β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 （A〉G） mutations is one of the three most common mutations in China and Southeast Asia ...patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor （BE） system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 （A〉G） mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 （A〉G） homozygous mutation. Data showed that base editor could precisely correct HBB -28 （A〉G） mutation in the patient＇s primary cells. To model homozygous mutation disease embryos, we consb＇ucted nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured （IVM） oocytes.Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.
Genome editing tools such as the clustered regularly interspaced short palindromic repeat （CRISPR）-associated system （Cas） have been widely used to modify genes in model systems including animal ...zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear （3PN） zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene （HBB）. However, the efficiency of homologous recombination directed repair （HDR） of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene （HBD）, which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
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•Single PI/rGO composite nanofiber by in-situ polymerization and in-situ thermal conversion.•Tensile strength of single nanofiber up to 4.2 GPa (PI/rGO-1.0% single nanofiber).•Modulus ...of single nanofiber up to 121 GPa (PI/rGO-1.2% single nanofiber).•Homogeneous dispersion of rGO by in-situ strategies.•In-situ strategies enhance the interfacial interaction between rGO and PI fibrous matrix.
High mechanical performance electrospun polymeric nanofibers are highly desired for practical applications, especially as reinforcements for composites. However, most of the electrospun polymeric nanofibers present tensile strength < 3 GPa. To overcome such limitation, this work successfully prepared single rGO reinforced polyimide composite nanofiber with tensile strength up to 4.2 GPa (PI/rGO-1.0%) and modulus up to 121 GPa (PI/rGO-1.2%) by applying in-situ polymerization, electrospinning, and in-situ thermal conversion. These mechanical properties are higher than other polymer-based electrospun nanofibers, and 45% and 236% higher than those of neat PI single nanofiber, respectively. The in-situ strategies provide the homogeneous dispersion of rGO in single electrospun nanofibers and enhance the interfacial interaction between rGO and PI. In addition, the PI/rGO composite nanofibers also present excellent thermal stability with glass transition temperature (Tg) > 295 °C, and the 5% thermal decomposition temperature (T5%) > 539 °C. This work would open a new route for the preparation of high performance electrospun nanofibers for composites.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Common polygenic diseases result from compounded risk contributed by multiple genetic variants, meaning that simultaneous correction or introduction of single nucleotide variants is required for ...disease modeling and gene therapy. Here, we show precise, efficient, and simultaneous multiplex base editing of up to three target sites across 11 genes/loci in cynomolgus monkey embryos using CRISPR-based cytidine- and adenine-base editors. Unbiased whole genome sequencing demonstrates high specificity of base editing in monkey embryos. Our data demonstrate feasibility of multiplex base editing for polygenic disease modeling in primate zygotes.
China’s rapid industrialization and urbanization have led to ecological and environmental problems, particularly air pollution. Digitalization has become a key component in high-quality development ...to overcome the previous high-energy and high-pollution development model. However, a certain degree of “green blindness” exists in the digital development process, and the impact on air pollution is not always positive. Therefore, the impact of the digital economy on air pollution is worth exploring. In this study, the authors examines the emissions increase and reduction effect mechanisms of the digital economy on air pollution using panel data in 30 provinces in China from 2011 to 2020. The two-tier stochastic frontier model showed that the interaction between the emissions increase effect and emissions reduction effect of the digital economy reduced the actual air pollution emissions level below the frontier level by 0.15%, on average. Overall air pollution level is characterized prominently by emission reduction, owing to the asymmetric bilateral effect of the digital economy. Second, the time trend characteristics of the net effect of the digital economy on air pollution emissions showed a wave-like change; the average values of the net effect in the three major regions (i.e., the east, central, and west) were all negative. Third, along with the development of digital economy, human capital levels, and general economic levels, the emissions reduction effect of the digital economy on air pollution has strengthened, such that the net effect became positive to negative. However, significant heterogeneous characteristics were noted in the effects of the digital economy on air pollution under different levels of digital economy development, human capital, and economic development. This study provides practical paths for air pollution management, strengthening inter-regional environmental synergy management and high-quality economic development.
