Programmable RNA 5-methylcytosine (m5C) modification of cellular RNAs by dCasRx conjugated methyltransferase and demethylase.

5-Methylcytosine (m5C), an abundant RNA modification, plays a crucial role in regulating RNA fate and gene expression. While recent progress has been made in understanding the biological roles of m5C, the inability to introduce m5C at specific sites within transcripts has hindered efforts to elucidate direct links between specific m5C and phenotypic outcomes. Here, we developed a CRISPR-Cas13d-based tool, named reengineered m5C modification system (termed 'RCMS'), for targeted m5C methylation and demethylation in specific transcripts. The RCMS editors consist of a nuclear-localized dCasRx conjugated to either a methyltransferase, NSUN2/NSUN6, or a demethylase, the catalytic domain of mouse Tet2 (ten-eleven translocation 2), enabling the manipulation of methylation events at precise m5C sites. We demonstrate that the RCMS editors can direct site-specific m5C incorporation and demethylation. Furthermore, we confirm their effectiveness in modulating m5C levels within transfer RNAs and their ability to induce changes in transcript abundance and cell proliferation through m5C-mediated mechanisms. These findings collectively establish RCMS editors as a focused epitranscriptome engineering tool, facilitating the identification of individual m5C alterations and their consequential effects.

Targeted mutagenesis in mice via an engineered AsCas12f1 system.

SpCas9 and AsCas12a are widely utilized as genome editing tools in human cells, but their applications are largely limited by their bulky size. Recently, AsCas12f1 protein, with a small size (422 amino acids), has been demonstrated to be capable of cleaving double-stranded DNA protospacer adjacent motif (PAM). However, low editing efficiency and large differences in activity against different genomic loci have been a limitation in its application. Here, we show that engineered AsCas12f1 sgRNA has significantly improved the editing efficiency in human cells and mouse embryos. Moreover, we successfully generated three stable mouse mutant disease models using the engineered CRISPR-AsCas12f1 system in this study. Collectively, our work uncovers the engineered AsCas12f1 system expands mini CRISPR toolbox, providing a remarkable promise for therapeutic applications.

Pre-diagnostic plasma advanced glycation end-products and soluble receptor for advanced glycation end-products and mortality in colorectal cancer patients.

Advanced glycation end-products (AGEs), formed endogenously or obtained exogenously from diet, may contribute to chronic inflammation, intracellular signaling alterations, and pathogenesis of several chronic diseases including colorectal cancer (CRC). However, the role of AGEs in CRC survival is less known. The associations of pre-diagnostic circulating AGEs and their soluble receptor (sRAGE) with CRC-specific and overall mortality were estimated using multivariable-adjusted Cox proportional hazards regression among 1369 CRC cases in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Concentrations of major plasma AGEs, Nε-[carboxy-methyl]lysine (CML), Nε-[carboxy-ethyl]lysine (CEL) and Nδ-[5-hydro-5-methyl-4-imidazolon-2-yl]-ornithine (MG-H1), were measured using ultra-performance liquid chromatography mass-spectrometry. sRAGE was assessed by enzyme-linked immunosorbent assay. Over a mean follow-up period of 96 months, 693 deaths occurred of which 541 were due to CRC. Individual and combined AGEs were not statistically significantly associated with CRC-specific or overall mortality. However, there was a possible interaction by sex for CEL (Pinteraction = .05). Participants with higher sRAGE had a higher risk of dying from CRC (HRQ5vs.Q1 = 1.67, 95% CI: 1.21-2.30, Ptrend = .02) or any cause (HRQ5vs.Q1 = 1.38, 95% CI: 1.05-1.83, Ptrend = .09). These associations tended to be stronger among cases with diabetes (Pinteraction = .03) and pre-diabetes (Pinteraction <.01) before CRC diagnosis. Pre-diagnostic AGEs were not associated with CRC-specific and overall mortality in individuals with CRC. However, a positive association was observed for sRAGE. Our findings may stimulate further research on the role of AGEs and sRAGE in survival among cancer patients with special emphasis on potential effect modifications by sex and diabetes.

