Polyoxometalates-engineered hydrogen generation rate and durability of Pt/CNT catalysts from ammonia borane

Heterogeneously catalyzed hydrolytic dehydrogenation of ammonia borane is a remarkable structure sensitive reaction. In this work, a strategy by using polyoxometalates(POMs) as the ligands is proposed to engineer the surface and electronic properties of Pt/CNT catalysts toward the enhanced hydrogen generation rate and durability. Three kinds of POMs, i.e., silicotungstic acid(STA), phosphotungstic acid(PTA)and molybdophosphoric acid(PMA), are comparatively studied, among which the STA shows positive effects on the catalytic activity and durability. A catalyst structure-performance relationship is established by a combination of kinetic and isotopic analyses with multiple characterization techniques, such as HAADF-STEM, EDS, Raman spectroscopy and XPS. It is shown that the STA compared to the other two POMs can increase the Pt binding energy and thus promote the reaction. The insights demonstrated here could open a new avenue for boosting the reaction by employing the POMs as the ligands to engineer the catalyst electronic properties.

炎性肠病易感基因GPR35在肠炎发生发展中的功能研究

炎性肠病在全球范围内发生极其普遍,具有反复发作、难以治愈的特点,也是诱发结直肠癌的高风险因素之一。肠炎的发生与遗传因素密切相关,有报道发现位于GPR35基因座上的多个单核苷酸多态性(single nucleotidepolymorphism,SNP)位点rs4676410、rs3749171和rs3749172与肠炎敏感性高度相关,但是GPR35基因在肠炎的发生发展进程中的功能及相关机制尚没有明确结论。为了研究GPR35在肠炎中的作用,首先通过CRISPR/Cas9技术构建Gpr35敲除小鼠,随后利用DSS诱导的肠炎模型评价Gpr35在肠炎发生中的作用,发现敲除小鼠在体重变化、DAI评分、肠上皮损伤以及炎性细胞浸润等肠炎相关指标显著低于野生型小鼠。为了研究肠炎相关SNP突变对GPR35活性的影响,首先根据rs3749171和rs3749172SNP位点突变信息构建GPR35-T108M和GPR35-S294R两种突变型受体,其次通过多种GPR35下游信号通路活性测试,发现两种突变均能够增强GPR35受体活性。最后通过Westernblotting分析发现相较于野生型小鼠,Gpr35敲除小鼠肠上皮Erk1/2磷酸化水平增加,表明Gpr35敲除后可能通过上调Erk1/2信号通路的方式抑制肠炎的发生发展。综上所述,本研究发现人类肠炎易感的rs3749171和rs3749172位点可能通过激活GPR35及下游信号通路的方式促进肠炎的发生发展,为炎性肠病的治疗提供了潜在的药物作用靶点。

Uhrf1对肠上皮发育的影响

作为一种常见的表观遗传修饰类型,DNA甲基化对哺乳动物发育起着重要作用。Uhrf1作为重要的表观遗传调控因子,在DNA合成过程中可结合半甲基化的DNA同时招募DNA甲基转移酶1参与DNA甲基化的维持,保证遗传信息在细胞分裂前后的稳定传递。目前关于Uhrf1介导的DNA甲基化是否影响肠上皮发育过程尚不清楚。为探索Uhrf1在肠上皮发育中的作用,本研究成功构建了肠上皮特异性敲除Uhrf1的小鼠模型,利用HE染色对肠上皮组织形态学观察发现,与正常小鼠相比,敲除Uhrf1的小鼠肠上皮发育异常,主要表现为绒毛变短,数量减少,隐窝萎缩;通过表型分析发现,在小鼠肠上皮中特异性敲除Uhrf1后,细胞增殖明显受到抑制、凋亡细胞增加、细胞分化异常,同时肠干细胞相关基因表达降低。进一步对可能的分子机制进行初步探索发现Uhrf1缺失后DNA甲基化水平大幅下降,诱发DNA损伤。本研究结果表明Uhrf1介导的DNA甲基化对肠上皮的正常发育成熟具有重要作用,有望丰富Uhrf1介导的DNA甲基化在体内的生物学功能,并为进一步明确Uhrf1介导的表观遗传调控机制提供实验依据。

Corrigendum to“Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration”[Journal of Genetics and Genomics(2022)49,1114-1126]

The authors regret that the grant number“21CJ1402200”in the Acknowledgments session should be replaced as“21JC1402200”.The corrected contents areprovided as follows.

Gene editing and its applications in biomedicine

The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats(CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.

Generation of obese rat model by transcription activator-like effector nucleases targeting the leptin receptor gene

The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator-like effector nucleases(TALENs) technology to generate Leptin receptor(Lepr) knockout rats on the Sprague Dawley(SD) genetic background. Through direct injection of in vitro transcribed m RNA of TALEN pairs into SD rat zygotes, somatic mutations were induced in two of three resulting pups. One of the founders carrying bi-allelic mutation exhibited early onset of obesity and infertility. The other founder carried a chimeric mutation which was efficiently transmitted to the progenies. Through phenotyping of the resulting three lines of rats bearing distinct mutations in the Lepr locus, we found that the strains with a frame-shifted or premature stop codon mutation led to obesity and metabolic disorders. However, no obvious defect was observed in a strain with an in-frame 57 bp deletion in the extracellular domain of Lepr. This suggests the deleted amino acids do not significantly affect Lepr structure and function. This is the first report of generating the Lepr mutant obese rat model in SD strain through a reverse genetic approach. This suggests that TALEN is an efficient and powerful gene editing technology for the generation of disease models.

