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Abstract R-loop-triggered collateral single-stranded DNA (ssDNA) nuclease activity within Class 1 Type I CRISPR-Cas systems holds immense potential for nucleic acid detection. However, the hyperactive ssDNase activity of Cas3 introduces unwanted noise and false-positive results. In this study, we identified a novel Type I-A Cas3 variant derived from Thermococcus siculi, which remains in an auto-inhibited state until it is triggered by Cascade complex and R-loop formation. This Type I-A CRISPR-Cas3 system not only exhibits an expanded protospacer adjacent motif (PAM) recognition capability but also demonstrates remarkable intolerance towards mismatched sequences. Furthermore, it exhibits dual activation modes—responding to both DNA and RNA targets. The culmination of our research efforts has led to the development of the Hyper-Active-Verification Establishment (HAVE, 惠父). This innovation enables swift and precise human papillomavirus (HPV) diagnosis in clinical samples, providing a robust molecular diagnostic tool based on the Type I-A CRISPR-Cas3 system. Our findings contribute to understanding type I-A CRISPR-Cas3 system regulation and facilitate the creation of advanced diagnostic solutions with broad clinical applicability.

Summary: IscBs, as hypercompact ancestry proteins of Cas9 nuclease, are suitable for in vivo gene editing via single adeno-associated virus (AAV) delivery. Due to the low activity of natural IscBs in eukaryotic cells, recent studies have been focusing on improving OgeuIscB’s gene editing efficiency via protein engineering. However, in vivo gene editing efficacy of IscBs for disease correction remained to be demonstrated. Here, we showed effective gene knockout and base editing in mouse embryos. To further improve IscB activity, we performed systematic engineering of IscB-associated ωRNA and identified a variant, ωRNA∗-v2, with enhanced gene editing efficiency. Furthermore, our study demonstrated the efficacy of an engineered IscB-ωRNA system for robust gene knockout and base editing in vivo. Single AAV delivery of IscB-derived cytosine and adenine base editors achieved disease correction in a mouse model of tyrosinemia. Therefore, our results indicated the great potential of miniature IscBs for developing single-AAV-based gene editing therapeutics.

The adaptive survival mechanisms of bacterial pathogens under host-induced stress are crucial for understanding pathogenesis. Recently, Uppalapati et al. revealed a unique dual function of the Gifsy-1 prophage terminase in Salmonella enterica: it acts as a transfer ribonuclease (tRNase) under oxidative stress. The Gifsy-1 prophage terminase targets and fragments tRNALeu to halt translation and temporarily impairs bacterial growth when exposed to high levels of ROS generated by the host immune cells. This response not only preserves genomic integrity by facilitating DNA repair but also inhibits prophage mobilization, thereby aiding in bacterial survival within vertebrate hosts. This study highlights a novel intersection between phage biology and bacterial adaptive strategies.

Abstract Heterochromatin is generally associated with the nuclear periphery, but how the spatial organization of heterochromatin is regulated to ensure epigenetic silencing remains unclear. Here we found that Sad1, an inner nuclear membrane SUN-family protein in fission yeast, interacts with histone H2A-H2B but not H3-H4. We solved the crystal structure of the histone binding motif (HBM) of Sad1 in complex with H2A-H2B, revealing the intimate contacts between Sad1HBM and H2A-H2B. Structure-based mutagenesis studies revealed that the H2A-H2B-binding activity of Sad1 is required for the dynamic distribution of Sad1 throughout the nuclear envelope (NE). The Sad1-H2A-H2B complex mediates tethering telomeres and the mating-type locus to the NE. This complex is also important for heterochromatin silencing. Mechanistically, H2A-H2B enhances the interaction between Sad1 and HDACs, including Clr3 and Sir2, to maintain epigenetic identity of heterochromatin. Interestingly, our results suggest that Sad1 exhibits the histone-enhanced liquid-liquid phase separation property, which helps recruit heterochromatin factors to the NE. Our results uncover an unexpected role of SUN-family proteins in heterochromatin regulation and suggest a nucleosome-independent role of H2A-H2B in regulating Sad1’s functionality.