To develop an efficient, low-cost and green adsorbent for removing heavy metals from acid mine drainage (AMD), bentonite and eggshell were used to prepare a novel granular composite (BEP) adsorbent. ...Upon coupling of bentonite and eggshell with one another, BEP had the structural characteristics and merits of both bentonite and component CaCO
3
of eggshell, and the interlayer spacing of Ca-bentonite increased from 1.46 to 1.78 nm by the addition of polyethylene glycol (PEG). Such characteristics made BEP superior to single montmorillonite or eggshell for significantly higher Pb ion removal ratio of 99.90%, adsorption amount of about 40 mg·g
−1
and granulation integrity of 96.90% after adsorption. More importantly, this granular adsorbent was able to enhance the pH value of acid mine drainage from 3.00 to 6.10, and thus the acid neutralization capability of BEP can reduce H
+
competitive adsorption. Moreover, the prepared material could overcome the negative effect of fine bentonite used in aqueous medium, and thereby realize the solid-liquid separation of adsorbent and wastewater. In addition, the Elovich kinetic model fitted well for the adsorption process of Pb by BEP, and the possible removal mechanisms included ion exchange, electrostatic adsorption and complexation effects between BEP and Pb.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The goal of the assisted reproductive treatment is to transfer one euploid blastocyst and to help infertile women giving birth one healthy neonate. Some algorithms have been used to assess the ploidy ...status of embryos derived from couples with normal chromosome, who subjected to preimplantation genetic testing for aneuploidy (PGT-A) treatment. However, it is currently unknown whether artificial intelligence model can be used to assess the euploidy status of blastocyst derived from populations with chromosomal rearrangement.
From February 2020 to May 2021, we collected the whole raw time-lapse videos at multiple focal planes from in vitro cultured embryos, the clinical information of couples, and the comprehensive chromosome screening results of those blastocysts that had received PGT treatment. Initially, we developed a novel deep learning model called the Attentive Multi-Focus Selection Network (AMSNet) to analyze time-lapse videos in real time and predict blastocyst formation. Building upon AMSNet, we integrated additional clinically predictive variables and created a second deep learning model, the Attentive Multi-Focus Video and Clinical Information Fusion Network (AMCFNet), to assess the euploidy status of embryos. The efficacy of the AMCFNet was further tested in embryos with parental chromosomal rearrangements. The receiver operating characteristic curve (ROC) was used to evaluate the superiority of the model.
A total of 4112 embryos with complete time-lapse videos were enrolled for the blastocyst formation prediction task, and 1422 qualified blastocysts received PGT-A ( n = 589) or PGT for chromosomal structural rearrangement (PGT-SR, n = 833) were enrolled for the euploidy assessment task in this study. The AMSNet model using seven focal raw time-lapse videos has the best real-time accuracy. The real-time accuracy for AMSNet to predict blastocyst formation reached above 70% on the day 2 of embryo culture, and then increased to 80% on the day 4 of embryo culture. Combing with 4 clinical features of couples, the AUC of AMCFNet with 7 focal points increased to 0.729 in blastocysts derived from couples with chromosomal rearrangement.
Integrating seven focal raw time-lapse images of embryos and parental clinical information, AMCFNet model have the capability of assessing euploidy status in blastocysts derived from couples with chromosomal rearrangement.
Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease- causing mutations in patients. However, ...problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical applicaUon of such approaches. Recently, a base editor （BE） system built on cytidine （C） deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine （T） efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high- fidelity version of base editor 2 （HF2-BE2）, and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.
Abstract
m
5
C is one of the longest-known RNA modifications, however, its developmental dynamics, functions, and evolution in mRNAs remain largely unknown. Here, we generate quantitative mRNA m
5
C ...maps at different stages of development in 6 vertebrate and invertebrate species and find convergent and unexpected massive methylation of maternal mRNAs mediated by NSUN2 and NSUN6. Using
Drosophila
as a model, we reveal that embryos lacking maternal mRNA m
5
C undergo cell cycle delays and fail to timely initiate maternal-to-zygotic transition, implying the functional importance of maternal mRNA m
5
C. From invertebrates to the lineage leading to humans, two waves of m
5
C regulatory innovations are observed: higher animals gain cis-directed NSUN2-mediated m
5
C sites at the 5' end of the mRNAs, accompanied by the emergence of more structured 5'UTR regions; humans gain thousands of trans-directed NSUN6-mediated m
5
C sites enriched in genes regulating the mitotic cell cycle. Collectively, our studies highlight the existence and regulatory innovations of a mechanism of early embryonic development and provide key resources for elucidating the role of mRNA m
5
C in biology and disease.
