Assessing the reliability of spike-in normalization for analyses of single-cell RNA sequencing data.

By profiling the transcriptomes of individual cells, single-cell RNA sequencing provides unparalleled resolution to study cellular heterogeneity. However, this comes at the cost of high technical noise, including cell-specific biases in capture efficiency and library generation. One strategy for removing these biases is to add a constant amount of spike-in RNA to each cell and to scale the observed expression values so that the coverage of spike-in transcripts is constant across cells. This approach has previously been criticized as its accuracy depends on the precise addition of spike-in RNA to each sample. Here, we perform mixture experiments using two different sets of spike-in RNA to quantify the variance in the amount of spike-in RNA added to each well in a plate-based protocol. We also obtain an upper bound on the variance due to differences in behavior between the two spike-in sets. We demonstrate that both factors are small contributors to the total technical variance and have only minor effects on downstream analyses, such as detection of highly variable genes and clustering. Our results suggest that scaling normalization using spike-in transcripts is reliable enough for routine use in single-cell RNA sequencing data analyses.This work was supported by Cancer Research UK (core funding to JCM, award no. A17197), the University of Cambridge and Hutchison Whampoa Limited. JCM was also supported by core funding from EMBL. LHV was supported by an EMBL Interdisciplinary Postdoctoral fellowship. Work in the G ottgens group was supported by Cancer Research UK, Bloodwise, the National Institute of Diabetes and Digestive and Kidney Diseases, the Leukemia and Lymphoma Society and core infrastructure grants from the Wellcome Trust and the Medical Research Council to the Cambridge Stem Cell Institute

Multi-omics of the esophageal microenvironment identifies signatures associated with progression of Barrett’s esophagus

Background: The enrichment of Gram-negative bacteria of oral origin in the esophageal microbiome has been associated with the development of metaplasia. However, to date, no study has comprehensively assessed the relationships between the esophageal microbiome and the host. Methods: Here, we examine the esophageal microenvironment in gastro-esophageal reflux disease and metaplasia using multi-omics strategies targeting the microbiome and host transcriptome, followed by targeted culture, comparative genomics, and host-microbial interaction studies of bacterial signatures of interest. Results: Profiling of the host transcriptome from esophageal mucosal biopsies revealed profound changes during metaplasia. Importantly, five biomarkers showed consistent longitudinal changes with disease progression from reflux disease to metaplasia. We showed for the first time that the esophageal microbiome is distinct from the salivary microbiome and the enrichment of Campylobacter species as a consistent signature in disease across two independent cohorts. Shape fitting and matrix correlation identified associations between the microbiome and host transcriptome profiles, with a novel co-exclusion relationship found between Campylobacter and napsin B aspartic peptidase. Targeted culture of Campylobacter species from the same cohort revealed a subset of isolates to have a higher capacity to survive within primary human macrophages. Comparative genomic analyses showed these isolates could be differentiated by specific genomic features, one of which was validated to be associated with intracellular fitness. Screening for these Campylobacter strain-specific signatures in shotgun metagenomics data from another cohort showed an increase in prevalence with disease progression. Comparative transcriptomic analyses of primary esophageal epithelial cells exposed to the Campylobacter isolates revealed expression changes within those infected with strains with high intracellular fitness that could explain the increased likelihood of disease progression. Conclusions: We provide a comprehensive assessment of the esophageal microenvironment, identifying bacterial strain-specific signatures with high relevance to progression of metaplasia

ngs_backbone: a pipeline for read cleaning, mapping and SNP calling using Next Generation Sequence

