Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver

In this study, the CasRx system was demonstrated to efficiently and functionally knock down genes related to metabolism functions, including Pten, Pcsk9 and lncLstr, in mouse hepatocytes. CasRx-mediated simultaneous knockdown of multiple genes was also achieved by sgRNA arrays, providing a useful strategy to modulate complex metabolism networks. Moreover, the AAV (adeno-associated virus)-mediated delivery of CasRx and Pcsk9 sgRNAs into mouse liver successfully decreased serum PCSK9, resulting in significant reduction of serum cholesterol levels. Importantly, CasRx-mediated knockdown of Pcsk9 is reversible and Pcsk9 could be repeatedly down-regulated, providing an effective strategy to reversibly modulate metabolic genes. The present work supplies a successful proof-of-concept trial that suggests efficient and regulatory knockdown of target metabolic genes for a designed metabolism modulation in the liver

Spatiotemporal Distribution of HIV Self-testing Kits Purchased on the Web and Implications for HIV Prevention in China: Population-Based Study

BackgroundHIV self-testing (HIVST) holds great promise for expanding HIV testing. Nonetheless, large-scale data on HIVST behavior are scant. Millions of HIVST kits are sold through e-commerce platforms each year. ObjectiveThis study aims to analyze the spatiotemporal distribution of the HIVST kit–purchasing population (HIVSTKPP) in China. MethodsDeidentified transaction data were retrieved from a leading e-commerce platform in China. A joinpoint regression model was used to examine annual trends of the HIVSTKPP rates by calculating average annual percentage change. Bayesian spatiotemporal analysis was performed to locate hot spots with HIVSTKPP rates. Spatial autocorrelation analysis and space-time cluster analysis were conducted to identify clusters of HIVSTKPP. High-high clusters of HIVSTKPP can be identified by spatial autocorrelation analysis, and high-high clusters indicate that a region and its surrounding region jointly had a higher-than-average HIVSTKPP rate. Spatial regression analysis was used to elucidate the association between the number of HIV testing facilities, urbanization ratio (the proportion of urban population in the total population), and gross domestic product per capita and the HIVSTKPP. ResultsBetween January 1, 2016, and December 31, 2019, a total of 2.18 million anonymous persons in China placed 4.15 million orders and purchased 4.51 million HIVST kits on the web. In each of these 4 years, the observed monthly size of the HIVSTKPP peaked in December, the month of World AIDS Day. HIVSTKPP rates per 100,000 population significantly increased from 20.62 in 2016 to 64.82 in 2019 (average annual percentage change=48.2%; P<.001). Hot spots were mainly located in municipalities, provincial capitals, and large cities, whereas high-high clusters and high-demand clusters were predominantly detected in cities along the southeast coast. We found positive correlations between a region’s number of HIV testing facilities, urbanization ratio, and gross domestic product per capita and the HIVSTKPP. ConclusionsOur study identified key areas with larger demand for HIVST kits for public health policy makers to reallocate resources and optimize the HIV care continuum. Further research combining spatiotemporal patterns of HIVST with HIV surveillance data is urgently needed to identify potential gaps in current HIV-monitoring practices

Enhanced C‐To‐T and A‐To‐G Base Editing in Mitochondrial DNA with Engineered DdCBE and TALED

Abstract Mitochondrial base editing with DddA‐derived cytosine base editor (DdCBE) is limited in the accessible target sequences and modest activity. Here, the optimized DdCBE tools is presented with improved editing activity and expanded C‐to‐T targeting scope by fusing DddA11 variant with different cytosine deaminases with single‐strand DNA activity. Compared to previous DdCBE based on DddA11 variant alone, fusion of the activation‐induced cytidine deaminase (AID) from Xenopus laevis not only permits cytosine editing of 5′‐GC‐3′ sequence, but also elevates editing efficiency at 5′‐TC‐3′, 5′‐CC‐3′, and 5′‐GC‐3′ targets by up to 25‐, 10‐, and 6‐fold, respectively. Furthermore, the A‐to‐G editing efficiency is significantly improved by fusing the evolved DddA6 variant with TALE‐linked deoxyadenosine deaminase (TALED). Notably, the authors introduce the reported high‐fidelity mutations in DddA and add nuclear export signal (NES) sequences in DdCBE and TALED to reduce off‐target editing in the nuclear and mitochondrial genome while improving on‐target editing efficiency in mitochondrial DNA (mtDNA). Finally, these engineered mitochondrial base editors are shown to be efficient in installing mtDNA mutations in human cells or mouse embryos for disease modeling. Collectively, the study shows broad implications for the basic study and therapeutic applications of optimized DdCBE and TALED

Single-cell RNA sequencing reveals cellular and molecular landscape of fetal cystic hygroma

