3D Chromatin Structures of Mature Gametes and Structural Reprogramming during Mammalian Embryogenesis.

High-order chromatin structure plays important roles in gene expression regulation. Knowledge of the dynamics of 3D chromatin structures during mammalian embryo development remains limited. We report the 3D chromatin architecture of mouse gametes and early embryos using an optimized Hi-C method with low-cell samples. We find that mature oocytes at the metaphase II stage do not have topologically associated domains (TADs). In sperm, extra-long-range interactions (>4 Mb) and interchromosomal interactions occur frequently. The high-order structures of both the paternal and maternal genomes in zygotes and two-cell embryos are obscure but are gradually re-established through development. The establishment of the TAD structure requires DNA replication but not zygotic genome activation. Furthermore, unmethylated CpGs are enriched in A compartment, and methylation levels are decreased to a greater extent in A compartment than in B compartment in embryos. In summary, the global reprogramming of chromatin architecture occurs during early mammalian development.

Transient upregulation of IRF1 during exit from naive pluripotency confers viral protection.

Stem cells intrinsically express a subset of genes which are normally associated with interferon stimulation and the innate immune response. However, the expression of these interferon-stimulated genes (ISG) in stem cells is independent from external stimuli such as viral infection. Here, we show that the interferon regulatory factor 1, Irf1, is directly controlled by the murine formative pluripotency gene regulatory network and transiently upregulated during the transition from naive to formative pluripotency. IRF1 binds to regulatory regions of a conserved set of ISGs and is required for their faithful expression upon exit from naive pluripotency. We show that in the absence of IRF1, cells exiting the naive pluripotent stem cell state are more susceptible to viral infection. Irf1 therefore acts as a link between the formative pluripotency network, regulation of innate immunity genes, and defense against viral infections during formative pluripotency.

Global evaluation of carbon neutrality and peak carbon dioxide emissions: current challenges and future outlook.

With the acceleration of urbanization and industrialization, carbon neutrality and peak carbon dioxide emissions have become common sustainability goals worldwide. However, there are few literature statistics and econometric analyses targeting carbon neutrality and peak carbon dioxide emissions, especially the publication trends, geographic distribution, citation literature, and research hotspots. To conduct an in-depth analysis of existing research fields and future perspectives in this research area, 1615 publications from the Web of Science Core Collection, between 2010 and 2020, were evaluated by using three analysis tools, under the framework of the bibliometrics method. These publications are distributed between the start-up (2010-2015) and the stable development (2016-2020) phases. Cluster analysis suggests three areas of ongoing research: energy-related carbon emissions, methane emissions, and energy biomass. Overall, future trends in this field include cumulative carbon emissions, the residential building sector, methane emission measurement, nitrogen fertilization, land degradation neutrality, and sciamachy satellite methane measurement. Finally, this paper further examines the most comprehensive coverage of nitrogen fertilization and the most recent research of the residential building sector. In view of the statistical clusters from 1615 publications, this paper provides new insights and perspectives for climate-environment-related researchers and policymakers. Specifically, countries could apply nitrogen fertilizer to crops according to the conditions of different regions. Additionally, experiences from developed countries could be learned from, including optimizing the energy supply structure of buildings and increasing the use of clean energy to reduce CO2 emissions from buildings.

CTCF, BEAF-32, and CP190 are not required for the establishment of TADs in early Drosophila embryos but have locus-specific roles.

The boundaries of topologically associating domains (TADs) are delimited by insulators and/or active promoters; however, how they are initially established during embryogenesis remains unclear. Here, we examined this during the first hours of Drosophila embryogenesis. DNA-FISH confirms that intra-TAD pairwise proximity is established during zygotic genome activation (ZGA) but with extensive cell-to-cell heterogeneity. Most newly formed boundaries are occupied by combinations of CTCF, BEAF-32, and/or CP190. Depleting each insulator individually from chromatin revealed that TADs can still establish, although with lower insulation, with a subset of boundaries (~10%) being more dependent on specific insulators. Some weakened boundaries have aberrant gene expression due to unconstrained enhancer activity. However, the majority of misexpressed genes have no obvious direct relationship to changes in domain-boundary insulation. Deletion of an active promoter (thereby blocking transcription) at one boundary had a greater impact than deleting the insulator-bound region itself. This suggests that cross-talk between insulators and active promoters and/or transcription might reinforce domain boundary insulation during embryogenesis.

