Comparison of clustering tools in R for medium-sized 10x Genomics single-cell RNA-sequencing data [version 1; referees: 1 approved, 2 approved with reservations]

Background: The commercially available 10x Genomics protocol to generate droplet-based single-cell RNA-seq (scRNA-seq) data is enjoying growing popularity among researchers. Fundamental to the analysis of such scRNA-seq data is the ability to cluster similar or same cells into non-overlapping groups. Many competing methods have been proposed for this task, but there is currently little guidance with regards to which method to use. Methods: Here we use one gold standard 10x Genomics dataset, generated from the mixture of three cell lines, as well as three silver standard 10x Genomics datasets generated from peripheral blood mononuclear cells to examine not only the accuracy but also robustness of a dozen methods. Results: We found that some methods, including Seurat and Cell Ranger, outperform other methods, although performance seems to be dependent on the complexity of the studied system. Furthermore, we found that solutions produced by different methods have little in common with each other. Conclusions: In light of this, we conclude that the choice of clustering tool crucially determines interpretation of scRNA-seq data generated by 10x Genomics. Hence practitioners and consumers should remain vigilant about the outcome of 10x Genomics scRNA-seq analysis

Electric-field Control of Magnetism with Emergent Topological Hall Effect in SrRuO3 through Proton Evolution

Ionic substitution forms an essential pathway to manipulate the carrier density and crystalline symmetry of materials via ion-lattice-electron coupling, leading to a rich spectrum of electronic states in strongly correlated systems. Using the ferromagnetic metal SrRuO3 as a model system, we demonstrate an efficient and reversible control of both carrier density and crystalline symmetry through the ionic liquid gating induced protonation. The insertion of protons electron-dopes SrRuO3, leading to an exotic ferromagnetic to paramagnetic phase transition along with the increase of proton concentration. Intriguingly, we observe an emergent topological Hall effect at the boundary of the phase transition as the consequence of the newly-established Dzyaloshinskii-Moriya interaction owing to the breaking of inversion symmetry in protonated SrRuO3 with the proton compositional film-depth gradient. We envision that electric-field controlled protonation opens a novel strategy to design material functionalities

Spatiotemporal transcriptomic maps of whole mouse embryos at the onset of organogenesis

Spatiotemporal orchestration of gene expression is required for proper embryonic development. The use of single-cell technologies has begun to provide improved resolution of early regulatory dynamics, including detailed molecular definitions of most cell states during mouse embryogenesis. Here we used Slide-seq to build spatial transcriptomic maps of complete embryonic day (E) 8.5 and E9.0, and partial E9.5 embryos. To support their utility, we developed sc3D, a tool for reconstructing and exploring three-dimensional ‘virtual embryos’, which enables the quantitative investigation of regionalized gene expression patterns. Our measurements along the main embryonic axes of the developing neural tube revealed several previously unannotated genes with distinct spatial patterns. We also characterized the conflicting transcriptional identity of ‘ectopic’ neural tubes that emerge in Tbx6 mutant embryos. Taken together, we present an experimental and computational framework for the spatiotemporal investigation of whole embryonic structures and mutant phenotypes

Effect of acupuncture on lung cancer-related fatigue: study protocol for a multi-center randomized controlled trial

BACKGROUND: Fatigue is one of the primary symptoms in lung cancer, with a prevalence of 88.0% in survivors of cancer, and an even higher prevalence post resection surgery. Effective fatigue control after lung cancer surgery is important for patient recovery and quality of life. Some studies have shown that acupuncture might be effective in treating cancer-related fatigue; however, randomized controlled trials (RCTs) of suitable sample size are limited. METHOD/DESIGN: This is a multi-center, patient-blinded RCT. A total of 320 eligible patients will be recruited in four centers and randomly assigned to either the acupuncture group or the sham acupuncture group in a 1:1 ratio. Treatment will be given twice per week for 12 sessions. Treatment will be given at acupoints GV20, GV29, CV12, CV6, CV4, and bilateral LI4, LR3, SP6, ST36. The primary outcome will be assessed using the Chinese version of The Brief Fatigue Inventory. The secondary outcomes will be measured using The European Organization for Research and The Treatment of Cancer Quality of Life Questionnaire, and the Hamilton Rating Scale for Depression. The primary outcome will be assessed at all main points (baseline, the 3rd week, the 6th week, and at follow up time points) and the secondary outcomes will be assessed at baseline and the 6th week. Intention-to-treat analysis will be used in this RCT. DISCUSSION: This trial protocol provides an example of the clinical application acupuncture treatment in the management of lung cancer-related fatigue. If the acupuncture treatment protocol confirms that acupuncture is an effective and safe option for lung cancer-related fatigue, it can be adopted as a standardized treatment. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR1900022831. Registered on 27 April 2019. URL: http://www.chictr.org.cn/showproj.aspx?proj=37823

Difference in regional neural fluctuations and functional connectivity in Crohn’s disease: a resting-state functional MRI study