Background: The possibilities offered by next generation sequencing (NGS) platforms are revolutionizing biotechnological laboratories. Moreover, the combination of NGS sequencing and affordable high-throughput genotyping technologies is facilitating the rapid discovery and use of SNPs in non-model species. However, this abundance of sequences and polymorphisms creates new software needs. To fulfill these needs, we have developed a powerful, yet easy-to-use application. Results: The ngs_backbone software is a parallel pipeline capable of analyzing Sanger, 454, Illumina and SOLiD (Sequencing by Oligonucleotide Ligation and Detection) sequence reads. Its main supported analyses are: read cleaning, transcriptome assembly and annotation, read mapping and single nucleotide polymorphism (SNP) calling and selection. In order to build a truly useful tool, the software development was paired with a laboratory experiment. All public tomato Sanger EST reads plus 14.2 million Illumina reads were employed to test the tool and predict polymorphism in tomato. The cleaned reads were mapped to the SGN tomato transcriptome obtaining a coverage of 4.2 for Sanger and 8.5 for Illumina. 23,360 single nucleotide variations (SNVs) were predicted. A total of 76 SNVs were experimentally validated, and 85% were found to be real. Conclusions: ngs_backbone is a new software package capable of analyzing sequences produced by NGS technologies and predicting SNVs with great accuracy. In our tomato example, we created a highly polymorphic collection of SNVs that will be a useful resource for tomato researchers and breeders. The software developed along with its documentation is freely available under the AGPL license and can be downloaded from http://bioinf. comav.upv.es/ngs_backbone/ or http://github.com/JoseBlanca/franklin.Blanca Postigo, JM.; Pascual Bañuls, L.; Ziarsolo Areitioaurtena, P.; Nuez Viñals, F.; Cañizares Sales, J. (2011). Ngs_backbone: a pipeline for read cleaning, mapping and SNP calling using Next Generation Sequence. BMC Genomics. 12:1-8. doi:10.1186/1471-2164-12-285S1812Metzker ML: Sequencing technologies - the next generation. Nature Reviews Genetics. 2010, 11 (1): 31-46. 10.1038/nrg2626.454 sequencing. [ http://www.454.com/ ]Illumina Inc. [ http://www.illumina.com/ ]Flicek P, Birney E: Sense from sequence reads: methods for alignment and assembly (vol 6, pg S6, 2009). 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[ http://1000genomes.org/wiki/doku.php?id=1000_genomes:analysis:vcf4.0 ]The seqanswers internet forum. [ http://seqanswers.com/ ]Blankenberg D, Taylor J, Schenck I, He JB, Zhang Y, Ghent M, Veeraraghavan N, Albert I, Miller W, Makova KD, Ross CH, Nekrutenko A: A framework for collaborative analysis of ENCODE data: Making large-scale analyses biologist-friendly. Genome Research. 2007, 17 (6): 960-964. 10.1101/gr.5578007.CloVR Automated Sequence Analysis from Your Desktop. [ http://clovr.org/ ]Papanicolaou A, Stierli R, Ffrench-Constant RH, Heckel DG: Next generation transcriptomes for next generation genomes using est2assembly. Bmc Bioinformatics. 2009, 10:Applied Biosystems by life technologies. [ http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing.html ]Wall PK, Leebens-Mack J, Chanderbali AS, Barakat A, Wolcott E, Liang HY, Landherr L, Tomsho LP, Hu Y, Carlson JE, Ma H, Schuster SC, Soltis DE, Soltis PS, Altman N, dePamphilis CW: Comparison of next generation sequencing technologies for transcriptome characterization. Bmc Genomics. 2009, 10:Murchison EP, Tovar C, Hsu A, Bender HS, Kheradpour P, Rebbeck CA, Obendorf D, Conlan C, Bahlo M, Blizzard CA, Pyecroft S, Kreiss A, Kellis M, Stark A, Harkins TT, Marshall Graves JA, Woods GM, Hanon GJ, Papenfuss AT: The Tasmanian Devil Transcriptome Reveals Schwann Cell Origins of a Clonally Transmissible Cancer. Science. 2010, 327 (5961): 84-87. 10.1126/science.1180616.Parchman TL, Geist KS, Grahnen JA, Benkman CW, Buerkle CA: Transcriptome sequencing in an ecologically important tree species: assembly, annotation, and marker discovery. Bmc Genomics. 2010, 11:Babik W, Stuglik M, Qi W, Kuenzli M, Kuduk K, Koteja P, Radwan J: Heart transcriptome of the bank vole (Myodes glareolus): towards understanding the evolutionary variation in metabolic rate. BMC Genomics. 2010, 11: 390-10.1186/1471-2164-11-390.Miller JC, Tanksley SD: RFLP analysis of phylogenetic-relationships and genetic-variation in the genus Lycopersicon. Theoretical and Applied Genetics. 1990, 80 (4): 437-448.Williams CE, Stclair DA: Phenetic relationships and levels of variability detected by restriction-fragment-length-polymorphism and random amplified polymorphic DNA analysis of cultivated and wild accessions of Lycopersicon-esculentum. Genome. 1993, 36 (3): 619-630. 10.1139/g93-083.Rick CM: Tomato, Lycopersicon esculentum (Solanaceae). Evolution of crop plants. Edited by: Simmonds NW. 1976, London: Longman Group, 268-273.Labate JA, Baldo AM: Tomato SNP discovery by EST mining and resequencing. Molecular Breeding. 2005, 16 (4): 343-349. 10.1007/s11032-005-1911-5.Yano K, Watanabe M, Yamamoto N, Maeda F, Tsugane T, Shibata D: Expressed sequence tags (EST) database of a miniature tomato cultivar, Micro-Tom. Plant and Cell Physiology. 2005, 46: S139-S139.Jimenez-Gomez JM, Maloof JN: Sequence diversity in three tomato species: SNPs, markers, and molecular evolution. Bmc Plant Biology. 2009, 9:Yang WC, Bai XD, Kabelka E, Eaton C, Kamoun S, van der Knaap E, Francis D: Discovery of single nucleotide polymorphisms in Lycopersicon esculentum by computer aided analysis of expressed sequence tags. Molecular Breeding. 2004, 14 (1): 21-34.Van Deynze A, Stoffel K, Buell CR, Kozik A, Liu J, van der Knaap E, Francis D: Diversity in conserved genes in tomato. Bmc Genomics. 2007, 8:Sim SC, Robbins MD, Chilcott C, Zhu T, Francis DM: Oligonucleotide array discovery of polymorphisms in cultivated tomato (Solanum lycopersicum L.) reveals patterns of SNP variation associated with breeding. 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Young people’s perceptions of smartphone-enabled self-testing and online care for sexually transmitted infections: qualitative interview study