Separable Microneedle for Integrated Hyperglycemia Sensing and Photothermal Responsive Metformin Release.

Microdevices that offer hyperglycemia monitoring and controllable drug delivery are urgently needed for daily diabetes management. Herein, a theranostic separable double-layer microneedle (DLMN) patch consisting of a swellable GelMA supporting base layer for glycemia sensing and a phase-change material (PCM) arrowhead layer for hyperglycemia regulation has been fabricated. The Cu-TCPP(Fe)/glucose oxidase composite and 3,3',5,5'-tetramethylbenzidine coembedded in the supporting base layer permit a visible color shift at the base surface in the presence of glucose via a cascade reaction, allowing for the in situ detection of glucose in interstitial fluid. The PCM arrowhead layer is encapsulated with water monodispersity melanin nanoparticles from Sepia officinalis and metformin that is imparted with a near-infrared ray photothermal response feature, which is beneficial to the controllable release of metformin for suppression of hyperglycemia. By applying the DLMN patch to the streptozotocin-induced type 2 diabetic Sprague-Dawley rat model, the results demonstrated that it can effectively extract dermal interstitial fluid, read out glucose levels, and regulate hyperglycemia. This DLMN-integrated portable colorimetric sensor and self-regulated glucose level hold great promise for daily diabetes management.

Advances in the Application of Microneedles in the Treatment of Local Organ Diseases.

In recent years, microneedles (MNs) have attracted a lot of attention due to their microscale sizes and high surface area (500-1000 µm in length), allowing pain-free and efficient drug delivery through the skin. In addition to the great success of MNs based transdermal drug delivery, especially for skin diseases, increasing studies have indicated the expansion of MNs to diverse nontransdermal applications, including the delivery of therapeutics for hair loss, ocular diseases, and oral mucosal. Here, the current treatment of hair loss, eye diseases, and oral disease is discussed and an overview of recent advances in the application of MNs is provided for these three noncutaneous localized organ diseases. Particular emphasis is laid on the future trend of MNs technology development and future challenges of expanding the generalizability of MNs.

The Cdkn2a gene product p19 alternative reading frame (p19ARF) is a critical regulator of IFNß-mediated Lyme arthritis.

Type I interferon (IFN) has been identified in patients with Lyme disease, and its abundant expression in joint tissues of C3H mice precedes development of Lyme arthritis. Forward genetics using C3H mice with severe Lyme arthritis and C57BL/6 (B6) mice with mild Lyme arthritis identified the Borrelia burgdorferi arthritis-associated locus 1 (Bbaa1) on chromosome 4 (Chr4) as a regulator of B. burgdorferi-induced IFNß expression and Lyme arthritis severity. B6 mice introgressed with the C3H allele for Bbaa1 (B6.C3-Bbaa1 mice) displayed increased severity of arthritis, which is initiated by myeloid lineage cells in joints. Using advanced congenic lines, the physical size of the Bbaa1 interval has been reduced to 2 Mbp, allowing for identification of potential genetic regulators. Small interfering RNA (siRNA)-mediated silencing identified Cdkn2a as the gene responsible for Bbaa1 allele-regulated induction of IFNß and IFN-stimulated genes (ISGs) in bone marrow-derived macrophages (BMDMs). The Cdkn2a-encoded p19 alternative reading frame (p19ARF) protein regulates IFNß induction in BMDMs as shown by siRNA silencing and overexpression of ARF. In vivo studies demonstrated that p19ARF contributes to joint-specific induction of IFNß and arthritis severity in B. burgdorferi-infected mice. p19ARF regulates B. burgdorferi-induced IFNß in BMDMs by stabilizing the tumor suppressor p53 and sequestering the transcriptional repressor BCL6. Our findings link p19ARF regulation of p53 and BCL6 to the severity of IFNß-induced Lyme arthritis in vivo and indicate potential novel roles for p19ARF, p53, and BCL6 in Lyme disease and other IFN hyperproduction syndromes.

Metaproteomics analysis of anaerobic digestion of food waste by the addition of calcium peroxide and magnetite.

