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Due to the sustainable and pollution-free characteristics, wind energy has been one of the fastest growing renewable energy sources. However, the intermittent and random fluctuation of wind speed presents many challenges for reliable wind power integration and normal operation of wind farm. Accurate wind speed prediction is the key to ensure the safe operation of power system and to develop wind energy resources. Therefore, this paper has presented a wavelet time series wind speed prediction model based on Lorenz disturbance. Therefore, in this paper, combined with the atmospheric dynamical system, a wavelet-time series improved wind speed prediction model based on Lorenz disturbance is proposed and the wind turbines of different climate types in Spain and China are used to simulate the disturbances of Lorenz equations with different initial values. The prediction results show that the improved model can effectively correct the preliminary prediction of wind speed, improving the prediction. In a word, the research work in this paper will be helpful to arrange the electric power dispatching plan and ensure the normal operation of the wind farm.

As conventional energy sources are non-renewable, the world's major countries are investing heavily in renewable energy research. Wind power represents the development trend of future energy, but the intermittent and volatility of wind energy are the main reasons that leads to the poor accuracy of wind power prediction. However, by analyzing the error level at different time points, it can be found that the errors of adjacent time are often approximately the same, the least square support vector machine (LS-SVM) model with error correction is used to predict the wind power in this paper. According to the simulation of wind power data of two wind farms, the proposed method can effectively improve the prediction accuracy of wind power, and the error distribution is concentrated almost without deviation. The improved method proposed in this paper takes into account the error correction process of the model, which improved the prediction accuracy of the traditional model (RBF, Elman, LS-SVM). Compared with the single LS-SVM prediction model in this paper, the mean absolute error of the proposed method had decreased by 52 percent. The research work in this paper will be helpful to the reasonable arrangement of dispatching operation plan, the normal operation of the wind farm and the large-scale development as well as fully utilization of renewable energy resources.

Abstract Photoacoustic tomography offers a powerful tool to visualize biologically relevant molecules and understand processes within living systems at high resolution in deep tissue, facilitated by the conversion of incident photons into low-scattering acoustic waves through non-radiative relaxation. Although current endogenous and exogenous photoacoustic contrast agents effectively enable molecular imaging within deep tissues, their broad absorption spectra in the visible to near-infrared (NIR) range limit photoacoustic multiplexed imaging. Here, we exploit the distinct ultrasharp NIR absorption peaks of lanthanides to engineer a series of NIR photoacoustic nanocrystals. This engineering involves precise host and dopant material composition, yielding nanocrystals with sharply peaked photoacoustic absorption spectra (~3.2 nm width) and a ~10-fold enhancement in NIR optical absorption for efficient deep tissue imaging. By combining photoacoustic tomography with these engineered nanocrystals, we demonstrate photoacoustic multiplexed differential imaging with substantially decreased background signals and enhanced precision and contrast.

Ying Yang,1– 3,* Bixia Zhou,1,2,* Min Li,4 Yishuai Sun,1,2 Xulei Jiang,1,2 Xinxin Zhou,1,2 Chengjun Hu,1,2 Dou Zhang,3 Hang Luo,3 Wuyuan Tan,1,2 Xinghua Yang,1,2 Shaorong Lei1,2 1Department of Plastic Surgery, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China; 2National Clinical Research Center for Geriatric Disorders, Xiangya Hospital Central South University, Changsha, Hunan, People’s Republic of China; 3State Key Laboratory of Powder Metallurgy, Central South University, Changsha, Hunan, People’s Republic of China; 4Department of Oncology, Changsha Central Hospital, University of South China, Changsha, Hunan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Shaorong Lei, Department of Plastic Surgery, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan, 410008, People’s Republic of China, Tel +86 73189753014, Email leishaorong@csu.edu.cn Xinghua Yang, Department of Plastic Surgery, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan, 410008, People’s Republic of China, Tel +86 73189753014, Email yxhua8805@sina.comPurpose: The treatment of craniofacial bone defects caused by trauma, tumors, and infectious and degenerative diseases is a significant issue in current clinical practice. Following the rapid development of bone tissue engineering (BTE) in the last decade, bioactive scaffolds coupled with multifunctional properties are in high demand with regard to effective therapy for bone defects. Herein, an innovative bone scaffold consisting of GO/Cu nanoderivatives and GelMA-based organic-inorganic hybrids was reported for repairing full-thickness calvarial bone defect.Methods: In this study, motivated by the versatile biological functions of nanomaterials and synthetic hydrogels, copper nanoparticle (CuNP)-decorated graphene oxide (GO) nanosheets (GO/Cu) were combined with methacrylated gelatin (GelMA)-based organic-inorganic hybrids to construct porous bone scaffolds that mimic the extracellular matrix (ECM) of bone tissues by photocrosslinking. The material characterizations, in vitro cytocompatibility, macrophage polarization and osteogenesis of the biohybrid hydrogel scaffolds were investigated, and two different animal models (BALB/c mice and SD rats) were established to further confirm the in vivo neovascularization, macrophage recruitment, biocompatibility, biosafety and bone regenerative potential.Results: We found that GO/Cu-functionalized GelMA/β-TCP hydrogel scaffolds exhibited evidently promoted osteogenic activities, M2 type macrophage polarization, increased secretion of anti-inflammatory factors and excellent cytocompatibility, with favorable surface characteristics and sustainable release of Cu2+. Additionally, improved neovascularization, macrophage recruitment and tissue integration were found in mice implanted with the bioactive hydrogels. More importantly, the observations of microCT reconstruction and histological analysis in a calvarial bone defect model in rats treated with GO/Cu-incorporated hydrogel scaffolds demonstrated significantly increased bone morphometric values and newly formed bone tissues, indicating accelerated bone healing.Conclusion: Taken together, this BTE-based bone repair strategy provides a promising and feasible method for constructing multifunctional GO/Cu nanocomposite-incorporated biohybrid hydrogel scaffolds with facilitated osteogenesis, angiogenesis and immunoregulation in one system, with the optimization of material properties and biosafety, it thereby demonstrates great application potential for correcting craniofacial bone defects in future clinical scenarios. Keywords: nanomaterials, hybrid hydrogel, bone scaffolds, craniofacial bone defects, bone regeneration

Yanwu Zhang,1,* Buasiyamu Abudunaibi,2,* Yunkang Zhao,2 Dongliang Zhang,1 Yanru Chu,1 Song Lei,1 Xiaomin Gu,1 Xuying Lao,1 Xianhao Wu,1 Weitao Yao,1 Yi Chen,1 Feng Tong1 1Department of Infectious Disease Control and Prevention, Ningbo Prefectural Center for Disease Control and Prevention, Ningbo, Zhejiang Province, People’s Republic of China; 2State Key Laboratory of Vaccines for Infectious Diseases, Xiang’ an Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen, Fujian Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Feng Tong; Yi Chen, Ningbo Prefectural Center for Disease Control and Prevention, Ningbo, Zhejiang Province, People’s Republic of China, Email tongfeng1031@163.com; 30279068@qq.comObjective: To conduct a comprehensive evaluation of the Dengue Fever Surveillance and Early Warning System deployed in Ningbo City during 2023, focusing on its capacity for timely identification and reporting of dengue fever cases, particularly imported cases from endemic regions.Methods: A detailed data of patient clinical features and blood profile trends was collected from clinical records and surveillance reports, focusing on the rapid diagnostic processes and surveillance rigor. This study assessed the effectiveness of the system in identifying and reporting dengue cases and identified the limitations of the existing framework through a basic statistical approach.Results: The system demonstrated timely identification and reporting of dengue fever cases, with a particular emphasis on imported cases. However, several limitations were identified, including the need for more precise monitoring criteria and improved coordination with medical entities.Conclusion: The study underscores the critical role of public health bodies in managing disease outbreaks and advocates for enhanced methodologies to refine epidemic control efforts. The findings contribute to the advancement of early warning mechanisms and the improvement of proactive infectious disease monitoring in metropolitan environments, providing valuable insights for fortifying the Dengue Fever Surveillance and Early Warning System in Ningbo City.Keywords: Dengue fever, Imported, Surveillance, Early-warning

Abstract Equitable and accessible education in life sciences, bioengineering, and synthetic biology is crucial for training the next generation of scientists, fostering transparency in public decision-making, and ensuring biotechnology can benefit a wide-ranging population. As a groundbreaking technology for genome engineering, CRISPR has transformed research and therapeutics. However, hands-on exposure to this technology in educational settings remains limited due to the extensive resources required for CRISPR experiments. Here, we develop CRISPRkit, an affordable kit designed for gene editing and regulation in high school education. CRISPRkit eliminates the need for specialized equipment, prioritizes biosafety, and utilizes cost-effective reagents. By integrating CRISPRi gene regulation, colorful chromoproteins, cell-free transcription-translation systems, smartphone-based quantification, and an in-house automated algorithm (CRISPectra), our kit offers an inexpensive (~$2) and user-friendly approach to performing and analyzing CRISPR experiments, without the need for a traditional laboratory setup. Experiments conducted by high school students in classroom settings highlight the kit’s utility for reliable CRISPRkit experiments. Furthermore, CRISPRkit provides a modular and expandable platform for genome engineering, and we demonstrate its applications for controlling fluorescent proteins and metabolic pathways such as melanin production. We envision CRISPRkit will facilitate biotechnology education for communities of diverse socioeconomic and geographic backgrounds.

Abstract The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelles, the sites of replication of viral genomic RNA (vgRNA). To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain numerous vgRNA molecules along with the replication enzymes and clusters of viral double-stranded RNA (dsRNA). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of endoplasmic reticulum (ER) markers and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are encapsulated into DMVs, which have membranes derived from the host ER. These organelles merge into larger vesicle packets as infection advances. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

The pleiotropic benefits of statins in cardiovascular diseases that are independent of their lipid-lowering effects have been well documented, but the underlying mechanisms remain elusive. Here we show that simvastatin significantly improves human induced pluripotent stem cell-derived endothelial cell functions in both baseline and diabetic conditions by reducing chromatin accessibility at transcriptional enhanced associate domain elements and ultimately at endothelial-to-mesenchymal transition (EndMT)-regulating genes in a yes-associated protein (YAP)-dependent manner. Inhibition of geranylgeranyltransferase (GGTase) I, a mevalonate pathway intermediate, repressed YAP nuclear translocation and YAP activity via RhoA signaling antagonism. We further identified a previously undescribed SOX9 enhancer downstream of statin-YAP signaling that promotes the EndMT process. Thus, inhibition of any component of the GGTase-RhoA-YAP-SRY box transcription factor 9 (SOX9) signaling axis was shown to rescue EndMT-associated endothelial dysfunction both in vitro and in vivo, especially under diabetic conditions. Overall, our study reveals an epigenetic modulatory role for simvastatin in repressing EndMT to confer protection against endothelial dysfunction.

Xuqing Zhou,1,2,* Xu Wang,1,2,* Jiaqi Li,3,* Mengxue Zhang,4 Yi Yang,1 Shihui Lei,1 Ying He,3 Hua Yang,3 Desheng Zhou,4 Chun Guo1,2 1Experiment Center of Medical Innovation, the First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan, 410007, People’s Republic of China; 2The First Clinical College of Traditional Chinese Medicine, Hunan University of Chinese Medicine, Changsha, Hunan, 410007, People’s Republic of China; 3College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan, 410128, People’s Republic of China; 4Department of Neurology, the First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan, 410007, People’s Republic of China*These authors contributed equally to this workCorrespondence: Chun Guo, Email chunguosm@163.comBackground: Annao Pingchong decoction (ANPCD) is a traditional Chinese decoction which has definite effects on treating intracerebral hemorrhage (ICH) validated through clinical and experimental studies. However, the impact of ANPCD on oxidative stress (OS) after ICH remains unclear and is worth further investigating.Aim: To investigate whether the therapeutic effects of ANPCD on ICH are related to alleviating OS damage and seek potential targets for its antioxidant effects.Materials and Methods: The therapeutic candidate genes of ANPCD on ICH were identified through a comparison of the target genes of ANPCD, target genes of ICH and differentially expressed genes (DEGs). Protein–protein interaction (PPI) network analysis and functional enrichment analysis were combined with targets-related literature to select suitable antioxidant targets. The affinity between ANPCD and the selected target was verified using macromolecular docking. Subsequently, the effects of ANPCD on OS and the selected target were further investigated through in vivo experiments.Results: Forty-eight candidate genes were screened, in which silent information regulator sirtuin 1 (SIRT1) is one of the core genes that has antioxidant effects and ICH significantly affected its expression. The good affinity between 6 compounds of ANPCD and SIRT1 was also demonstrated by macromolecular docking. The results of in vivo experiments demonstrated that ANPCD significantly decreased modified neurological severity scoring (mNSS) scores and serum MDA and 8-OHdG content in ICH rats, while significantly increasing serum SOD and CAT activity, complicated with the up-regulation of ANPCD on SIRT1, FOXO1, PGC-1α and Nrf2. Furthermore, ANPCD significantly decreased the apoptosis rate and the expression of apoptosis-related proteins (P53, cytochrome c and caspase-3).Conclusion: ANPCD alleviates OS damage and apoptosis after ICH in rats. As a potential therapeutic target, SIRT1 can be effectively regulated by ANPCD, as are its downstream proteins. Keywords: intracerebral hemorrhage, Annao Pingchong decoction, oxidative stress, network pharmacology, vivo experiments, SIRT1