Background Control of sexually transmitted infections (STI) is a global public health priority. Despite the UK’s free, confidential sexual health clinical services, those at greatest risk of STIs, including young people, report barriers to use. These include: embarrassment regarding face-to-face consultations; the time-commitment needed to attend clinic; privacy concerns (e.g. being seen attending clinic); and issues related to confidentiality. A smartphone-enabled STI self-testing device, linked with online clinical care pathways for treatment, partner notification, and disease surveillance, is being developed by the eSTI2 consortium. It is intended to benefit public health, and could do so by increasing testing among populations which underutilise existing services and/or by enabling rapid provision of effective treatment. We explored its acceptability among potential users. Methods In-depth interviews were conducted in 2012 with 25 sexually-experienced 16–24 year olds, recruited from Further Education colleges in an urban, high STI prevalence area. Thematic analysis was undertaken. Results Nine females and 16 males participated. 21 self-defined as Black; three, mixed ethnicity; and one, Muslim/Asian. 22 reported experience of STI testing, two reported previous STI diagnoses, and all had owned smartphones. Participants expressed enthusiasm about the proposed service, and suggested that they and their peers would use it and test more often if it were available. Utilizing sexual healthcare was perceived to be easier and faster with STI self-testing and online clinical care, which facilitated concealment of STI testing from peers/family, and avoided embarrassing face-to-face consultations. Despite these perceived advantages to privacy, new privacy concerns arose regarding communications technology: principally the risk inherent in having evidence of STI testing or diagnosis visible or retrievable on their phone. Some concerns arose regarding the proposed self-test’s accuracy, related to self-operation and the technology’s novelty. Several expressed anxiety around the possibility of being diagnosed and treated without any contact with healthcare professionals. Conclusions Remote STI self-testing and online care appealed to these young people. It addressed barriers they associated with conventional STI services, thus may benefit public health through earlier detection and treatment. Our findings underpin development of online care pathways, as part of ongoing research to create this complex e-health intervention