Enhanced genome editing to ameliorate a genetic metabolic liver disease through co-delivery of adeno-associated virus receptor

Genome editing through adeno-associated viral(AAV) vectors is a promising gene therapy strategy for various diseases,especially genetic disorders. However, homologous recombination(HR) efficiency is extremely low in adult animal models. We assumed that increasing AAV transduction efficiency could increase genome editing activity, especially HR efficiency, for in vivo gene therapy. Firstly, a mouse phenylketonuria(PKU) model carrying a pathogenic R408W mutation in phenylalanine hydroxylase(Pah) was generated. Through co-delivery of the general AAV receptor(AAVR), we found that AAVR could dramatically increase AAV transduction efficiency in vitro and in vivo. Furthermore, co-delivery of SaCas9/sgRNA/donor templates with AAVR via AAV8 vectors increased indel rate over 2-fold and HR rate over 15-fold for the correction of the single mutation in Pah;mice. Moreover, AAVR co-injection successfully increased the site-specific insertion rate of a 1.4 kb Pah cDNA by 11-fold, bringing the HR rate up to 7.3% without detectable global off-target effects. Insertion of Pah cDNA significantly decreased the Phe level and ameliorated PKU symptoms. This study demonstrates a novel strategy to dramatically increase AAV transduction which substantially enhanced in vivo genome editing efficiency in adult animal models, showing clinical potential for both conventional and genome editing-based gene therapy.

In vivo target protein degradation induced by PROTACs based on E3 ligase DCAF15

Dear Editor,As an emerging drug discovery paradigm,Proteolysis targeting chimeras(PROTACs)facilitate ubiquitination and degradation of the targets by cellular endogenous proteasome system.Compared with traditional small molecular inhibitors,PROTACs work like enzymes and can operate with or without functional inhibition of the targets,which endows this strategy to degrade drug targets,especially useful for“undruggable”ones.

Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration

CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications.However,it is still infeasible because homologous recombination(HR)is inefficient,especially for nondividing cells.To overcome the challenge,we report that a homology-independent targeted integration(HITI)strategy is used for permanent integration of high-specificity-activity Factor IX variant(F9 Padua,R338L)at the albumin(Alb)locus in a novel hemophilia B(HB)rat model.The knock-in efficiency reaches 3.66%,as determined by droplet digital PCR(dd PCR).The clotting time is reduced to a normal level four weeks after treatment,and the circulating factor IX(FIX)level is gradually increased up to 52%of the normal level over nine months even after partial hepatectomy,demonstrating the amelioration of hemophilia.Through primer-extension-mediated sequencing(PEM-seq),no significant off-target effect is detected.This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.

Long-term amelioration of an early-onset familial atrial fibrillation model with AAV-mediated in vivo gene therapy

Atrial fibrillation(AF)is a common cardiac disease with high prevalence in the general population.Despite a mild manifestation at the onset stage,it causes serious consequences,including sudden death,when the disease progresses to the late stage.Most available treatments of AF focus on symptom management or alleviation,due to a lack of fundamental knowledge and the fact that considerable variations of AF exist.With the popularisation of the next-generation sequencing technology,several causal genetic factors,including MYL4,have been discovered to contribute to AF,giving hope to developing its gene therapies.In this study,we attempted to treat a previously established rat AF model,which carried Myl4E11K/E11K loss of function mutation,via overexpression of exogenous wild-type Myl4 by AAV9 vectors.Our results showed that delivery of Myl4 expressing AAV9 to postnatal rat models rescued the symptoms of AF,indicating the therapeutic potential that early gene therapy intervention can achieve long-term effects in treating cardiac arrhythmias caused by gene mutations.

The effect of nano-calcium carbonate onβ-glucosidase immobilized by alginate and chitosan