Is embryo vitrification associated with a higher risk of adverse perinatal outcomes than slow-freezing?
Embryo vitrification was not associated with increased risks of adverse perinatal outcomes of ...pre-term birth (PTB), low birthweight (LBW), small for gestational age (SGA), large for gestational age (LGA) and macrosomia, as compared to slow-freezing.
Vitrification is becoming a widely adopted technology for embryo cryopreservation with higher embryo survival rate and live birth rate than the slow-freezing technique. However, limited data are currently available on risks of adverse perinatal outcomes following vitrification as compared to that of slow-freezing. The impact of vitrification on perinatal outcomes remains further to be elucidated.
Six large reproductive medical centers in Guangdong province, Southeast of China, took part in this multicenter retrospective cohort study. Cohorts of 3199 live born singletons after Day 3 frozen-thawed embryo transfer (FET) cycles with either vitrification or slow-freezing between January 2011 and December 2015 were included in the study. Each patient only contributed one cycle per cohort and vanishing twins were excluded. Propensity score (PS) matching was used to control for potential confounding factors.
All live-born singletons following either a vitrified or a slow-frozen cleavage FET cycle during the period from 2011 to 2015 were analyzed. Perinatal outcomes of PTB, LBW, macrosomia, SGA and LGA were compared. The vitrified and slow-frozen cohorts were matched by propensity scores with a 1:1 ratio accounting for potential confounding factors associated with perinatal outcomes. These variables included baseline demographics (maternal age, BMI, education level, parity, type of infertility and cause of infertility), as well as IVF characteristics (insemination method, endometrial preparation protocol and embryo cryopreservation duration).
A total of 2858 cases from vitrified embryo transfer (ET) and 341 babies from the slow-freezing group were included. After PS matching, 297 pairs of newborns were generated for comparison. The median gestational age was 39 weeks for both cohorts and the birthweights were comparable (3187.7 ± 502.1 g in the vitrified group vs. 3224.6 ± 483.6 in the slow-freezing group, P>0.05). There were no significant differences between the two groups on the incidence of PTB (5.4% vs. 7.7%), LBW (6.7% vs. 5.7%), macrosomia (5.7% vs. 6.1%), SGA (12.5% vs. 8.4%) and LGA (6.4% vs. 8.1%). Parallel logistic regression analysis indicated that vitrification was non-inferior to slow-freezing method in terms of the occurrence of PTB (OR, 0.68 95% CI, 0.35, 1.31), LBW (OR, 1.190.61-2.32), macrosomia (OR, 0.94 0.48-1.86), SGA (1.550.91-2.64) and LGA (0.780.42-1.45), P>0.05. Sex-stratified PS matching models with multivariable regression analysis further confirmed that vitrification did not increase the risks of above-mentioned adverse perinatal outcomes in either the male or female infant cohort.
Although the analysis was adjusted for a number of important confounders, the hospital dataset did not contain other potential confounders such as the medical history and obstetrics outcomes of women during pregnancy to allow adjustment. In addition, the current findings are only applicable to cleavage stage FET, but not pronuclei stage or blastocyst stage ET.
Vitrified ET, in comparison with slow-frozen ET, was not associated with increased risks of adverse neonatal outcomes. With its superiority on live birth rates and non-inferiority on safety perinatal outcomes, transition from slow-freezing to the use of vitrification for embryo cryopreservation is reassuring. Nonetheless, future research is needed for the long-term effects of vitrification method on offspring's health outcomes.
The study was funded by the National Key Research and Development Program (2016YFC100205), Guangzhou Science and Technology Project (201804020087), Guangdong Province Science and Technology Project (2016A020218008) and Guangdong Provincial Key Laboratory of Reproductive Medicine (2012A061400003). The authors have no conflicts of interest to declare.
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