Identification of TREM2-positive tumor-associated macrophages in esophageal squamous cell carcinoma: implication for poor prognosis and immunotherapy modulation.

Background: It is now understood that the effectiveness of checkpoint immunotherapy can be impaired by immunosuppressive tumor-associated macrophages (TAMs). Nonetheless, the impact of different TAM subpopulations on the antitumor immune response remains unclear, mainly due to their heterogeneity. Herein, we identified a novel TAM subpopulation in esophageal squamous cell carcinoma (ESCC) that might contribute to poor clinical outcomes and immunotherapy modulation. Methods and results: We analyzed two single-cell RNA sequencing (scRNA-seq) datasets (GSE145370 and GSE160269) of esophageal squamous cell carcinoma to identify a novel TREM2-positive TAM subpopulation characterized by upregulation of TREM2, C1QC, C1QB, C1QA, SPP1, and APOE. Quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that these genes were significantly overexpressed in ESCC. Multiplex immunofluorescence validated the infiltration of TREM2+ TAMs in ESCC tissues, which correlated with poorer overall survival (OS). The scRNA-seq analysis in dataset GSE120575 indicated significant enrichment of TREM2+ TAMs in melanoma patients (n=48) with poor immunotherapy response, which had an identical gene signature with TREM2+ TAMs from ESCC. Analysis of 29 bulk-RNA melanoma samples from dataset GSE78220 revealed that a gene signature of 40 genes associated with TREM2+ TAMs was upregulated in the transcriptome of melanomas that did not respond to anti-PD1 therapy. Validation in the TCGA ESCC cohort (n=80) showed that a high enrichment score of the TREM2+ TAM was associated with poor prognosis. In addition, 10 ESCC patients treated with anti-PD1 therapy suggested that patients who are not sensitive to immunotherapy have higher density of TREM2+TAMs infiltration. Conclusion: Overall, TREM2+ TAM infiltration in ESCC is associated with poor prognosis and may serve as a biomarker for predicting outcomes and immunotherapy modulation in this patient population. modulation; single-cell RNA sequencing.

Sustainable development and health assessment model of higher education in India: A mathematical modeling approach.

The Coronavirus Disease 2019 has resulted in a transition from physical education to online learning, leading to a collapse of the established educational order and a wisdom test for the education governance system. As a country seriously affected by the pandemic, the health of the Indian higher education system urgently requires assessment to achieve sustainable development and maximize educational externalities. This research systematically proposes a health assessment model from four perspectives, including educational volume, efficiency, equality, and sustainability, by employing the Technique for Order Preference by Similarity to an Ideal Solution Model, Principal Component Analysis, DEA-Tobit Model, and Augmented Solow Model. Empirical results demonstrate that India has high efficiency and an absolute health score in the higher education system through multiple comparisons between India and the other selected countries while having certain deficiencies in equality and sustainability. Additionally, single-target and multiple-target path are simultaneously proposed to enhance the Indian current education system. The multiple-target approach of the India-China-Japan-Europe-USA process is more feasible to achieve sustainable development, which would improve the overall health score from .351 to .716. This finding also reveals that the changes are relatively complex and would take 91.5 years considering the relationship between economic growth rates and crucial indicators. Four targeted policies are suggested for each catching-up period, including expanding and increasing the social funding sources, striving for government expenditure support to improve infrastructures, imposing gender equality in education, and accelerating the construction of high-quality teachers.

Construction and optimization of a base editor based on the MS2 system.