Abstract Background The molecular mechanism of fetal cystic hygroma (CH) is still unclear, and no study has previously reported the transcriptome changes of single cells in CH. In this study, single-cell transcriptome sequencing (scRNA-seq) was used to investigate the characteristics of cell subsets in the lesion tissues of CH patients. Methods Lymphoid tissue collected from CH patients and control donors for scRNA-seq analysis. Differentially expressed gene enrichment in major cell subpopulations as well as cell-cell communication were analyzed. At the same time, the expression and interactions of important VEGF signaling pathway molecules were analyzed, and potential transcription factors that could bind to KDR (VEGFR2) were predicted. Results The results of scRNA-seq showed that fibroblasts accounted for the largest proportion in the lymphatic lesions of CH patients. There was a significant increase in the proportion of lymphatic endothelial cell subsets between the cases and controls. The VEGF signaling pathway is enriched in lymphatic endothelial cells and participates in the regulation of cell-cell communication between lymphatic endothelial cells and other cells. The key regulatory gene KDR in the VEGF signaling pathway is highly expressed in CH patients and interacts with other differentially expressed EDN1, TAGLN, and CLDN5 Finally, we found that STAT1 could bind to the KDR promoter region, which may play an important role in promoting KDR up-regulation. Conclusion Our comprehensive delineation of the cellular composition in tumor tissues of CH patients using single-cell RNA-sequencing identified the enrichment of lymphatic endothelial cells in CH and highlighted the activation of the VEGF signaling pathway in lymphoid endothelial cells as a potential modulator. Simple summary The molecular and cellular pathogenesis of fetal cystic hygroma (CH) remains largely unknown. This study examined the distribution and gene expression signature of each cell subpopulation and the possible role of VEGF signaling in lymphatic endothelial cells in regulating the progression of CH by single-cell transcriptome sequencing. The enrichment of lymphatic endothelial cells in CH and the activation of the VEGF signaling pathway in lymphatic endothelial cells provide some clues to the pathogenesis of CH from the perspective of cell subpopulations

Engineering a transposon-associated TnpB-ωRNA system for efficient gene editing and phenotypic correction of a tyrosinaemia mouse model

Abstract Transposon-associated ribonucleoprotein TnpB is known to be the ancestry endonuclease of diverse Cas12 effector proteins from type-V CRISPR system. Given its small size (408 aa), it is of interest to examine whether engineered TnpB could be used for efficient mammalian genome editing. Here, we showed that the gene editing activity of native TnpB from Deinococcus radiodurans (ISDra2 TnpB) in mouse embryos was already higher than previously identified small-sized Cas12f1. Further stepwise engineering of noncoding RNA (ωRNA or reRNA) component of TnpB significantly elevated the nuclease activity of TnpB. Notably, an optimized TnpB-ωRNA system could be efficiently delivered in vivo with single adeno-associated virus (AAV) and corrected the disease phenotype in a tyrosinaemia mouse model. Thus, the engineered miniature TnpB system represents a new addition to the current genome editing toolbox, with the unique feature of the smallest effector size that facilitate efficient AAV delivery for editing of cells and tissues

Neutrophil to Lymphocyte Ratio Predicts Adverse Cardiovascular Outcome in Peritoneal Dialysis Patients Younger than 60 Years Old

Background. Neutrophil to lymphocyte ratio (NLR) is a new inflammatory marker; the relationship between NLR and adverse cardiovascular (CV) prognosis has been gradually emphasized in the general population. However, their association in peritoneal dialysis (PD) patients remains unclear. Methods. From January 1, 2010, to May 31, 2017, a total of 1652 patients were recruited. NLR was categorized in triplicates: NLR≤2.74, 2.743.96. Kaplan-Meier cumulative incidence curve and multivariable COX regression analysis were used to determine the relationship between NLR and the incidence of adverse CV outcome, while a competitive risk model was applied to assess the effects of other outcomes on adverse CV prognosis. Besides, forest plot was investigated to analyze the adverse CV prognosis in different subgroups. Results. During follow-up, 213 new-onset CV events and 153 CV disease (CVD) deaths were recorded. Multivariable COX regression models showed that the highest tertile of NLR level was associated with increased risk of CV events (HR=1.39, 95%CI=1.01‐1.93, P=0.046) and CVD mortality (HR=1.81, 95%CI=1.22‐2.69, P=0.003), while compared to the lowest tertile. Competitive risk models showed that the differences in CV event (P<0.001) and CVD mortality (P=0.004) among different NLR groups were still significant while excluding the effects of other outcomes. In subgroups, with each 1 increased in the NLR level, adjusted HR of new-onset CV event was 2.02 (95%CI=1.26−3.23, P=0.003) and CVD mortality was 2.98 (95%CI=1.58−5.62, P=0.001) in the younger group (age<60 years). Conclusions. NLR is an independent risk factor for adverse CV prognosis in PD patients younger than 60 years old