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RNA editing is a post-transcriptional modification with a cell-specific manner and important biological implications. Although single-cell RNA-seq (scRNA-seq) is an effective method for studying cellular heterogeneity, it is difficult to detect and study RNA editing events from scRNA-seq data because of the low sequencing coverage. To overcome this, we develop a computational method to systematically identify RNA editing sites of cell types from scRNA-seq data. To demonstrate its effectiveness, we apply it to scRNA-seq data of human hematopoietic stem/progenitor cells (HSPCs) with an annotated lineage differentiation relationship according to previous research and study the impacts of RNA editing on hematopoiesis. The dynamic editing patterns reveal the relevance of RNA editing on different HSPCs. For example, four microRNA (miRNA) target sites on 3ʹ UTR of EIF2AK2 are edited across all HSPC populations, which may abolish the miRNA-mediated inhibition of EIF2AK2. Elevated EIF2AK2 may thus activate the integrated stress response (ISR) pathway to initiate global translational attenuation as a protective mechanism to maintain cellular homeostasis during HSPCs' differentiation. Besides, our findings also indicate that RNA editing plays an essential role in the coordination of lineage commitment and self-renewal of hematopoietic stem cells (HSCs). Taken together, we demonstrate the capacity of scRNA-seq data to exploit RNA editing events of cell types, and find that RNA editing may exert multiple modules of regulation in hematopoietic processes. [ABSTRACT FROM AUTHOR]

Background: Hematopoietic stem cells (HSCs) from different sources show varied repopulating capacity, and HSCs lose their stemness after long‐time ex vivo culture. A deep understanding of these phenomena may provide helpful insights for HSCs. Methods: Here, we applied single‐cell RNA‐seq (scRNA‐seq) to analyse the naïve and stimulated human CD34+ cells from cord blood (CB) and mobilised peripheral blood (mPB). Results: We collected over 16 000 high‐quality single‐cell data to construct a comprehensive inference map and characterised the HSCs under a quiescent state on the hierarchy top. Then, we compared HSCs in CB with those in mPB and HSCs of naïve samples to those of cultured samples, and identified stemness‐related genes (SRGs) associated with cell source (CS‐SRGs) and culture time (CT‐SRGs), respectively. Interestingly, CS‐SRGs and CT‐SRGs share genes enriched in the signalling pathways such as mRNA catabolic process, translational initiation, ribonucleoprotein complex biogenesis and cotranslational protein targeting to membrane, suggesting dynamic protein translation and processing may be a common requirement for stemness maintenance. Meanwhile, CT‐SRGs are enriched in pathways involved in glucocorticoid and corticosteroid response that affect HSCs homing and engraftment. In contrast, CS‐SRGs specifically contain genes related to purine and ATP metabolic process, which is crucial for HSC homeostasis in the stress settings. Particularly, when CT‐SRGs are used as reference genes for the construction of the development trajectory of CD34+ cells, lymphoid and myeloid lineages are clearly separated after HSCs/MPPs. Finally, we presented an application through a small‐scale drug screening using Connectivity Map (CMap) against CT‐SRGs. A small molecule, cucurbitacin I, was found to efficiently expand HSCs ex vivo while maintaining its stemness. Conclusions: Our findings provide new perspectives for understanding HSCs, and the strategy to identify candidate molecules through SRGs may be applicable to study other stem cells. [ABSTRACT FROM AUTHOR]

β-thalassemia, caused by mutations in the human hemoglobin β (HBB) gene, is one of the most common genetic diseases in the world. The HBB −28(A>G) mutation is one of the five most common mutations in Chinese patients with β-thalassemia. However, few studies have been conducted to understand how this mutation affects the expression of pathogenesis-related genes, including globin genes, due to limited homozygote clinical materials. Therefore, we developed an efficient technique using CRISPR/Cas9 combined with asymmetric single-stranded oligodeoxynucleotides (assODNs) to generate a K562 cell model with HBB −28(A>G) named K562–28(A>G). Then, we systematically analyzed the differences between K562–28(A>G) and K562 at the transcriptome level by high-throughput RNA-seq before and after erythroid differentiation. We found that the HBB −28(A>G) mutation not only disturbed the transcription of HBB , but also decreased the expression of HBG , which may further aggravate the thalassemia phenotype and partially explain the more severe clinical outcome of β-thalassemia patients with the HBB −28(A>G) mutation. Moreover, we found that the K562–28(A>G) cell line is more sensitive to hypoxia and shows a defective erythrogenic program compared with K562 before differentiation. Importantly, all abovementioned abnormalities in K562–28(A>G) were reversed after correction of this mutation with CRISPR/Cas9 and assODNs, confirming the specificity of these phenotypes. Overall, this is the first time to analyze the effects of the HBB −28(A>G) mutation at the whole-transcriptome level based on isogenic cell lines, providing a landscape for further investigation of the mechanism of β-thalassemia with the HBB −28(A>G) mutation. [ABSTRACT FROM AUTHOR]

Human mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from a three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from a monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSC spheroids but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stem medium (MiPS) adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR-derived spheroids. Interestingly, genes related to inflammatory response, immune response, and angiogenesis were upregulated in spheroids at day 6 and qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were replated in normal FBS medium, cells formed a typical spindle-shaped morphology and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSC spheroids for clinical research and therapy. [ABSTRACT FROM AUTHOR]

The live replication-competent modified vaccinia virus Tiantan (MVTT) is an attractive vaccine vector, yet little is known about its tissue tropism and pathology in vivo. Recently, we demonstrated that a recombinant MVTT expressing the spike glycoprotein of SARS-CoV (namely MVTT-S) is superior to the non-replicating modified vaccinia Ankara (MVA-S) for inducing high level of neutralizing antibodies through mucosal vaccination. In this study, we further determined the tissue tropism and safety of MVTT-S after the vaccine was administrated through various routes including: intramuscular (i.m.), intranasal (i.n.), and intravaginal (i.vag.) inoculations, respectively. Using real-time PCR, nested PCR, immunohistochemistry and in situ hybridization assays, we found that MVTT-S was able to produce a transient infection in all cases within 48 hr post-inoculation, yet the major site of viral replication in various tissues or organs was dependent on the route of viral administration. We demonstrated that i.m. injection of MVTT-S primarily targeted draining inguinal lymph nodes, whereas mucosal inoculation had broader range of tissue infections. i.n. inoculation involved infections in lungs, kidneys, spleens and cervix lymph nodes while i.vag. administration targeted uteruses, ovaries, kidneys and spleens. Critically, the infection did not cause severe pathogenic consequences in infected tissues, which was consistent to the attenuated phenotype of MVTT-S. Our findings have implications for the optimization of vaccination route and for studies on the correlation between the magnitude of immune responses and the extent of tissue involvement in vivo. J. Med. Virol. 82: 727-734, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Ocean wave spectrum and surface currents can be determined from a series of spatial wave images recorded by an X-band marine radar. In the absence of a surface current, the three-dimensional spectral energy found by using the series of images will be confined to a trajectory defined by the still water dispersion relationship. The presence of a surface current will make the three-dimensional spectral energy show a corresponding Doppler shift which may determine the current using the least squares method and obtain the directional wave spectrum. On the basis of conventional wave spectrum and directional function, the paper emulates a series of X-band radar images considering shadowing modulation and simulates numerically the three-dimensional image spectrum both with and without a surface current, calculates the current velocity by virtue of the Doppler shift, and obtains the two-dimensional image spectrum. Finally the paper analyzes measured wave level elevation—a function of time t to obtain one-dimensional image spectrum, and the data comes from an X-band radar in McMaster University. [ABSTRACT FROM AUTHOR]