Patients with Crohn’s disease (CD) are shown to have abnormal changes in brain structures. This study aimed to further investigate whether these patients have abnormal brain activities and network connectivity. Sixty patients with CD and 40 healthy controls (HCs) underwent resting-state functional magnetic resonance imaging (fMRI) scans. Amplitude of low-frequency fluctuation (ALFF) and seed-based functional connectivity (FC) were used to assess differences in spontaneous regional brain activity and functional connectivity. Compared to the HCs, patients with CD showed significantly higher ALFF values in hippocampus and parahippocampus (HIPP/paraHIPP), anterior cingulate cortex, insula, superior frontal cortex and precuneus. The ALFF values were significantly lower in secondary somatosensory cortex (S2), precentral gyrus, and medial prefrontal cortex. Functional connectivities between left HIPP and left inferior temporal cortex, and right middle cingulate cortex, HIPP, and fusiform area were significantly lower. The functional connectivities between right HIPP and right inferior orbitofrontal cortex and left HIPP were also significantly lower. Patients with CD showed higher or lower spontaneous activity in multiple brain regions. Altered activities in these brain regions may collectively reflect abnormal function and regulation of visceral pain and sensation, external environmental monitoring, and cognitive processing in these patients. Lower functional connectivity of the hippocampus-limbic system was observed in these patients. These findings may provide more information to elucidate the neurobiological mechanisms of the disease

Different brain responses to electro-acupuncture and moxibustion treatment in patients with Crohn's disease

This study aimed to investigate changes in resting state brain activity in remissive Crohn's Disease (CD) patients after electro-acupuncture or moxibustion treatment. Fifty-two CD patients and 36 healthy subjects were enrolled, and 36 patients were equally and randomly assigned to receive either electro-acupuncture or moxibustion treatment for twelve weeks. We used resting state functional magnetic resonance imaging to assess Regional Homogeneity (ReHo) levels, and Crohn's Disease Activity Index (CDAI) and Inflammatory Bowel Disease Questionnaire (IBDQ) scores to evaluate disease severity and quality of life. The results show that (i) The ReHo levels in CD patients were significantly increased in cortical but decreased in subcortical areas, and the coupling between them was declined. (ii) Both treatments decreased CDAI, increased IBDQ scores, and normalized the ReHo values of the cortical and subcortical regions. (iii) ReHo changes in multiple cortical regions were significantly correlated with CDAI score decreases. ReHo changes in several subcortical regions in the electro-acupuncture group, and those of several cortical regions in the moxibustion group, were correlated with reduced CDAI. These findings suggest that both treatments improved cortex-subcortical coupling in remissive CD patients, but electro-acupuncture regulated homeostatic afferent processing network, while moxibustion mainly regulated the default mode network of the brain

Reversible manipulation of the magnetic state in SrRuO3 through electric-field controlled proton evolution

Ionic substitution forms an essential pathway to manipulate the structural phase, carrier density and crystalline symmetry of materials via ion-electron-lattice coupling, leading to a rich spectrum of electronic states in strongly correlated systems. Using the ferromagnetic metal SrRuO3 as a model system, we demonstrate an efficient and reversible control of both structural and electronic phase transformations through the electric-field controlled proton evolution with ionic liquid gating. The insertion of protons results in a large structural expansion and increased carrier density, leading to an exotic ferromagnetic to paramagnetic phase transition. Importantly, we reveal a novel protonated compound of HSrRuO3 with paramagnetic metallic as ground state. We observe a topological Hall effect at the boundary of the phase transition due to the proton concentration gradient across the film-depth. We envision that electric-field controlled protonation opens up a pathway to explore novel electronic states and material functionalities in protonated material systems

Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia.

Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance

New protocols and computational tools for scRNAseq analysis

© 2020 Luyi TianThe fast development of single cell RNA sequencing (scRNAseq) presents new challenges in data analysis and opportunities for protocol development. To address challenges in data preprocessing, I developed scPipe, an R/Bioconductor package that integrates barcode demultiplexing, read alignment, UMI-aware gene-level quantification and quality control of raw sequencing data generated by multiple protocols. Results from scPipe can be used as input for downstream analyses and can be easily incorporated into R-based pipelines with other tools. In order to compare different computational methods for scRNAseq data, I generated a realistic benchmark experiment that included single cells and admixtures of cells or RNA to create `pseudo cells' from up to five distinct cancer cell lines. Multiple datasets were generated using both droplet and plate-based scRNAseq protocols and processed by scPipe. We found pipelines suited to different types of data for different tasks. Our data and analysis provide a comprehensive framework for benchmarking most common scRNA-seq analysis steps. Finally, I developed single cell full-length transcript sequencing by sampling (FLT-seq), together with the computational pipeline FLAMES to perform isoform discovery and quantification, splicing analysis and mutation detection in single cells. With FLT-seq and FLAMES, I performed a comprehensive characterization of the full-length isoform landscape in single cells of different types and species and found conserved functional modules that were enriched for alternative transcript usage in different cell populations, including ribosome biogenesis and mRNA splicing. The datasets, protocols and tools that I developed and generated are useful resources for the single cell research community