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

Dissipative dynamics of superconducting hybrid qubit systems

We perform a theoretical study of composite superconducting qubit systems for the case of a coupled qubit configuration based on a hybrid qubit circuit made of both charge and phase qubits, which are coupled via a sigma(x)xsigma(z) interaction. We compute the system's eigen-energies in terms of the qubit transition frequencies and the strength of the inter-qubit coupling, and describe the sensitivity of the energy crossing/anti-crossing features to such coupling. We compute the hybrid system's dissipative dynamics for the cases of i) collective and ii) independent decoherence, whereby the system interacts with one common and two different baths of harmonic oscillators, respectively. The calculations have been performed within the Bloch-Redfield formalism and we report the solutions for the populations and the coherences of the system's reduced density matrix. The dephasing and relaxation rates are explicitly calculated as a function of the heat bath temperature.Comment: To appear in J. Phys.: Conf. Series (2009

Mathematical analysis of a low cost mechanical ventilator respiratory dynamics enhanced by a sensor transducer (st) based in nanostructures of anodic aluminium oxide (aao)

Mechanical ventilation systems require a device for measuring the air flow provided to a patient in order to monitor and ensure the correct quantity of air proportionated to the patient, this device is the air flow sensor. At the beginning of the COVID-19 pandemic, flow sensors were not available in Peru because of the international supply shortage. In this context, a novel air flow sensor based on an orifice plate and an intelligent transducer was developed to form an integrated device. The proposed design was focused on simple manufacturing requirements for mass production in a developing country. CAD and CAE techniques were used in the design stage, and a mathematical model of the device was proposed and calibrated experimentally for the measured data transduction. The device was tested in its real working conditions and was therefore implemented in a breathing circuit connected to a low-cost mechanical ventilation system. Results indicate that the designed air flow sensor/transducer is a low-cost complete medical device for mechanical ventilators that is able to provide all the ventilation parameters by an equivalent electrical signal to directly display the following factors: air flow, pressure and volume over time. The evaluation of the designed sensor transducer was performed according to sundry transducer parameters such as geometrical parameters, material parameters and adaptive coefficients in the main transduction algorithm; in effect, the variety of the described results were achieved by the faster response time and robustness proportionated by transducers of nanostructures based on Anodic Aluminum Oxide (AAO), which enhanced the designed sensor/transducer (ST) during operation in intricate geographic places, such as the Andes mountains of Peru

Phase II randomized, double-blind, placebo-controlled study of tivantinib in men with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC)

Background Tivantinib is a non-ATP competitive inhibitor of c-MET receptor tyrosine kinase that may have additional cytotoxic mechanisms including tubulin inhibition. Prostate cancer demonstrates higher c-MET expression as the disease progresses to more advanced stages and to a castration resistant state. Methods 80 patients (pts) with asymptomatic or minimally symptomatic mCRPC were assigned (2:1) to either tivantinib 360 mg PO BID or placebo (P). The primary endpoint was progression free survival (PFS). Results Of the 80 pts. enrolled, 78 (52 tivantinib, 26 P) received treatment and were evaluable. Median follow up is 8.9 months (range: 2.3 to 19.6 months). Patients treated with tivantinib had significantly better PFS vs. those treated with placebo (medians: 5.5 mo vs 3.7 mo, respectively; HR = 0.55, 95% CI: 0.33 to 0.90; p = 0.02). Grade 3 febrile neutropenia was seen in 1 patient on tivantinib while grade 3 and 4 neutropenia was recorded in 1 patient each on tivantinib and placebo. Grade 3 sinus bradycardia was recorded in two men on the tivantinib arm. Conclusions Tivantinib has mild toxicity and improved PFS in men with asymptomatic or minimally symptomatic mCRPC

Tracking early mammalian organogenesis – prediction and validation of differentiation trajectories at whole organism scale

Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-h intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled timecourse of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-seq to transcriptionally determine cell fates of grafted primitive streak regions after 24 h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair

Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations.

Heterogeneity within the self-renewal durability of adult hematopoietic stem cells (HSCs) challenges our understanding of the molecular framework underlying HSC function. Gene expression studies have been hampered by the presence of multiple HSC subtypes and contaminating non-HSCs in bulk HSC populations. To gain deeper insight into the gene expression program of murine HSCs, we combined single-cell functional assays with flow cytometric index sorting and single-cell gene expression assays. Through bioinformatic integration of these datasets, we designed an unbiased sorting strategy that separates non-HSCs away from HSCs, and single-cell transplantation experiments using the enriched population were combined with RNA-seq data to identify key molecules that associate with long-term durable self-renewal, producing a single-cell molecular dataset that is linked to functional stem cell activity. Finally, we demonstrated the broader applicability of this approach for linking key molecules with defined cellular functions in another stem cell system.Work in the author’s laboratory is supported by grants from the Leukaemia and Lymphoma Research, the Medical Research Council, Cancer Research UK, Biotechnology and Biological Sciences Research Council, Leukemia Lymphoma Society, and the National Institute for Health Research Cambridge Biomedical Research Centre and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute. D.G.K. is the recipient of a Canadian Institutes of Health Research Postdoctoral Fellowship. F.B. and F.J.T. are funded by the European Research Council (starting grant “LatentCauses”). For funding for the open access charge, the core support grant was provided by the Wellcome Trust-MRC Cambridge Stem Cell Institute. We acknowledge the support of the University of Cambridge, Cancer Research UK Institute (core grant C14303/A17197), and Hutchison Whampoa Limited.This is the final published version. It first appeared at http://www.cell.com/cell-stem-cell/abstract/S1934-5909%2815%2900162-9