Risk factors associated with fatal pulmonary hemorrhage in locally advanced non-small cell lung cancer treated with chemoradiotherapy

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to identify the risk factors associated with fatal pulmonary hemorrhage (PH) in patients with locally advanced non-small cell lung cancer (NSCLC), treated with chemoradiotherapy.</p> <p>Methods</p> <p>The medical records of 583 patients with locally advanced NSCLC, who were treated with chemoradiotherapy between July 1992 and December 2009 were reviewed. Fatal PH was defined as PH leading to death within 24 h of its onset. Tumor cavitation size was defined by the cavitation diameter/tumor diameter ratio and was classified as minimum (< 0.25), minor (≥ 0.25, but < 0.5), and major (≥ 0.5).</p> <p>Results</p> <p>Of the 583 patients, 2.1% suffered a fatal PH. The numbers of patients with minimum, minor, and major cavitations were 13, 11, and 14, respectively. Among the 38 patients with tumor cavitation, all 3 patients who developed fatal PH had major cavitations. On multivariate analysis, the presence of baseline major cavitation (odds ratio, 17.878), and a squamous cell histology (odds ratio, 5.491) proved to be independent significant risk factors for fatal PH. Interestingly, all patients with fatal PH and baseline major cavitation were found to have tumors with squamous cell histology, and the occurrence of fatal PH in patients having both risk factors was 33.3%.</p> <p>Conclusions</p> <p>Patients at high risk of fatal PH could be identified using a combination of independent risk factors.</p

FISim: A new similarity measure between transcription factor binding sites based on the fuzzy integral

Background Regulatory motifs describe sets of related transcription factor binding sites (TFBSs) and can be represented as position frequency matrices (PFMs). De novo identification of TFBSs is a crucial problem in computational biology which includes the issue of comparing putative motifs with one another and with motifs that are already known. The relative importance of each nucleotide within a given position in the PFMs should be considered in order to compute PFM similarities. Furthermore, biological data are inherently noisy and imprecise. Fuzzy set theory is particularly suitable for modeling imprecise data, whereas fuzzy integrals are highly appropriate for representing the interaction among different information sources.Results We propose FISim, a new similarity measure between PFMs, based on the fuzzy integral of the distance of the nucleotides with respect to the information content of the positions. Unlike existing methods, FISim is designed to consider the higher contribution of better conserved positions to the binding affinity. FISim provides excellent results when dealing with sets of randomly generated motifs, and outperforms the remaining methods when handling real datasets of related motifs. Furthermore, we propose a new cluster methodology based on kernel theory together with FISim to obtain groups of related motifs potentially bound by the same TFs, providing more robust results than existing approaches.Conclusion FISim corrects a design flaw of the most popular methods, whose measures favour similarity of low information content positions. We use our measure to successfully identify motifs that describe binding sites for the same TF and to solve real-life problems. In this study the reliability of fuzzy technology for motif comparison tasks is proven.This work has been carried out as part of projects P08-TIC-4299 of J. A., Sevilla and TIN2006-13177 of DGICT, Madrid

Monitoring credit risk in the social economy sector by means of a binary goal programming model

