A Nested Semiparametric Method for Case-control study with missingness

We propose a nested semiparametric model to analyze a case-control study where genuine case status is missing for some individuals. The concept of a noncase is introduced to allow for the imputation of the missing genuine cases. The odds ratio parameter of the genuine cases compared to controls is of interest. The imputation procedure predicts the probability of being a genuine case compared to a noncase semiparametrically in a dimension reduction fashion. This procedure is flexible, and vastly generalizes the existing methods. We establish the root-n asymptotic normality of the odds ratio parameter estimator. Our method yields stable odds ratio parameter estimation owing to the application of an efficient semiparametric sufficient dimension reduction estimator. We conduct finite sample numerical simulations to illustrate the performance of our approach, and apply it to a dilated cardiomyopathy study

Determinants of mRNA recognition and translation regulation by Lin28

Lin28 is critical for stem cell maintenance and is also associated with advanced human malignancies. Our recent genome-wide studies mark Lin28 as a master post-transcriptional regulator of a subset of messenger RNAs important for cell growth and metabolism. However, the molecular basis underpinning the selective mRNA target regulation is unclear. Here, we provide evidence that Lin28 recognizes a unique motif in multiple target mRNAs, characterized by a small but critical ‘A’ bulge flanked by two G:C base pairs embedded in a complex secondary structure. This motif mediates Lin28-dependent stimulation of translation. As Lin28 is also known to inhibit the biogenesis of a cohort of miRNAs including let-7, we propose that Lin28 binding to different RNA types (precursor miRNAs versus mRNAs) may facilitate recruitment of different co-factors, leading to distinct regulatory outcomes. Our findings uncover a putative yet unexpected motif that may constitute a mechanistic base for the multitude of functions regulated by Lin28 in both stem cells and cancer cells

High-Throughput Single-Cell Labeling (Hi-SCL) for RNA-Seq Using Drop-Based Microfluidics

The importance of single-cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a micro-fluidics chip. High-throughput Single-Cell Labeling (Hi-SCL) in drops is a novel method that uses drop-based libraries of oligonucleotide barcodes to index individual cells in a population. The use of drops as containers, and a microfluidics platform to manipulate them en-masse, yields a highly scalable methodological framework. Once tagged, labeled molecules from different cells may be mixed without losing the cell-of-origin information. Here we demonstrate an application of the method for generating RNA-sequencing data for multiple individual cells within a population. Barcoded oligonucleotides are used to prime cDNA synthesis within drops. Barcoded cDNAs are then combined and subjected to second generation sequencing. The data are deconvoluted based on the barcodes, yielding single-cell mRNA expression data. In a proof-of-concept set of experiments we show that this method yields data comparable to other existing methods, but with unique potential for assaying very large numbers of cells

A global sampling approach to designing and reengineering RNA secondary structures

The development of algorithms for designing artificial RNA sequences that fold into specific secondary structures has many potential biomedical and synthetic biology applications. To date, this problem remains computationally difficult, and current strategies to address it resort to heuristics and stochastic search techniques. The most popular methods consist of two steps: First a random seed sequence is generated; next, this seed is progressively modified (i.e. mutated) to adopt the desired folding properties. Although computationally inexpensive, this approach raises several questions such as (i) the influence of the seed; and (ii) the efficiency of single-path directed searches that may be affected by energy barriers in the mutational landscape. In this article, we present RNA-ensign, a novel paradigm for RNA design. Instead of taking a progressive adaptive walk driven by local search criteria, we use an efficient global sampling algorithm to examine large regions of the mutational landscape under structural and thermodynamical constraints until a solution is found. When considering the influence of the seeds and the target secondary structures, our results show that, compared to single-path directed searches, our approach is more robust, succeeds more often and generates more thermodynamically stable sequences. An ensemble approach to RNA design is thus well worth pursuing as a complement to existing approaches. RNA-ensign is available at http://csb.cs.mcgill.ca/RNAensign.National Science Foundation (U.S.). Graduate Research Fellowship ProgramNatural Sciences and Engineering Research Council of Canada (NSERC) (RGPIN ) (386596-10)Fonds québécois de la recherche sur la nature et les technologies (PR-146375)National Institutes of Health (U.S.) (Grant GM081871)Natural Sciences and Engineering Research Council of Canada (NSERC)National Institutes of Health (U.S.