Adding trace calcium peroxide and magnetite into a semi-continuous digester is a new method to effectively improve the anaerobic digestion of food waste. However, the microbial mechanism in this system has not been fully explored. Metaproteomics further revealed that the most active and significantly regulated genus u_p_Chloroflexi had formed a good cooperative relationship with Methanomicrobiales and Methanothrix in the system. u_p_Chloroflexi decomposed more organic compounds into CO2, acetate, amino acids, and other substances by alternating between short aerobic-anaerobic respiration. It perceived and adapted to the surrounding environment by producing biofilm, extracellular enzymes, and accelerating substrate transport, formed a respiratory barrier, and enhanced iron transport capacity by using highly expressed cytochrome C. The methanogens formed reactive oxygen species scavengers and reduced iron transport to prevent oxidative damage. This study provides new insight for improving the efficiency of anaerobic digestion of food waste and identifying key microorganisms and their regulated functional proteins in the calcium peroxide-magnetite digestion system.IMPORTANCEPrevious study has found that the combination of calcium peroxide and magnetite has a good promoting effect on the anaerobic digestion process of food waste. Through multiple omics approaches, information such as microbial population structure and changes in metabolites can be further analyzed. This study can help researchers gain a deeper understanding of the digestion pathway of food waste under the combined action of calcium peroxide and magnetite, further elucidate the impact mechanisms of calcium peroxide and magnetite at the microbial level, and provide theoretical guidance to improve the efficiency and stability of anaerobic digestion of food waste, as well as reduce operational costs. This research contributes to improving energy recovery efficiency, promoting sustainable management and development of food waste, and is of great significance to environmental protection.

Optical fiber sensing probe for detecting a carcinoembryonic antigen using a composite sensitive film of PAN nanofiber membrane and gold nanomembrane.

An optical fiber sensing probe using a composite sensitive film of polyacrylonitrile (PAN) nanofiber membrane and gold nanomembrane is presented for the detection of a carcinoembryonic antigen (CEA), a biomarker associated with colorectal cancer and other diseases. The probe is based on a tilted fiber Bragg grating (TFBG) with a surface plasmon resonance (SPR) gold nanomembrane and a functionalized polyacrylonitrile (PAN) PAN nanofiber coating that selectively binds to CEA molecules. The performance of the probe is evaluated by measuring the spectral shift of the TFBG resonances as a function of CEA concentration in buffer. The probe exhibits a sensitivity of 0.46 dB/(µg/ml), a low limit of detection of 505.4 ng/mL in buffer, and a good selectivity and reproducibility. The proposed probe offers a simple, cost-effective, and a novel method for CEA detection that can be potentially applied for clinical diagnosis and monitoring of CEA-related diseases.

Artificial intelligence enhances whole-slide interpretation of PD-L1 CPS in triple-negative breast cancer: A multi-institutional ring study.

BACKGROUND AND AIMS: Evaluation of the programmed cell death ligand-1 (PD-L1) combined positive score (CPS) is vital to predict the efficacy of the immunotherapy in triple-negative breast cancer (TNBC), but pathologists show substantial variability in the consistency and accuracy of the interpretation. It is of great importance to establish an objective and effective method which is highly repeatable. METHODS: We proposed a model in a deep learning-based framework, which at the patch level incorporated cell analysis and tissue region analysis, followed by the whole-slide level fusion of patch results. Three rounds of ring studies (RSs) were conducted. Twenty-one pathologists of different levels from four institutions evaluated the PD-L1 CPS in TNBC specimens as continuous scores by visual assessment and our artificial intelligence (AI)-assisted method. RESULTS: In the visual assessment, the interpretation results of PD-L1 (Dako 22C3) CPS by different levels of pathologists have significant differences and showed weak consistency. Using AI-assisted interpretation, there were no significant differences between all pathologists (P = 0.43), and the intraclass correlation coefficient (ICC) value was increased from 0.618 [95% confidence interval (CI) = 0.524-0.719] to 0.931 (95% CI = 0.902-0.955). The accuracy of interpretation result is further improved to 0.919 (95% CI = 0.886-0.947). Acceptance of AI results by junior pathologists was the highest among all levels, and 80% of the AI results were accepted overall. CONCLUSION: With the help of the AI-assisted diagnostic method, different levels of pathologists achieved excellent consistency and repeatability in the interpretation of PD-L1 (Dako 22C3) CPS. Our AI-assisted diagnostic approach was proved to strengthen the consistency and repeatability in clinical practice.