The final publication is available at Springer via http://dx.doi.org/10.1007/s11628-012-0173-7Monitoring the credit risk of firms in the social economy sector presents a considerable challenge, since it is difficult to calculate ratings with traditional methods such as logit or discriminant analysis, due to the relatively small number of firms in the sector and the low default rate among cooperatives. This paper intro- duces a goal programming model to overcome such constraints and to successfully manage credit risk using economic and financial information, as well as expert advice. After introducing the model, its application to a set of Spanish cooperative societies is described.García García, F.; Guijarro Martínez, F.; Moya Clemente, I. (2013). Monitoring credit risk in the social economy sector by means of a binary goal programming model. 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Effects of protein–carbohydrate supplementation on immunity and resistance training outcomes: a double-blind, randomized, controlled clinical trial

Purpose: To examine the impact of ingesting hydrolyzed beef protein, whey protein, and carbohydrate on resistance training outcomes, body composition, muscle thickness, blood indices of health and salivary human neutrophil peptides (HNP1-3), as reference of humoral immunity followed an 8-week resistance training program in college athletes. Methods: Twenty-seven recreationally physically active males and females (n = 9 per treatment) were randomly assigned to one of the three groups: hydrolyzed beef protein, whey protein, or non-protein isoenergetic carbohydrate. Treatment consisted of ingesting 20 g of supplement, mixed with orange juice, once a day immediately post-workout or before breakfast on non-training days. Measurements were performed pre- and post-intervention on total load (kg) lifted at the first and last workout, body composition (via plethysmography) vastus medialis thickness (mm) (via ultrasonography), and blood indices of health. Salivary HNP1-3 were determined before and after performing the first and last workout. Results: Salivary concentration and secretion rates of the HNP1-3 decreased in the beef condition only from pre-first-workout (1.90 ± 0.83 μg/mL; 2.95 ± 2.83 μg/min, respectively) to pre-last-workout (0.92 ± 0.63 μg/mL, p = 0.025, d = 1.03; 0.76 ± 0.74 μg/min, p = 0.049, d = 0.95), and post-last-workout (0.95 ± 0.60 μg/mL, p = 0.032, d = 1.00; 0.59 ± 0.52 μg/min, p = 0.027, d = 1.02). No other significant differences between groups were observed. Conclusions: Supplementation with a carbohydrate–protein beverage may support resistance training outcomes in a comparable way as the ingestion of only carbohydrate. Furthermore, the ingestion of 20 g of hydrolyzed beef protein resulted in a decreased level and secretion rates of the HNP1-3 from baseline with no negative effect on blood indices of health

Targeted calcium influx boosts cytotoxic T lymphocyte function in the tumour microenvironment

Adoptive cell transfer utilizing tumour-targeting cytotoxic T lymphocytes (CTLs) is one of the most effective immunotherapies against haematological malignancies, but significant clinical success has not yet been achieved in solid tumours due in part to the strong immunosuppressive tumour microenvironment. Here, we show that suppression of CTL killing by CD4+CD25+Foxp+ regulatory T cell (Treg) is in part mediated by TGFβ-induced inhibition of inositol trisphosphate (IP3) production, leading to a decrease in T cell receptor (TCR)-dependent intracellular Ca2+ response. Highly selective optical control of Ca2+ signalling in adoptively transferred CTLs enhances T cell activation and IFN-γ production in vitro, leading to a significant reduction in tumour growth in mice. Altogether, our findings indicate that the targeted optogenetic stimulation of intracellular Ca2+ signal allows for the remote control of cytotoxic effector functions of adoptively transferred T cells with outstanding spatial resolution by boosting T cell immune responses at the tumour sites

Secreted Human Amyloid Precursor Protein Binds Semaphorin 3a and Prevents Semaphorin-Induced Growth Cone Collapse

The amyloid precursor protein (APP) is well known for giving rise to the amyloid-β peptide and for its role in Alzheimer's disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα695 (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα695 binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα695 inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (Kd≤8·10−9 M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins