal mammary gland, the effect of the menstrual cycle and hormonal contraceptives on DNA expression in the normal breast epithelium was examined. Methods Using normal breast tissue from 20 premenopausal donors to KTB, the changes in the mRNA of the normal breast epithelium as a function of phase of the menstrual cycle and hormonal contraception were assayed using next-generation whole transcriptome sequencing (RNA-Seq). Results In total, 255 genes representing 1.4% of all genes were deemed to have statistically significant differential expression between the two phases of the menstrual cycle. The overwhelming majority (221; 87%) of the genes have higher expression during the luteal phase. These data provide important insights into the processes occurring during each phase of the menstrual cycle. There was only a single gene significantly differentially expressed when comparing the epithelium of women using hormonal contraception to those in the luteal phase. Conclusions We have taken advantage of a unique research resource, the KTB, to complete the first-ever next-generation transcriptome sequencing of the epithelial compartment of 20 normal human breast specimens. This work has produced a comprehensive catalog of the differences in the expression of protein-coding genes as a function of the phase of the menstrual cycle. These data constitute the beginning of a reference data set of the normal mammary gland, which can be consulted for comparison with data developed from malignant specimens, or to mine the effects of the hormonal flux that occurs during the menstrual cycle

Sacubitril/valsartan versus valsartan in regressing myocardial fibrosis in hypertension: a prospective, randomized, open-label, blinded endpoint clinical trial protocol

BackgroundDiffuse interstitial myocardial fibrosis is a key common pathological manifestation in hypertensive heart disease (HHD) progressing to heart failure (HF). Angiotensin receptor–neprilysin inhibitors (ARNi), now a front-line treatment for HF, confer benefits independent of blood pressure, signifying a multifactorial mode of action beyond hemodynamic regulation. We aim to test the hypothesis that compared with angiotensin II receptor blockade (ARB) alone, ARNi is more effective in regressing diffuse interstitial myocardial fibrosis in HHD.MethodsRole of ARNi in Ventricular Remodeling in Hypertensive LVH (REVERSE-LVH) is a prospective, randomized, open-label, blinded endpoint (PROBE) clinical trial. Adults with hypertension and left ventricular hypertrophy (LVH) according to Asian sex- and age-specific thresholds on cardiovascular magnetic resonance (CMR) imaging are randomized to treatment with either sacubitril/valsartan (an ARNi) or valsartan (an ARB) in 1:1 ratio for a duration of 52 weeks, at the end of which a repeat CMR is performed to assess differential changes from baseline between the two groups. The primary endpoint is the change in CMR-derived diffuse interstitial fibrosis volume. Secondary endpoints include changes in CMR-derived left ventricular mass, volumes, and functional parameters. Serum samples are collected and stored to assess the effects of ARNi, compared with ARB, on circulating biomarkers of cardiac remodeling. The endpoints will be analyzed with reference to the corresponding baseline parameters to evaluate the therapeutic effect of sacubitril/valsartan vs. valsartan.DiscussionREVERSE-LVH will examine the anti-fibrotic potential of sacubitril/valsartan and will offer mechanistic insights into the clinical benefits of sacubitril/valsartan in hypertension in relation to cardiac remodeling. Advancing the knowledge of the pathophysiology of HHD will consolidate effective risk stratification and personalized treatment through a multimodal manner integrating complementary CMR and biomarkers into the conventional care approach.Clinical Trial Registration: ClinicalTrials.gov, identifier, NCT03553810

RA-MAP, molecular immunological landscapes in early rheumatoid arthritis and healthy vaccine recipients

Rheumatoid arthritis (RA) is a chronic inflammatory disorder with poorly defined aetiology characterised by synovial inflammation with variable disease severity and drug responsiveness. To investigate the peripheral blood immune cell landscape of early, drug naive RA, we performed comprehensive clinical and molecular profiling of 267 RA patients and 52 healthy vaccine recipients for up to 18 months to establish a high quality sample biobank including plasma, serum, peripheral blood cells, urine, genomic DNA, RNA from whole blood, lymphocyte and monocyte subsets. We have performed extensive multi-omic immune phenotyping, including genomic, metabolomic, proteomic, transcriptomic and autoantibody profiling. We anticipate that these detailed clinical and molecular data will serve as a fundamental resource offering insights into immune-mediated disease pathogenesis, progression and therapeutic response, ultimately contributing to the development and application of targeted therapies for RA.</p

Discovery of common and rare genetic risk variants for colorectal cancer.

To further dissect the genetic architecture of colorectal cancer (CRC), we performed whole-genome sequencing of 1,439 cases and 720 controls, imputed discovered sequence variants and Haplotype Reference Consortium panel variants into genome-wide association study data, and tested for association in 34,869 cases and 29,051 controls. Findings were followed up in an additional 23,262 cases and 38,296 controls. We discovered a strongly protective 0.3% frequency variant signal at CHD1. In a combined meta-analysis of 125,478 individuals, we identified 40 new independent signals at P < 5 × 10-8, bringing the number of known independent signals for CRC to ~100. New signals implicate lower-frequency variants, Krüppel-like factors, Hedgehog signaling, Hippo-YAP signaling, long noncoding RNAs and somatic drivers, and support a role for immune function. Heritability analyses suggest that CRC risk is highly polygenic, and larger, more comprehensive studies enabling rare variant analysis will improve understanding of biology underlying this risk and influence personalized screening strategies and drug development.Goncalo R Abecasis has received compensation from 23andMe and Helix. He is currently an employee of Regeneron Pharmaceuticals. Heather Hampel performs collaborative research with Ambry Genetics, InVitae Genetics, and Myriad Genetic Laboratories, Inc., is on the scientific advisory board for InVitae Genetics and Genome Medical, and has stock in Genome Medical. Rachel Pearlman has participated in collaborative funded research with Myriad Genetics Laboratories and Invitae Genetics but has no financial competitive interest

Les droits disciplinaires des fonctions publiques : « unification », « harmonisation » ou « distanciation ». A propos de la loi du 26 avril 2016 relative à la déontologie et aux droits et obligations des fonctionnaires

The production of tt‾ , W+bb‾ and W+cc‾ is studied in the forward region of proton–proton collisions collected at a centre-of-mass energy of 8 TeV by the LHCb experiment, corresponding to an integrated luminosity of 1.98±0.02 fb−1 . The W bosons are reconstructed in the decays W→ℓν , where ℓ denotes muon or electron, while the b and c quarks are reconstructed as jets. All measured cross-sections are in agreement with next-to-leading-order Standard Model predictions.The production of $t\overline{t}$, $W+b\overline{b}$ and $W+c\overline{c}$ is studied in the forward region of proton-proton collisions collected at a centre-of-mass energy of 8 TeV by the LHCb experiment, corresponding to an integrated luminosity of 1.98 $\pm$ 0.02 \mbox{fb}^{-1}. The $W$ bosons are reconstructed in the decays $W\rightarrow\ell\nu$, where $\ell$ denotes muon or electron, while the $b$ and $c$ quarks are reconstructed as jets. All measured cross-sections are in agreement with next-to-leading-order Standard Model predictions