HECTD2 as a target for veratric acid in the regulation of ferroptosis in renal cell carcinoma.

Function of HECTD2 in renal cell carcinoma malignant progression is undefined. Molecular mechanism behind anti-cancer effects of veratric acid (VA) from traditional Chinese medicine (TCM) is underexplored. The Cancer Genome Atlas was leveraged to study HECTD2 expression in renal cell carcinoma and its relationship with histological grading. Kaplan-Meier survival analysis was performed. HECTD2 expression was detected in cancer cells and tissues via qRT-PCR and immunohistochemistry. GPX4 and SLC7A11 expression in tumor samples with high or low HECTD2 expression was examined by immunohistochemistry, cell viability by CCK8, cell proliferation by colony formation assay, lipid ROS and mitochondrial superoxide levels by flow cytometry, Fe2+ and MDA content by assay kits, and GPX4 and SLC7A11 proteins by western blot. SeeSAR software screened TCM small molecule compounds with highest affinity to HECTD2, confirmed with cellular thermal shift assay. VA IC50 was measured by CCK8. Xenograft model was developed and treated with VA. Tumor size and weight were monitored, with immunohistochemistry to detect HECTD2 expression in tumors and assess ferroptosis-related markers. HECTD2 was overexpressed in tumor tissues and cells, which positively correlated with histological grading. HECTD2 depletion inhibited cell vitality and proliferation, raised intracellular lipid ROS, mitochondrial superoxide, Fe2+, and MDA. HECTD2 was a target with highest VA affinity. In vitro and vivo experiments concurred that VA treatment hindered malignancy of renal cell carcinoma and enhanced its susceptibility to ferroptosis. HECTD2 supports ferroptosis resistance in renal cell carcinoma, but VA, through its targeting of HECTD2, initiates ferroptosis, showcasing its anti-cancer efficacy.

Metal-Organic-Framework-Derived Bromine and Nitrogen Dual-Doped Porous Carbon for CO2 Photocycloaddition Reaction.

The cycloaddition of CO2 with epoxides driven by light irradiation is an intriguing approach to preparing cyclic carbonates. However, it remains a great challenge to achieve high photocatalytic efficiency in the absence of a cocatalyst. Herein, we explored a metal-organic-framework (MOF)-templated pyrolysis strategy to prepare uniform bromine ions/nitrogen-codoped carbon materials (Br-CN) as low-cost photocatalysts for CO2 cycloaddition. The optimal catalyst Br-CN-1-550 can be used as a photocatalyst to catalyze CO2 cycloaddition, remarkably reducing the energy consumption. As a result of its benefits of high photothermal efficiency and rich nucleophilic sites (Br ions), BN-CN-1-550 affords a 9 times higher yield of 4-(chloromethyl)-1,3-dioxolan-2-one than that of the ZIF-8-derived CN under cocatalyst-free conditions and light irradiation (300 mW·cm-2 full-spectrum irradiation, 10 h). This strategy provides a cost-effective way to obtain cyclic carbonate under cocatalyst-free conditions.

Pd Nanoparticle-Modified BiOBr/CdS S-Scheme Photocatalyst for Enhanced Conversion of CO2.

S-scheme heterojunction photocatalyst-coupled plasma-resonance effect can enhance the response range and absorption of light and charge transfer, and, at the same time, obtain strong redox ability, which is an effective way to improve CO2 conversion. In this work, plasma S-scheme heterojunctions of Pd/BiOBr/CdS with heterogeneous interfaces have been successfully constructed by a simple hydrothermal method. The possible reaction mechanism was proposed by in situ infrared, ultraviolet-visible spectroscopy (UV-vis), electron paramagnetic resonance (ESR), density functional theory (DFT), and electrochemical techniques. It was proved that the plasma S-scheme heterojunction can enhance the charge separation efficiency and improve the photocatalytic activity. When the loading ratio is Pd0.6-10%-BiOBr/CdS, it has the best performance, and the CO yield is 30.24 µmol/g, which is 15 and 30 times that of pure BiOBr and CdS, respectively. The results show that with the strong absorption of photon energy and the special electron transfer mode of S-scheme heterojunction, the charge can be effectively separated and transferred, and the photocatalytic activity is significantly improved. This study provides a useful strategy for charge transfer kinetics of plasma S-scheme heterojunction photocatalysts.