Abstract Type I CRISPR-Cas systems employ multi-subunit effector Cascade and helicase-nuclease Cas3 to target and degrade foreign nucleic acids, representing the most abundant RNA-guided adaptive immune systems in prokaryotes. Their ability to cause long fragment deletions have led to increasing interests in eukaryotic genome editing. While the Cascade structures of all other six type I systems have been determined, the structure of the most evolutionarily conserved type I-B Cascade is still missing. Here, we present two cryo-EM structures of the Synechocystis sp. PCC 6714 (Syn) type I-B Cascade, revealing the molecular mechanisms that underlie RNA-directed Cascade assembly, target DNA recognition, and local conformational changes of the effector complex upon R-loop formation. Remarkably, a loop of Cas5 directly intercalated into the major groove of the PAM and facilitated PAM recognition. We further characterized the genome editing profiles of this I-B Cascade-Cas3 in human CD3+ T cells using mRNA-mediated delivery, which led to unidirectional 4.5 kb deletion in TRAC locus and achieved an editing efficiency up to 41.2%. Our study provides the structural basis for understanding target DNA recognition by type I-B Cascade and lays foundation for harnessing this system for long range genome editing in human T cells.

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.

Primary microcephaly is a clinical feature of several human telomere disorder syndromes. Here the authors reveal a role of Microcephalin 1 in promoting telomere replication and repair.

Background: Feline panleukopenia virus (FPV) is a widespread and highly infectious pathogen in cats with a high mortality rate. Although Yanji has a developed cat breeding industry, the variation of FPV locally is still unclear. Objectives: This study aimed to isolate and investigate the epidemiology of FPV in Yanji between 2021 and 2022. Methods: A strain of FPV was isolated from F81 cells. Cats suspected of FPV infection (n = 80) between 2021 and 2022 from Yanji were enrolled in this study. The capsid protein 2 (VP2) of FPV was amplified. It was cloned into the pMD-19T vector and transformed into a competent Escherichia coli strain. The positive colonies were analyzed via VP2 Sanger sequencing. A phylogenetic analysis based on a VP2 coding sequence was performed to identify the genetic relationships between the strains. Results: An FPV strain named YBYJ-1 was successfully isolated. The virus diameter was approximately 20-24 nm, 50% tissue culture infectious dose = 1 × 10-4.94/mL, which caused cytopathic effect in F81 cells. The epidemiological survey from 2021 to 2022 showed that 27 of the 80 samples were FPV-positive. Additionally, three strains positive for CPV-2c were unexpectedly found. Phylogenetic analysis showed that most of the 27 FPV strains belonged to the same group, and no mutations were found in the critical amino acids. Conclusions: A local FPV strain named YBYJ-1 was successfully isolated. There was no critical mutation in FPV in Yanji, but some cases with CPV-2c infected cats were identified. [ABSTRACT FROM AUTHOR]

BackgroundPulmonary arterial hypertension (PAH) is a serious condition characterized by elevated pulmonary artery pressure, leading to right heart failure and increased mortality. This study investigates the link between PAH and genes associated with hypoxia and cuproptosis.MethodsWe utilized expression profiles and single-cell RNA-seq data of PAH from the GEO database and genecad. Genes related to cuproptosis and hypoxia were identified. After normalizing the data, differential gene expression was analyzed between PAH and control groups. We performed clustering analyses on cuproptosis-related genes and constructed a weighted gene co-expression network (WGCNA) to identify key genes linked to cuproptosis subtype scores. KEGG, GO, and DO enrichment analyses were conducted for hypoxia-related genes, and a protein–protein interaction (PPI) network was created using STRING. Immune cell composition differences were examined between groups. SingleR and Seurat were used for scRNA-seq data analysis, with PCA and t-SNE for dimensionality reduction. We analyzed hub gene expression across single-cell clusters and built a diagnostic model using LASSO and random forest, optimizing parameters with 10-fold cross-validation. A total of 113 combinations of 12 machine learning algorithms were employed to evaluate model accuracy. GSEA was utilized for pathway enrichment analysis of AHR and FAS, and a Nomogram was created to assess risk impact. We also analyzed the correlation between key genes and immune cell types using Spearman correlation.ResultsWe identified several diagnostic genes for PAH linked to hypoxia and cuproptosis. PPI networks illustrated relationships among these hub genes, with immune infiltration analysis highlighting associations with monocytes, macrophages, and CD8 T cells. The genes AHR, FAS, and FGF2 emerged as key markers, forming a robust diagnostic model (NaiveBayes) with an AUC of 0.9.ConclusionAHR, FAS, and FGF2 were identified as potential biomarkers for PAH, influencing cell proliferation and inflammatory responses, thereby offering new insights for PAH prevention and treatment.

Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated wRNA plays the combined role of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) to guide double-stranded DNA (dsDNA) cleavage. Here we report a 2.78-angstrom cryo- electron microscopy structure of IscB-wRNA bound to a dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 ribonucleoproteins. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. wRNA plays the equivalent function of REC domains in Cas9 and contacts the RNA-DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the wRNA and introducing protein domain replacements. [ABSTRACT FROM AUTHOR]

Abstract This paper addresses a two-stage stochastic-robust model for the day-ahead self-scheduling problem of an aggregator considering uncertainties. The aggregator, which integrates power and capacity of small-scale prosumers and flexible community-owned devices, trades electric energy in the day-ahead (DAM) and real-time energy markets (RTM), and trades reserve capacity and deployment in the reserve capacity (RCM) and reserve deployment markets (RDM). The ability of the aggregator providing reserve service is constrained by the regulations of reserve market rules, including minimum offer/bid size and minimum delivery duration. A combination approach of stochastic programming (SP) and robust optimization (RO) is used to model different kinds of uncertainties, including those of market price, power/demand and reserve deployment. The risk management of the aggregator is considered through conditional value at risk (CVaR) and fluctuation intervals of the uncertain parameters. Case studies numerically show the economic revenue and the energy-reserve schedule of the aggregator with participation in different markets, reserve regulations, and risk preferences.

Background: White coat hypertension (WCH) and masked hypertension (MH) can increase the risk of target organ damage. Home blood pressure monitoring is an important method for detecting WCH and MH. However, the prevalence and related factors of WCH and MH in China have been rarely reported.Objective: To explore the prevalence and related factors associated with white coat hypertension (WCH) and masked hypertension (MH) in Shunde District, Southern China.Methods: This study recruited subjects from the Physical Examination Center in Shunde Hospital, Southern Medical University. Office blood pressure and home blood pressure values were collected using the home blood pressure monitor with telemedicine device and office blood pressure monitor, and the prevalence of WCH and MH was calculated by the values. Multivariate logistic regression was used to explore the related factors for WCH and MH.Results: Four-hundred and sixty-one participants (61% male), with an average age of 49 years, were included. The prevalence of WCH and MH was 5.1 and 15.2%, respectively. Multivariate logistic regression analysis showed that smoking (OR = 4.71, 95% CI = 1.05–21.15) and family history of coronary heart disease (OR = 4.51, 95% CI = 1.08–18.93) were associated with higher odds of WCH. The associated factors for higher odds of MH were smoking (OR = 2.83, 95% CI = 1.11–7.23), family history of hypertension (OR = 2.17, 95% CI = 1.11–4.26) and family history of coronary heart disease (OR = 2.82, 95% CI = 1.07–7.45).Conclusion: WCH and MH are highly prevalent in the Physical Examination Center in Shunde Hospital, Southern Medical University. We found smoking and family history of coronary heart disease were related factors for WCH, and smoking, family history of hypertension and coronary heart disease were associated with the odds of MH. Home blood pressure monitoring with a telemedicine device should be recommended to identity abnormal BP phenotype.

ObjectiveGlucagon-like peptide-1 receptor agonists (GLP-1 RAs) and sodium-glucose cotransporter-2 (SGLT-2) inhibitors reduce glycaemia and weight and improve insulin resistance (IR) via different mechanisms. We aim to evaluate and compare the ability of GLP-1 RAs and SGLT-2 inhibitors to ameliorate the IR of nonalcoholic fatty liver disease (NAFLD) patients.Data SynthesisThree electronic databases (Medline, Embase, PubMed) were searched from inception until March 2021. We selected randomized controlled trials comparing GLP-1 RAs and SGLT-2 inhibitors with control in adult NAFLD patients with or without T2DM. Network meta-analyses were performed using fixed and random effect models, and the mean difference (MD) with corresponding 95% confidence intervals (CI) were determined. The within-study risk of bias was assessed with the Cochrane collaborative risk assessment tool RoB.Results25 studies with 1595 patients were included in this network meta-analysis. Among them, there were 448 patients, in 6 studies, who were not comorbid with T2DM. Following a mean treatment duration of 28.86 weeks, compared with the control group, GLP-1 RAs decreased the HOMA-IR (MD [95%CI]; -1.573[-2.523 to -0.495]), visceral fat (-0.637[-0.992 to -0.284]), weight (-2.394[-4.625 to -0.164]), fasting blood sugar (-0.662[-1.377 to -0.021]) and triglyceride (- 0.610[-1.056 to -0.188]). On the basis of existing studies, SGLT-2 inhibitors showed no statistically significant improvement in the above indicators. Compared with SGLT-2 inhibitors, GLP-1 RAs decreased visceral fat (-0.560[-0.961 to -0.131]) and triglyceride (-0.607[-1.095 to -0.117]) significantly.ConclusionsGLP-1 RAs effectively improve IR in NAFLD, whereas SGLT-2 inhibitors show no apparent effect.Systematic Review RegistrationPROSPERO https://www.crd.york.ac.uk/PROSPERO/, CRD42021251704