Nanotechnology has become the most promising domain to boost the efficiency of enzymes.Enzymes are vital as a green catalyst in many industries,food,pharmaceutical and biomedical,etc.The immobilization process of the enzyme increases its catalytic properties.In this research,a novel method is presented to describe the effect of nano-calcium carbonate on the characteristics of immobilizedβ-glucosidase,which was extracted from the Agrocybe aegirit.The nano-CaCO_(3)was produced using the eco-friendly natural deep eutectic solvent.The pure nano-CaCO_(3)was observed as vaterite,with a size of about 300 nm.The nano-calcium carbonate was coated by a natural polymer sodium alginate compound and then adsorbed chitosan.Further,this obtained composite is cross-linked by the bioactive genipin to immobilize theβ-glucosidase.The enzyme/protein loading ratio to the supports was 1:4,respectively.The recovery efficiency of immobilizedβ-glucosidase was 89.3%,and immobilization yield was 96.452%.Chitosan-coated nano-CaCO_(3)was used as a carrier for immobilization ofβ-glucosidase to improve its stability and reusability.In addition to stability and reusability,pH tolerance,temperature tolerance,and enzyme kinetics are the significant parameters that illustrate the proficiency of an immobilized enzyme.The measured optimal enzymatic reaction conditions for the immobilizedβ-glucosidase were 50℃and pH 6.Furthermore,it has shown noticeable improvements in thermo-stability and pH tolerance.Temperature tolerance was observed 50%to the initial activity of the immobilized enzyme even after the 3 h of incubation at 50℃,while pH tolerance was noticed more than 50%and 40%at pH 7 and 8,respectively.The K_(m)and V_(max)values of free and immobilizedβ-glucosidase to 4-nitrophenylβ-D-glucopyranoside were 1.549μmol/L/min,0.346 mmol/L and 0.532μmol/L/min,0.080 mmol/L,respectively.The immobilizedβ-glucosidase retains its storability 80%even after 30 days of storage at 4℃and maintains 93.1%of its residual activity by reusing up to 10 cycles.

A de novo missense mutation in MPP2 confers an increased risk of Vogt–Koyanagi–Harada disease as shown by trio-based whole-exome sequencing

Vogt–Koyanagi–Harada(VKH)disease is a leading cause of blindness in young and middle-aged people.However,the etiology of VKH disease remains unclear.Here,we performed the first trio-based whole-exome sequencing study,which enrolled 25 VKH patients and 50 controls,followed by a study of 2081 VKH patients from a Han Chinese population to uncover detrimental mutations.A total of 15 de novo mutations in VKH patients were identified,with one of the most important being the membrane palmitoylated protein 2(MPP2)p.K315N(MPP2-N315)mutation.The MPP2-N315 mutation was highly deleterious according to bioinformatic predictions.Additionally,this mutation appears rare,being absent from the 1000 Genome Project and Genome Aggregation Database,and it is highly conserved in 10 species,including humans and mice.Subsequent studies showed that pathological phenotypes and retinal vascular leakage were aggravated in MPP2-N315 mutation knock-in or MPP2-N315 adeno-associated virus-treated mice with experimental autoimmune uveitis(EAU).In vitro,we used clustered regularly interspaced short palindromic repeats(CRISPR‒Cas9)gene editing technology to delete intrinsic MPP2 before overexpressing wild-type MPP2 or MPP2-N315.Levels of cytokines,such as IL-1β,IL-17E,and vascular endothelial growth factor A,were increased,and barrier function was destroyed in the MPP2-N315 mutant ARPE19 cells.Mechanistically,the MPP2-N315 mutation had a stronger ability to directly bind to ANXA2 than MPP2-K315,as shown by LC‒MS/MS and Co-IP,and resulted in activation of the ERK3/IL-17E pathway.Overall,our results demonstrated that the MPP2-K315N mutation may increase susceptibility to VKH disease.

CRISPR/Cas9 system： a powerful technology for in vivo and ex vivo gene therapy

CRISPR/Cas9 is a versatile genome-editing tool which is widely used for modifying the genome of both prokaryotic and eukaryotic organisms for basic research and applications. An increasing number of reports have demonstrated that CRISPR/Cas9-mediated genome editing is a powerful technology for gene therapy. Here, we review the recent advances in CRISPR/Cas9-mediated gene therapy in animal models via different strategies and discuss the challenges as well as future prospects.

Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants

Dear Editor, The clustered regularly interspaced short palindromic repeat （CRISPR） system has been widely adapted to genome editing to either introduce or correct genetic mutations （Wang et al., 2016）. However, due to competition with the dominant non-homologous end-joining （NHEJ） pathway, precise genome modifications through Cas9-stimulated homologous recombination （HR） is inefficient. Through fusion of cytidine deaminases, APOBEC1 （apolipoprotein B editing complex 1） or AID （activation-induced deaminase）, with Cas9 variants, several groups have developed the cytidine base editor （BE） systems （Komor et al., 2016; Li et al., 2018; Nishida et al., 2016）. The BE system achieves programmable conversion of C-G base pairs to T.A without double-stranded DNA cleavage （Zhou et al., 2017）. More recently, adenine base editors （ABEs）, which efficiently convert A-T base pairs to G-C in genomic DNA, have been developed via fusion of an engineered tRNA adenosine deaminase （ecTadA from Escherichia coh） with nCas9 （Gaudelli et al., 2017）. The ABE system has quickly been adapted to generate disease models and correction of genetic disease in mice （Ryu et al., 2017; Liu et al, 2018）. However, whether the editing efficiency and the targeting scope of ABE could be improved is largely unexplored. In this study, we describe the efficient generation of base-edi- ted mice and rats modeling human diseases through ABEs with highest efficiency up to 100%. We also demonstrate an increase of ABE activity through injection of chemically modified tracrRNA and crRNA in mouse zygotes, and the expansion of editing scope by fusion of an ecTadA mutant to SaCas9n-KKH and Casgn-VQR variants in both cells and embryos. Our study suggests that the ABE system is a powerful and convenient tool to introduce precise base conversions in rodents.

Correction to: Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants

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