Detection and removal of barcode swapping in single-cell RNA-seq data.

Barcode swapping results in the mislabelling of sequencing reads between multiplexed samples on patterned flow-cell Illumina sequencing machines. This may compromise the validity of numerous genomic assays; however, the severity and consequences of barcode swapping remain poorly understood. We have used two statistical approaches to robustly quantify the fraction of swapped reads in two plate-based single-cell RNA-sequencing datasets. We found that approximately 2.5% of reads were mislabelled between samples on the HiSeq 4000, which is lower than previous reports. We observed no correlation between the swapped fraction of reads and the concentration of free barcode across plates. Furthermore, we have demonstrated that barcode swapping may generate complex but artefactual cell libraries in droplet-based single-cell RNA-sequencing studies. To eliminate these artefacts, we have developed an algorithm to exclude individual molecules that have swapped between samples in 10x Genomics experiments, allowing the continued use of cutting-edge sequencing machines for these assays

European Network of Breast Development and Cancer turned 10 years: a growing family of mammary gland researchers

The European Network for Breast Development and Cancer (ENBDC), a worldwide network (http://www.enbdc.org/), celebrated its tenth anniversary with a fantastic meeting last March 15-17, 2018 in Weggis with 76 attendees

Pooling across cells to normalize single-cell RNA sequencing data with many zero counts.

Normalization of single-cell RNA sequencing data is necessary to eliminate cell-specific biases prior to downstream analyses. However, this is not straightforward for noisy single-cell data where many counts are zero. We present a novel approach where expression values are summed across pools of cells, and the summed values are used for normalization. Pool-based size factors are then deconvolved to yield cell-based factors. Our deconvolution approach outperforms existing methods for accurate normalization of cell-specific biases in simulated data. Similar behavior is observed in real data, where deconvolution improves the relevance of results of downstream analyses.All authors were supported by core funding from Cancer Research UK (code: SW73).This is the final version of the article. It first appeared from Biomed Central via https://doi.org/10.1186/s13059-016-0947-

Effect of flosulide, a selective cyclooxygenase 2 inhibitor, on passive Heymann nephritis in the rat

Effect of flosulide, a selective cyclooxygenase 2 inhibitor, on passive Heymann nephritis in the rat.BackgroundNonsteroidal anti-inflammatory drugs (NSAIDs) induce an inhibition of cyclooxygenase (COX), an enzyme that makes prostaglandins. Two isoforms of COX exist: COX-1 represents the constitutively expressed enzyme, whereas COX-2 is the inducible isoform. This study investigated the role of COX-2 in the inflammatory processes of the kidneys of rats with passive Heymann nephritis (PHN), and focused of the effect of a selective COX-2 inhibitor, flosulide. COX-2–selective inhibitors are thought to represent potent anti-inflammatory agents without major renal side effects.MethodsPHN was induced by injecting heterologous Fx1A antiserum into female Wistar rats. Two treatment groups, each consisting of 12 rats with PHN, received either 3 or 9 mg of flosulide/kg body wt/day and were compared with untreated controls. After four weeks, the generation of thromboxane B2 (TxB2) and 6-keto-PGF1α were determined in renal tissue and in urine. COX-2 protein expression was investigated by Western blotting using a selective antibody.ResultsRats with PHN exhibited a marked proteinuria of 71 ± 8 mg/24 hr as compared with 2.0 ± 0.3 mg/24 hr in healthy controls (P < 0.01). Treatment with flosulide reduced the proteinuria to 26.1 ± 9 mg/24 hr at 3 mg flosulide/kg body wt/day and 35.5 ± 6 mg/24 hr at 9 mg/kg body wt/day, which was equivalent to a reduction of proteinuria by a maximum of 65% (P < 0.05). This was accompanied by an increase in glomerular TxB2 from 3073 ± 355 to 5255 ± 1041 pg/mg glomerular protein and 6-keto-PGF1α from 1702 ± 161 to 2724 ± 770 pg/mg glomerular protein in rats with PHN. COX-2 protein expression was also highly elevated in comparison to healthy controls. Low-dose flosulide treatment had no effect on COX protein expression and renal prostaglandin formation. High-dose flosulide treatment reduced renal prostaglandin production and caused a marked decline in COX-1 and COX-2 protein expression. Urine prostanoid excretion remained unchanged in all therapeutic groups. There was a small though significant reduction in renal creatinine clearance from 0.86 ml ± 0.2/min in untreated controls to 0.6 ml ± 0.1/min in flosulide-treated rats with PHN (P < 0.01) after four weeks.ConclusionsUnder the influence of flosulide, a highly COX-2–selective inhibitor, we observed an antiproteinuric drug effect. The inflammation in PHN induced COX-2 protein expression that was not affected by low-dose flosulide. COX-1 and COX-2 protein expression was affected by high-dose flosulide, which therefore might lose its selectivity. High-dose flosulide induced a decrease in glomerular prostanoid production possibly because of COX-1 inhibition. Our results suggest that the therapeutic use of flosulide in proteinuria seems advantageous and deserves further studies because the basal prostaglandin levels remain unchanged in the low-dose–treated group, indicating that the compensatory capacity of prostaglandin production, which is essential for the regulation of renal hemodynamics, is maintained

Transcriptional changes in the mammary gland during lactation revealed by single cell sequencing of cells from human milk.