Self-correction of cycle threshold values by a normal distribution-based process to improve accuracy of quantification in real-time digital PCR.

The digital polymerase chain reaction (dPCR) is a new and developing nucleic acid detection technology with high sensitivity that can realize the absolute quantitative analysis of samples. In order to improve the accuracy of quantitative results, real-time digital PCR emphasizes the kinetic information during amplification to identify prominent abnormal data. However, it is challenging to use a unified standard to accurately classify the amplification curve of each well as negative and positive, due to the interference caused by various factors in the experiment. In this work, a normal distribution-based cycle threshold value self-correcting model (NCSM) was established, which focused on the feature of the cycle threshold values in amplification curves and conducted continuous detection and correction on the whole. The cycle threshold value distribution was closer to the ideal normal distribution to avoid the influence of interference. Thus, the model achieves a more accurate classification between positive and negative results. The corrective process was applied to plasmid samples and resulted in an accuracy improvement from 92 to 99%. The coefficient of variation was below 5% when considering the quantitation of a range between 100 and 10,000 copies. At the same time, by utilizing this model, the distribution of cycle threshold values at the endpoint can be predicted with fewer thermal cycles, which can reduce the cycling time by around 25% while maintaining a consistency of more than 98%. Therefore, using the NCSM can effectively enhance the quantitative accuracy and increase the detection efficiency based on the real-time dPCR platform.

A deuterohemin peptide protects cerebral ischemia-reperfusion injury by preventing oxidative stress in vitro and in vivo.

Cerebral ischemia-reperfusion injury (CIRI) is a brain injury that usually occurs during thrombolytic therapy for acute ischemic stroke and impacts human health. Oxidative stress is one of the major causative factors of CIRI. DhHP-3 is a novel peroxidase-mimicking enzyme that exhibits robust reactive oxygen species (ROS) scavenging ability in vitro. Here, we established in vitro and in vivo models of cerebral ischemia-reperfusion to mechanistically investigate whether DhHP-3 can alleviate CIRI. DhHP-3 could reduce ROS, down-regulate apoptotic proteins, suppress p53 phosphorylation, attenuate the DNA damage response (DDR), and inhibit apoptosis in SH-SY5Y cells subjected to oxygen-glucose deprivation/re-oxygenation (OGD/R) and in the brain of Sprague Dawley rats subjected to transient middle cerebral artery occlusion. In conclusion, DhHP-3 has bioactivity of CIRI inhibition through suppression of the ROS-induced apoptosis.

Development of a highly efficient prime editor system in mice and rabbits.

The recently developed prime-editing (PE) technique is more precise than previously available techniques and permits base-to-base conversion, replacement, and insertions and deletions in the genome. However, previous reports show that the efficiency of prime editing is insufficient to produce genome-edited animals. In fact, prime-guide RNA (pegRNA) designs have posed a challenge in achieving favorable editing efficiency. Here, we designed prime binding sites (PBS) with a melting temperature (Tm) of 42 °C, leading to optimal performance in cells, and we found that the optimal Tm was affected by the culture temperature. In addition, the ePE3max system was developed by updating the PE architecture to PEmax and expressing engineered pegRNA (epegRNA) based on the original PE3 system. The updated ePE3max system can efficiently induce gene editing in mouse and rabbit embryos. Furthermore, we successfully generated a Hoxd13 (c. 671 G > T) mutation in mice and a Tyr (c. 572 del) mutation in rabbits by ePE3max. Overall, the editing efficiency of modified ePE3max systems is superior to that of the original PE3 system in producing genome-edited animals, which can serve as an effective and versatile genome-editing tool for precise genome modification in animal models.