BACKGROUND: Catalytic defect Cas9-cytosine deaminase fusion is widely used in base editing. The Multiple copy numbers of the MS2 binding site (MBS) can recruit multiple MS2 coat proteins (MCPs), which are usually applied to amplify signals. Our study aimed to apply the MS2 signal amplification system to the base editing system in order to achieve simultaneous mutations of multiple bases at the target genome site. METHODS: Multiple copy numbers of the MS2 were ligated to the 3'-end of sgRNA, and MCP was fused to the 5'-end of cytosine deaminases. The MS2 was recognized by MCP to recruit cytosine deaminase for base substitutions (C-T) at the target site. Different Cas9 variants, different cytosine deaminases and different copy numbers of MS2 were tested in this system, and the different versions of base editors were compared by editing efficiency and window. RESULTS: In this study, dCas9, nCas9 (D10A) and nCas9 (H840A) were used. Among these 3 Cas9 variants, dCas9 exhibited higher base mutation efficiency. Two cytosine deaminases were then applied and the efficiency of rAPOBEC1 deaminase was found to be higher than AID. We also increased the copy numbers of MS2 linked to sgRNA from 2 to 12. Disappointingly, the sgRNA-12x MS2 did not improve the editing efficiency or increase the editing window. CONCLUSION: An optimal version of base editor based on the MS2 system, MS2-BE-rAPOBEC1 (sgRNA-2x MS2, MCP-rAPOBEC1 and dCas9), was obtained. This tool can simultaneously mutate multiple bases at the target site, providing a new approach for the study of genome functions.

BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity.

Base editor (BE), containing a cytidine deaminase and catalytically defective Cas9, has been widely used to perform base editing. However, the narrow editing window of BE limits its utility. Here, we developed a new editing technology named as base editor for programming larger C to U (T) scope (BE-PLUS) by fusing 10 copies of GCN4 peptide to nCas9(D10A) for recruiting scFv-APOBEC-UGI-GB1 to the target sites. The new system achieves base editing with a broadened window, resulting in an increased genome-targeting scope. Interestingly, the new system yielded much fewer unwanted indels and non-C-to-T conversions. We also demonstrated its potential use in gene disruption across the whole genome through induction of stop codons (iSTOP). Taken together, the BE-PLUS system offers a new editing tool with increased editing window and enhanced fidelity.

Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing.

A recently developed adenine base editor (ABE) efficiently converts A to G and is potentially useful for clinical applications. However, its precision and efficiency in vivo remains to be addressed. Here we achieve A-to-G conversion in vivo at frequencies up to 100% by microinjection of ABE mRNA together with sgRNAs. We then generate mouse models harboring clinically relevant mutations at Ar and Hoxd13, which recapitulates respective clinical defects. Furthermore, we achieve both C-to-T and A-to-G base editing by using a combination of ABE and SaBE3, thus creating mouse model harboring multiple mutations. We also demonstrate the specificity of ABE by deep sequencing and whole-genome sequencing (WGS). Taken together, ABE is highly efficient and precise in vivo, making it feasible to model and potentially cure relevant genetic diseases.

Generation of isogenic single and multiplex gene knockout mice by base editing-induced STOP.

Although CRISPR/Cas9 has been widely used to generate knockout mice, two major limitations remain: the founders usually carry a mixture of genotypes, and mosaicism harboring multiple genotypes. Therefore, it takes a long time to get homozygous mutants. Recently developed base editing (BE) system, which introduces C-to-T conversion without double strand DNA cleavage, has been used to introduce artificial stop codons (i-STOP) to prematurely terminate translation, providing a cleaner strategy for genome engineering. Using this strategy, we generated CD160 KO and VISTA/CD160 double KO mice by microinjection of a single sgRNA targeting CD160 and a mixture of sgRNAs targeting VISTA and CD160, respectively. The BE system induced STOP efficiently in mouse embryos and consequently in founder mice without detectable off-target. Most interestingly, the majority of the mutants harbor same genetic modifications, indicating we generated isogenic single and multiplex gene mutant mice by BE-induced STOP. We also obtained homozygous mutant mouse in F1 mice, demonstrating the accelerated strategy in generating animal models.