Funder: Wellcome TrustUnder normal conditions, the most significant expansion and differentiation of the adult mammary gland occurs in response to systemic reproductive hormones during pregnancy and lactation to enable milk synthesis and secretion to sustain the offspring. However, human mammary tissue remodelling that takes place during pregnancy and lactation remains poorly understood due to the challenge of acquiring samples. We report here single-cell transcriptomic analysis of 110,744 viable breast cells isolated from human milk or non-lactating breast tissue, isolated from nine and seven donors, respectively. We found that human milk largely contains epithelial cells belonging to the luminal lineage and a repertoire of immune cells. Further transcriptomic analysis of the milk cells identified two distinct secretory cell types that shared similarities with luminal progenitors, but no populations comparable to hormone-responsive cells. Taken together, our data offers a reference map and a window into the cellular dynamics that occur during human lactation and may provide further insights on the interplay between pregnancy, lactation and breast cancer.UKRI-MRC project grant ((MR/S036059/1) UKRI-BBSRC project grant (BB/S006745/1) Breast Cancer Now Project Grant (2017MayPR907) CRUK Programme Foundation Award (DCRPGF\100010

Single-cell RNAseq reveals seven classes of colonic sensory neuron.

OBJECTIVE: Integration of nutritional, microbial and inflammatory events along the gut-brain axis can alter bowel physiology and organism behaviour. Colonic sensory neurons activate reflex pathways and give rise to conscious sensation, but the diversity and division of function within these neurons is poorly understood. The identification of signalling pathways contributing to visceral sensation is constrained by a paucity of molecular markers. Here we address this by comprehensive transcriptomic profiling and unsupervised clustering of individual mouse colonic sensory neurons. DESIGN: Unbiased single-cell RNA-sequencing was performed on retrogradely traced mouse colonic sensory neurons isolated from both thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglia associated with lumbar splanchnic and pelvic spinal pathways, respectively. Identified neuronal subtypes were validated by single-cell qRT-PCR, immunohistochemistry (IHC) and Ca2+-imaging. RESULTS: Transcriptomic profiling and unsupervised clustering of 314 colonic sensory neurons revealed seven neuronal subtypes. Of these, five neuronal subtypes accounted for 99% of TL neurons, with LS neurons almost exclusively populating the remaining two subtypes. We identify and classify neurons based on novel subtype-specific marker genes using single-cell qRT-PCR and IHC to validate subtypes derived from RNA-sequencing. Lastly, functional Ca2+-imaging was conducted on colonic sensory neurons to demonstrate subtype-selective differential agonist activation. CONCLUSIONS: We identify seven subtypes of colonic sensory neurons using unbiased single-cell RNA-sequencing and confirm translation of patterning to protein expression, describing sensory diversity encompassing all modalities of colonic neuronal sensitivity. These results provide a pathway to molecular interrogation of colonic sensory innervation in health and disease, together with identifying novel targets for drug development

The submarine Azores Plateau : Evidence for a waning mantle plume?

The submarine Azores Plateau in the Central Northern Atlantic has generally been considered to represent a large igneous plateau formed some 10 Ma by widespread volcanism, however a lack of age progression amongst the younger submarine and subaerial volcanism, an irregular distribution of platform-related magmas east and west of the Mid-Atlantic Ridge, a strong tectonic stress regime, and a lack of abundant tholeiitic compositions that reflect initial, high degrees of melting is not easily explainable in the framework of a classic, long-lived mantle plume model. Here, we present new bathymetric and seismic data from the submarine Azores Plateau obtained during cruises M113/1 and M128 with the German R/V Meteor. Our new data combined with prior geochemical and petrological studies indicate that the majority of the western Azores Plateau may indeed have formed during the arrival of a short-lived mantle melting anomaly at 10 Ma. However, our new data also indicate that volcanismPeer reviewe

Diagrama de Fluxo das Excretas como instrumento integrador da governança de ecossistemas de esgotamento sanitário aplicado à realidade brasileira

O presente trabalho apresenta uma reflexão sobre utilização do diagrama de fluxo das excretas (SFD) sob o ponto de vista da governança aplicada aos sistemas de esgotamento sanitário. A utilização do SFD explicita conexões entre diferentes modais de esgotamento sanitário. Com o auxílio do SFD pode-se identificar que mesmo locais com elevados índices de cobertura podem carecer de serviços adequados em alguma etapa da cadeia de serviço. A ferramenta permite a comparação de diferentes modelos de esgotamento, resumindo a informação em um índice de gerenciamento seguro das excretas, considerando a coexistência de soluções, com e sem rede, evidenciando que o debate entre sistemas centralizados e descentralizados pode ser considerado obsoleto, já que na maioria dos casos se percebe a formação de um complexo ecossistema com diversos serviços ao longo da cadeia, englobando elementos estruturais e estruturantes