ACE2 mediates tryptophan alleviation on diarrhea by repairing intestine barrier involved mTOR pathway.

The membrane-delimited receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), angiotensin-converting enzyme 2 (ACE2), which is expressed in the intestine, collaborates with broad neutral amino acid transporter 1 (B0AT1). Tryptophan (Trp) is transported into intestinal epithelial cells by ACE2 and B0AT1. However, whether ACE2 and its binding protein B0AT1 are involved in Trp-mediated alleviation of intestinal injury is largely unknown. Here, we used weaned piglets and IPEC-J2 cells as models and found that ACE2/B0AT1 alleviated lipopolysaccharide (LPS)-induced diarrhea and promoted intestinal barrier recovery via transport of Trp. The levels of the aryl hydrocarbon receptor (AhR) and mechanistic target of rapamycin (mTOR) pathways were altered by ACE2. Dietary Trp supplementation in LPS-treated weaned piglets revealed that Trp alleviated diarrhea by promoting ACE2/B0AT1 expression, and examination of intestinal morphology revealed that the damage to the intestinal barrier was repaired. Our study demonstrated that ACE2 accompanied by B0AT1 mediated the alleviation of diarrhea by Trp through intestinal barrier repair via the mTOR pathway.

Retinopathy as a predictive indicator for significant hepatic fibrosis according to T2DM status: A cross-sectional study based on the national health and nutrition examination survey data.

INTRODUCTION AND OBJECTIVES: Type 2 Diabetes Mellitus (T2DM), a prevalent metabolic disorder, often coexists with a range of complications, with retinopathy being particularly common. Recent studies have shed light on a potential connection between diabetic retinopathy (DR) and hepatic fibrosis, indicating a possible shared pathophysiological foundation in T2DM. This study investigates the correlation between retinopathy and hepatic fibrosis among individuals with T2DM, as well as evaluates the diagnostic value of DR for significant hepatic fibrosis. MATERIALS AND METHODS: Our cross-sectional analysis incorporated 5413 participants from the National Health and Nutrition Examination Survey (NHANES) 2005-2008. The Fibrosis-4 score (FIB-4) classified hepatic fibrosis into different grades (F0-F4), with significant hepatic fibrosis marked as F2 or higher. Retinopathy severity was determined using retinal imaging and categorized into four levels. The analysis of variance or Chi-square tests facilitated group comparisons. Additionally, the receiver operating characteristic (ROC) analysis appraised the predictive accuracy of retinopathy for significant hepatic fibrosis in the T2DM population. RESULTS: Among 5413 participants, the mean age was 59.56 ± 12.41, with 50.2% male. And 20.6% were diagnosed with T2DM. Hepatic fibrosis grading was positively associated with retinopathy severity (OR [odds ratio]: 1.521, 95%CI [confidence interval]: 1.152-2.008, P = 0.003) across the entire population. The association was amplified in the T2DM population according to Pearson's analysis results. The ROC curve demonstrated retinopathy's diagnostic capacity for significant hepatic fibrosis in the T2DM population (AUC [area under curve] = 0.72, 95%CI: 0.651-0.793, P < 0.001). CONCLUSIONS: Retinopathy could serve as an independent predictor of significant hepatic fibrosis in T2DM population. Ophthalmologists are advised to closely monitor T2DM patients with retinopathy.

Urinary phenols and parabens exposure in relation to urinary incontinence in the US population.

