Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to ...manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.
Circular RNAs (circRNAs) are a new class of non-polyadenylated non-coding RNAs that may play important roles in many biological processes. Here we develop a single-cell universal poly(A)-independent ...RNA sequencing (SUPeR-seq) method to sequence both polyadenylated and non-polyadenylated RNAs from individual cells. This method exhibits robust sensitivity, precision and accuracy. We discover 2891 circRNAs and 913 novel linear transcripts in mouse preimplantation embryos and further analyze the abundance of circRNAs along development, the function of enriched genes, and sequence features of circRNAs. Our work is key to deciphering regulation mechanisms of circRNAs during mammalian early embryonic development.
Stable water-in-oil emulsion is essential to digital PCR and many other bioanalytical reactions that employ droplets as microreactors. We developed a novel technology to produce monodisperse emulsion ...droplets with high efficiency and high throughput using a bench-top centrifuge. Upon centrifugal spinning, the continuous aqueous phase is dispersed into monodisperse droplet jets in air through a micro-channel array (MiCA) and then submerged into oil as a stable emulsion. We performed dPCR reactions with a high dynamic range through the MiCA approach, and demonstrated that this cost-effective method not only eliminates the usage of complex microfluidic devices and control systems, but also greatly suppresses the loss of materials and cross-contamination. MiCA-enabled highly parallel emulsion generation combines both easiness and robustness of picoliter droplet production, and breaks the technical challenges by using conventional lab equipment and supplies.
Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman ...scattering within the Raman‐silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label‐free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state‐of‐the‐art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non‐invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live‐cell imaging.
Raman imaging: In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman‐silent region of a cell (see picture). Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label‐free microscopies.
Single-ceU genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome ...regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple om- ics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations （CNVs）, DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs gen- erally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells.
This study aimed to evaluate the global scientific output of research on pain catastrophizing and explore the hotspots and frontiers from 2010 to 2020 using bibliometric methods.
Publications ...regarding pain catastrophizing published from 2010 to 2020 were extracted from the Web of Science Core Collection. CiteSpace was used to analyze the number of publications, countries, institutions, journals, authors, cited references, and keywords using standard bibliometric indicators.
A total of 1,576 publications on pain catastrophizing were retrieved from 2010 to December 31, 2020. The number and rate of the annual publications gradually increased totally. Pain (130) was the most productive journal. Meanwhile, Pain ranked first in the frequency (1,432) and centrality (0.31) of the cited journals. The most productive country and institution in this frequency field were the United States (642) and the University of Washington (73), respectively. Jensen MP (34) was the most prolific author, and Sullivan MJL (1,196) ranked first among the cited authors. In the ranking of frequency in the cited references, the first article was a critical review about pain catastrophizing published by Quartana (100). The keyword "Low back pain" had the highest frequency (556). "Total hip" was identified as a frontier research item for 2016-2020.
The findings of this bibliometric study provide the current status and trends in the clinical research of pain catastrophizing and may help researchers to identify hot topics and explore new research directions in this field.
Whole-genome amplification (WGA) for next-generation sequencing has seen wide applications in biology and medicine when characterization of the genome of a single cell is required. High uniformity ...and fidelity of WGA is needed to accurately determine genomic variations, such as copy number variations (CNVs) and single-nucleotide variations (SNVs). Prevailing WGA methods have been limited by fluctuation of the amplification yield along the genome, as well as false-positive and -negative errors for SNV identification. Here, we report emulsion WGA (eWGA) to overcome these problems. We divide single-cell genomic DNA into a large number (10⁵) of picoliter aqueous droplets in oil. Containing only a few DNA fragments, each droplet is led to reach saturation of DNA amplification before demulsification such that the differences in amplification gain among the fragments are minimized. We demonstrate the proof-of-principle of eWGA with multiple displacement amplification (MDA), a popular WGA method. This easy-to-operate approach enables simultaneous detection of CNVs and SNVs in an individual human cell, exhibiting significantly improved amplification evenness and accuracy.
The ability to manipulate and sort droplets is a fundamental issue in droplet-based microfluidics. Various lab-on-a-chip applications can only be realized if droplets are systematically categorized ...and sorted. These micron-sized droplets act as ideal reactors which compartmentalize different biological and chemical reagents. Array processing of these droplets hinges on the competence of the sorting and integration into the fluidic system. Recent technological advances only allow droplets to be actively sorted at the rate of kilohertz or less. In this review, we present state-of-the-art technologies which are implemented to efficiently sort droplets. We classify the concepts according to the type of energy implemented into the system. We also discuss various key issues and provide insights into various systems.
This review presents the fundamentals of different active methods for sorting droplets in microfluidics.
Obesity and related metabolic diseases are becoming worldwide epidemics that lead to increased death rates and heavy health care costs. Effective treatment options have not been found yet. Here, ...based on the observation that baicalin, a flavonoid from the herbal medicine Scutellaria baicalensis, has unique antisteatosis activity, we performed quantitative chemoproteomic profiling and identified carnitine palmitoyltransferase 1 (CPT1), the controlling enzyme for fatty acid oxidation, as the key target of baicalin. The flavonoid directly activated hepatic CPT1 with isoform selectivity to accelerate the lipid influx into mitochondria for oxidation. Chronic treatment of baicalin ameliorated diet-induced obesity (DIO) and hepatic steatosis and led to systemic improvement of other metabolic disorders. Disruption of the predicted binding site of baicalin on CPT1 completely abolished the beneficial effect of the flavonoid. Our discovery of baicalin as an allosteric CPT1 activator opens new opportunities for pharmacological treatment of DIO and associated sequelae.
Somatic mutations that accumulate in normal tissues are associated with ageing and disease.sup.1,2. Here we performed a comprehensive genomic analysis of 1,737 morphologically normal tissue biopsies ...of 9 organs from 5 donors. We found that somatic mutation accumulations and clonal expansions were widespread, although to variable extents, in morphologically normal human tissues. Somatic copy number alterations were rarely detected, except for in tissues from the oesophagus and cardia. Endogenous mutational processes with the SBS1 and SBS5 mutational signatures are ubiquitous among normal tissues, although they exhibit different relative activities. Exogenous mutational processes operate in multiple tissues from the same donor. We reconstructed the spatial somatic clonal architecture with sub-millimetre resolution. In the oesophagus and cardia, macroscopic somatic clones that expanded to hundreds of micrometres were frequently seen, whereas in tissues such as the colon, rectum and duodenum, somatic clones were microscopic in size and evolved independently, possibly restricted by local tissue microstructures. Our study depicts a body map of somatic mutations and clonal expansions from the same individual.
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