Sizing and configuring community-shared energy storage according to the actual demand of community users is important for the development of user-side energy storage. To solve this problem, this paper first proposes a community energy storage cooperative sharing mode containing multiple transaction types and then establishes a sizing and configuration model of community-shared energy storage based on a cooperative game among community users and energy storage operators, in which the loss caused by the capacity decay of energy storage is quantified by a dynamic power loss cost factor. To improve the solving efficiency, a distributed and cooperating solving method based on ADMM is used to solve the sizing and configuration model. On this basis, the bilateral Shapley method is used to allocate the total annual cost according to the marginal expected cost brought by each user. Compared with existing strategies, this paper calculates the economic benefits of community-shared energy storage based on several typical days of each year and quantifies the capacity decay of energy storage by a dynamic power loss cost factor which increases year by year to be closer to the real situation. Finally, the simulation verifies that the model proposed in this paper can be used for the sizing and configuration of community-shared energy storage. Compared with the original annual cost, the total annual cost of the community is reduced by 3.92%, and the annual operation cost of the community which equals annual electricity purchasing cost minus annual electricity selling income plus annual power loss cost is reduced by 25.6%.

Inter-regional and transnational grid interconnection is necessary for energy development. Xinjiang, which is rich in renewable energy resources, is adjacent to countries in Central Asia and has great potential for interconnection with its neighbors. This paper outlines China’s relevant policies for transnational power interconnection, and introduces the energy structure, load demand endowments, and power supply status of Xinjiang, Pakistan, and five Central Asian countries. Further, it analyzes the advantages of the multinational power interconnection from the aspects of power supply and load complementation. Finally, from the perspective of technical support and practical basis, the feasibility of interconnection between Xinjiang, Pakistan, and five Central Asian countries have been analyzed. This paper provides a theoretical basis for promoting and implementing China’s “Belt and Road” power transnational interconnected development strategy. Keywords: Interconnected power grid, Renewable energy resources, Complementary features, Belt and Road

Background: Hypertension, as a predominant risk factor for cardiovascular disease, is a severe public health burden in China. Home blood pressure monitoring (HBPM) is an important tool in the detection and management of hypertension. However, there is a lack of HBPM data from prospective cohorts in China. Hence, we designed this study to investigate the impact of HBPM on major health outcomes in Chinese population participating in regular health check-ups. Methods: Leveraging telemedicine technology, the open prospective, multicenter, HBPM-iCloud (Home Blood Pressure Monitoring Based on an Intelligent Cloud Platform) cohort study will recruit participants from three participating health check-up centers in southern China to participate in cloud-based HBPM for 1 week. The prevalence of sustained hypertension, white coat hypertension (WCH), masked hypertension (MH), white coat uncontrolled hypertension (WUCH), and masked uncontrolled hypertension (MUCH) will be defined by a combination of average readings of home-based and office-based blood pressure (BP). Cardiovascular risk factors and subclinical target organ damage will be recorded. Participants will be followed-up for 5 years to examine the incidence and associated risk factors of composite major adverse cardiovascular and cerebrovascular event. Conclusion: The study will help to determine the best way to implement telemedicine technology in BP control for better prevention and treatment of hypertension. Results will provide data for a Chinese population to aid in the construction of screening, risk stratification, and intervention strategies for abnormal BP phenotypes, including WCH, MH, WUCH, and MUCH.