Clusterin is an extracellular chaperone that specifically interacts with slowly aggregating proteins on their off-folding pathway

Clusterin is an extracellular mammalian chaperone protein which inhibits stress-induced precipitation of many different proteins. The conformational state(s) of proteins that interact with clusterin and the stage(s) along the folding and off-folding (precipitation-bound) pathways where this interaction occurs were previously unknown. We investigated this by examining the interactions of clusterin with different structural forms of α-lactalbumin, γ-crystallin and lysozyme. When assessed by ELISA and native gel electrophoresis, clusterin did not bind to various stable, intermediately folded states of α-lactalbumin nor to the native form of this protein, but did bind to and inhibit the slow precipitation of reduced α-lactalbumin. Reduction-induced changes in the conformation of α-lactalbumin, in the absence and presence of clusterin, were monitored by real-time 1H NMR spectroscopy. In the absence of clusterin, an intermediately folded form of α-lactalbumin, with some secondary structure but lacking tertiary structure, aggregated and precipitated. In the presence of clusterin, this form of α-lactalbumin was stabilised in a non-aggregated state, possibly via transient interactions with clusterin prior to complexation. Additional experiments demonstrated that clusterin potently inhibited the slow precipitation, but did not inhibit the rapid precipitation, of lysozyme and γ-crystallin induced by different stresses. These results suggest that clusterin interacts with and stabilises slowly aggregating proteins but is unable to stabilise rapidly aggregating proteins. Collectively, our results suggest that during its chaperone action, clusterin preferentially recognises partly folded protein intermediates that are slowly aggregating whilst venturing along their irreversible off-folding pathway towards a precipitated protein

Emergent molecular traits of lettuce and tomato grown under wavelength-selective solar cells

The integration of semi-transparent organic solar cells (ST-OSCs) in greenhouses offers new agrivoltaic opportunities to meet the growing demands for sustainable food production. The tailored absorption/transmission spectra of ST-OSCs impacts the power generated as well as crop growth, development and responses to the biotic and abiotic environments. To characterize crop responses to ST-OSCs, we grew lettuce and tomato, traditional greenhouse crops, under three ST-OSC filters that create different light spectra. Lettuce yield and early tomato development are not negatively affected by the modified light environment. Our genomic analysis reveals that lettuce production exhibits beneficial traits involving nutrient content and nitrogen utilization while select ST-OSCs impact regulation of flowering initiation in tomato. These results suggest that ST-OSCs integrated into greenhouses are not only a promising technology for energy-neutral, sustainable and climate-change protected crop production, but can deliver benefits beyond energy considerations

DNA methylation landscapes of prostate cancer brain metastasis are shaped by early driver genetic alterations.

Metastases from primary prostate cancers to rare locations, such as the brain, are becoming more common due to longer life expectancy resulting from improved treatments. Epigenetic dysregulation is a feature of primary prostate cancer, and distinct DNA methylation profiles have been shown to be associated with the mutually exclusive SPOP mutant or TMPRSS2-ERG fusion genetic backgrounds. Using a cohort of prostate cancer brain metastases (PCBM) from 42 patients, with matched primary tumors for 17 patients, we carried out a DNA methylation analysis to examine the epigenetic distinction between primary prostate cancer and PCBM, the association between epigenetic alterations and mutational background, and particular epigenetic alterations that may be associated with PCBM. Multiregion sampling of PCBM revealed epigenetic stability within metastases. Aberrant methylation in PCBM was associated with mutational background and PRC2 complex activity, an effect that is particularly pronounced in SPOP mutant PCBM. While PCBM displayed a CpG island hypermethylator phenotype, hypomethylation at the promoters of genes involved in neuroactive ligand-receptor interaction and cell adhesion molecules such as GABRB3, CLDN8, and CLDN4 was also observed, suggesting that cells from primary tumors may require specific reprogramming to form brain metastasis. This study revealed the DNA methylation landscapes of PCBM and the potential mechanisms and effects of PCBM-associated aberrant DNA methylation