BACKGROUND: Our study aimed to investigate the impact of urinary concentrations of personal care products (PCPs)-related phenols (PNs) and parabens (PBs), including Triclosan (TCS), Bisphenol A (BPA), Benzophenone-3 (BP-3), Butylparaben (BPB), Ethylparaben (EPB), Methylparaben (MPB), and Propylparaben (PPB), on urinary incontinence (UI) occurrence. METHOD: We conducted a cross-sectional analysis using data from the National Health and Nutrition Examination Survey (NHANES) spanning the years 2007 to 2016. Regression analysis was employed to investigate the relationship between exposure to PCPs-related substances, various levels of exposure, and UI within both the general population and the female demographic. Additionally, the Bayesian Kernel Machine Regression (BKMR) model was used to assess the effects of mixtures on UI. RESULTS: Our analysis comprised 7,690 participants who self-reported their diagnosis. Among them, 12.80% experienced stress urinary incontinence (SUI), 11.80% reported urge urinary incontinence (UUI), and 10.22% exhibited mixed urinary incontinence (MUI). In our fully adjusted multivariable models, BP-3 exposure exhibited a positive association with SUI (OR 1.07, 95% CI 1.02-1.14, p = 0.045). BPA exposure correlated with an increased risk of UUI (OR 1.21, 95% CI 1.01-1.44, p = 0.046) and MUI (OR 1.26, 95% CI 1.02-1.54, p = 0.029). TCS exposure displayed a negative correlation with the incidence of MUI (OR 0.87, 95% CI 0.79-0.97, p = 0.009). No significant links were observed between parabens and urinary incontinence. Notably, among the female population, our investigation revealed that BPA exposure heightened the risk of MUI (OR 1.28, 95% CI 1.01-1.63, p = 0.043). Participants in the highest tertile of BP-3 exposure demonstrated elevated likelihoods of SUI and MUI compared to those in the lowest tertile. In the BKMR analysis, negative trends were observed between the mixture and the risks of UUI and MUI when the mixture ranged from the 25th to the 40th and 35th to the 40th percentiles or above, respectively. Additionally, a positive trend was identified between the mixture and MUI when it was in the 40th to 55th percentile. CONCLUSION: In conclusion, our findings suggest that exposure to BPA, TCS, and BP-3 may contribute to the development of urinary incontinence.

Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope.

BACKGROUND: Nme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme2Cas9 base editors to bring the deaminase domain closer to the nontarget strand to improve editing efficiency. RESULTS: Our results demonstrated that Nme2ABE8e-797 with adenine deaminase inserted between amino acids 797 and 798 has a significantly increased editing efficiency with a wide editing window ranging from 4 to 18 bases in mammalian cells, especially at the sites that were difficult to edit by Nme2ABE8e. In addition, by swapping the PAM-interacting domain of Nme2ABE8e-797 with that of SmuCas9 or introducing point mutations of eNme2-C in Nme2ABE8e-797, we created Nme2ABE8e-797Smu and Nme2ABE8e-797-C, respectively, which exhibited robust activities at a wide range of sites with N4CN PAMs in human cells. Moreover, the modified domain-inlaid Nme2ABE8e can efficiently restore or install disease-related loci in Neuro-2a cells and mice. CONCLUSIONS: These novel Nme2ABE8es with increased on-target DNA editing and expanded PAM compatibility will expand the base editing toolset for efficient gene modification and therapeutic applications.

A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9.

BACKGROUND: Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-associated virus (AAV) during preclinical applications. Despite a number of approaches having previously been attempted to overcome that challenge, including split Cas9-derived and numerous domain-deleted versions of editors, whether base editor (BE) and prime editor (PE) systems can also allow deletion of those domains remains to be proven. In this study, we present a new small ABE (sABE) with significantly reduced size. RESULTS: We discovered that ABE8e can tolerate large single deletions in the REC2 (Δ174-296) and HNH (Δ786-855) domains of SpCas9, and these deletions can be stacked together to create a new sABE. The sABE showed higher precision than the original ABE8e, with proximally shifted protospacer adjacent motif (PAM) editing windows (A3- A15), and comparable editing efficiencies to 8e-SaCas9-KKH. The sABE system efficiently generated A-G mutations at disease-relevant loci (T1214C in GAA and A494G in MFN2) in HEK293T cells and several canonical Pcsk9 splice sites in N2a cells. Moreover, the sABE enabled in vivo delivery in a single adeno-associated virus (AAV) vector with slight efficiency. Furthermore, we also successfully edited the genome of mouse embryos by microinjecting mRNA and sgRNA of sABE system into zygotes. CONCLUSIONS: We have developed a substantially smaller sABE system that expands the targeting scope and offers higher precision of genome editing. Our findings suggest that the sABE system holds great therapeutic potential in preclinical applications.