miR-122 activates hepatitis C virus translation by a specialized mechanism requiring particular RNA components

In animals, microRNAs (miRNAs) generally repress gene expression by binding to sites in the 3′-untranslated region (UTR) of target mRNAs. miRNAs have also been reported to repress or activate gene expression by binding to 5′-UTR sites, but the extent of such regulation and the factors that govern these different responses are unknown. Liver-specific miR-122 binds to sites in the 5′-UTR of hepatitis C virus (HCV) RNA and positively regulates the viral life cycle, in part by stimulating HCV translation. Here, we characterize the features that allow miR-122 to activate translation via the HCV 5′-UTR. We find that this regulation is a highly specialized process that requires uncapped RNA, the HCV internal ribosome entry site (IRES) and the 3′ region of miR-122. Translation activation does not involve a previously proposed structural transition in the HCV IRES and is mediated by Argonaute proteins. This study provides an important insight into the requirements for the miR-122–HCV interaction, and the broader consequences of miRNAs binding to 5′-UTR sites

DroID 2011: a comprehensive, integrated resource for protein, transcription factor, RNA and gene interactions for Drosophila

DroID (http://droidb.org/), the Drosophila Interactions Database, is a comprehensive public resource for Drosophila gene and protein interactions. DroID contains genetic interactions and experimentally detected protein–protein interactions curated from the literature and from external databases, and predicted protein interactions based on experiments in other species. Protein interactions are annotated with experimental details and periodically updated confidence scores. Data in DroID is accessible through user-friendly, intuitive interfaces that allow simple or advanced searches and graphical visualization of interaction networks. DroID has been expanded to include interaction types that enable more complete analyses of the genetic networks that underlie biological processes. In addition to protein–protein and genetic interactions, the database now includes transcription factor–gene and regulatory RNA–gene interactions. In addition, DroID now has more gene expression data that can be used to search and filter interaction networks. Orthologous gene mappings of Drosophila genes to other organisms are also available to facilitate finding interactions based on gene names and identifiers for a number of common model organisms and humans. Improvements have been made to the web and graphical interfaces to help biologists gain a comprehensive view of the interaction networks relevant to the genes and systems that they study

The presentation, clinical features, complications, and treatment of congenital dacryocystocele

Purpose To determine the incidence and presenting features of congenital dacryocystocele in the United Kingdom. To report on those cases complicated by dacryocystitis, respiratory compromise, and the treatment undertaken. Methods A prospective observational study of cases of congenital dacryocystocele presenting in the United Kingdom between September 2014 and October 2015. Infants <3 months of age presenting with a cystic swelling in the medial canthal area were included. Cases were identified via the British Ophthalmology Surveillance Unit (BOSU) reporting system. Results A total of 49 cases were reported during the study period. This gives an incidence of 1 in 18 597 live births. There was a 71% response rate to the questionnaire. The average age at presentation was 16.94 days. Dacryocystoceles were unilateral in 91% of cases. Dacryocystitis was a complicating factor in 49% of patients and 17% had respiratory distress. Uncomplicated dacryocystocele responded well to conservative measures in 86%. Surgical intervention was required in 23% of patients. Those cases complicated by dacryocystitis (29%) and nasal obstruction (17%) were more likely to require surgical intervention compared to those with dacryocystocele alone (14%). Digital massage appears to reduce the likelihood of requiring surgical intervention. The mean time to resolution was 19 days. Conclusions Congenital dacryocystocele is a rare presentation in the United Kingdom. Dacryocystitis and respiratory compromise commonly complicate a dacryocystocele. The use of digital massage as an early intervention is advocated and conservative measures may be sufficient in cases of uncomplicated dacryocystocele

Evolutionary Changes of the Target Sites of Two MicroRNAs Encoded in the Hox Gene Cluster of Drosophila and Other Insect Species

MicroRNAs (miRs) are noncoding RNAs that regulate gene expression at the post-transcriptional level. In animals, the target sites of a miR are generally located in the 3′ untranslated regions (UTRs) of messenger RNAs. However, how the target sites change during evolution is largely unknown. MiR-iab-4 and miR-iab-4as are known to regulate the expression of two Hox genes, Abd-A and Ubx, in Drosophila melanogaster. We have therefore studied the evolutionary changes of these two miR genes and their target sites of the Hox genes in Drosophila, other insect species, and Daphnia. Our homology search identified a single copy of each miR gene located in the same genomic position of the Hox gene cluster in all species examined. The seed nucleotide sequence was also the same for all species. Searching for the target sites in all Hox genes, we found several target sites of miR-iab-4 and miR-iab-4as in Antp in addition to Abd-A and Ubx in most insect species examined. Our phylogenetic analysis of target sites in Abd-A, Ubx, and Antp showed that the old target sites, which existed before the divergence of the 12 Drosophila species, have been well maintained in most species under purifying selection. By contrast, new target sites, which were generated during Drosophila evolution, were often lost in some species and mostly located in unalignable regions of the 3′ UTRs. These results indicate that these regions can be a potential source of generating new target sites, which results in multiple target genes for each miR in animals

Computational analysis of noncoding RNAs

Noncoding RNAs have emerged as important key players in the cell. Understanding their surprisingly diverse range of functions is challenging for experimental and computational biology. Here, we review computational methods to analyze noncoding RNAs. The topics covered include basic and advanced techniques to predict RNA structures, annotation of noncoding RNAs in genomic data, mining RNA-seq data for novel transcripts and prediction of transcript structures, computational aspects of microRNAs, and database resources.Austrian Science Fund (Schrodinger Fellowship J2966-B12)German Research Foundation (grant WI 3628/1-1 to SW)National Institutes of Health (U.S.) (NIH award 1RC1CA147187

Health and population effects of rare gene knockouts in adult humans with related parents.

Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3222 British adults of Pakistani heritage with high parental relatedness, discovering 1111 rare-variant homozygous genotypes with predicted loss of function (knockouts) in 781 genes. We observed 13.7% fewer homozygous knockout genotypes than we expected, implying an average load of 1.6 recessive-lethal-equivalent loss-of-function (LOF) variants per adult. When genetic data were linked to the individuals' lifelong health records, we observed no significant relationship between gene knockouts and clinical consultation or prescription rate. In this data set, we identified a healthy PRDM9-knockout mother and performed phased genome sequencing on her, her child, and control individuals. Our results show that meiotic recombination sites are localized away from PRDM9-dependent hotspots. Thus, natural LOF variants inform on essential genetic loci and demonstrate PRDM9 redundancy in humans.The study was funded by the Wellcome Trust (WT102627 and WT098051), Barts Charity (845/1796), Medical Research Council (MR/M009017/1). This paper presents independent research funded by the National Institute for Health Research (NIHR) under its Collaboration for Applied Health Research and Care (CLAHRC) for Yorkshire and Humber. Core support for Born in Bradford is also provided by the Wellcome Trust (WT101597). V.N. was supported by the Wellcome Trust PhD Studentship (WT099769). D.G.M. and K.K. were supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM104371. E.R.M. is funded by NIHR Cambridge Biomedical Research Centre. H.H. is supported by awards to establish the Farr Institute of Health Informatics Research, London, from the Medical Research Council, Arthritis Research UK, British Heart Foundation, Cancer Research UK, Chief Scientist Office, Economic and Social Research Council, Engineering and Physical Sciences Research Council, NIHR, National Institute for Social Care and Health Research, and Wellcome Trust.This is the author accepted manuscript. The final version is available from the American Association for the Advancement of Science via https://doi.org/10.1126/science.aac862