Yue Wu,1 Siqi Cheng,1 Shaoyuan Lei,2 Dongxiao Li,3 Zhongzhong Li,4 Yansu Guo1,2,5 1Beijing Geriatric Healthcare Center, Xuanwu Hospital, Capital Medical University, Beijing, People’s Republic of China; 2Department of Evidence-Based Medicine, Xuanwu Hospital, Capital Medical University, Beijing, People’s Republic of China; 3Department of Neurology, Hebei Provincial Hospital of Traditional Chinese Medicine, Shijiazhuang, People’s Republic of China; 4Department of Neurology, The Second Hospital of Hebei Medical University, Shijiazhuang, People’s Republic of China; 5Beijing Municipal Geriatric Medical Research Center, Beijing, People’s Republic of ChinaCorrespondence: Yansu Guo, Beijing Geriatric Healthcare Center, Xuanwu Hospital, Capital Medical University, Changchun Street 45, Beijing, People’s Republic of China, Email gys188@163.comBackground: There is growing evidence that an antioxidant diet is a protective factor against frailty. However, few studies have examined the effect of comprehensive dietary antioxidants on frailty symptoms. The aim of this study was to examine the relationships between the composite dietary antioxidant index (CDAI) and frailty and the underlying mechanisms involved.Methods: Based on the National Health and Nutrition Survey (NHANES) 2003– 2018, this study included 11,277 older persons aged ≥ 60 years. In this study, frailty was defined as having a total score > 0.21 on the 49-item frailty index. Six dietary antioxidants were selected for use in calculating the CDAI. A weighted multiple logistic regression model with subgroup analysis and restricted cubic splines (RCSs) were used to examine the association between the CDAI and frailty. To examine the role of oxidative stress, mediation analyses were also conducted.Results: The association between the CDAI score and frailty risk was significant according to the multivariate model. Compared with participants in tertile 1, participants in both tertile 2 and tertile 3 had lower odds of developing frailty symptoms (OR=0.86; 95% CI=0.75– 0.97; P=0.02; and OR=0.81; 95% CI=0.70– 0.93; P=0.003). According to the subgroup analyses, the differences in interactions were not statistically significant. There was also a potential nonlinear relationship between the CDAI score and frailty risk. The serum albumin concentration and uric acid concentration had significant mediating effects on the association between the CDAI score and frailty index, with 19.25% (P=0.002) and 21.26% (P < 0.001) of the total, respectively.Conclusion: Frailty is negatively associated with the CDAI score, which may be partially mediated by oxidative stress.Keywords: frailty, CDAI, NHANES, elderly adults

Many novel CRISPR-based genome-editing tools, with a wide variety of applications, have been developed in the past few years. The original CRISPR-Cas9 system was developed as a tool to alter genomic sequences in living organisms in a simple way. However, the functions of new CRISPR tools are not limited to conventional genome editing mediated by non-homologous end-joining or homology-directed repair but expand into gene-expression control, epigenome editing, single-nucleotide editing, RNA editing and live-cell imaging. Furthermore, genetic perturbation screening by multiplexing guide RNAs is gaining popularity as a method to identify causative genes and pathways in an unbiased manner. New CRISPR tools can also be applied to ex vivo or in vivo therapeutic genome editing for the treatment of conditions such as hyperlipidaemia. In this Review, we first provide an overview of the diverse new CRISPR tools that have been developed to date. Second, we summarize how these new CRISPR tools are being used to study biological processes and disease mechanisms in cardiovascular research and medicine. Finally, we discuss the prospect of therapeutic genome editing by CRISPR tools to cure genetic cardiovascular diseases.

Stress urinary incontinence (SUI) and pelvic floor disorder (PFD) are common conditions with limited treatment options in women worldwide. Regenerative therapy to restore urethral striated and pelvic floor muscles represents a valuable therapeutic approach. We aim to determine the CRISPR interference-mediated gene silencing effect of the nonviral delivery of nuclease-deactivated dCas9 ribonucleoprotein (RNP) complex on muscle regeneration at the cellular and molecular level. We designed four myostatin (MSTN)-targeting sgRNAs and transfected them into rat myoblast L6 cells together with the dCas9 protein. Myogenesis assay and immunofluorescence staining were performed to evaluate muscle differentiation, while CCK8 assay, cell cycle assay, and 5-ethynyl-2'-deoxyuridine staining were used to measure muscle proliferation. Reverse transcription-polymerase chain reaction and Western blotting were also performed to examine cellular signaling. Myogenic factors (including myosin heavy chain, MSTN, myocardin, and serum response factor) increased significantly after day 5 during myogenesis. MSTN was efficiently silenced after transfecting the dCas9 RNP complex, which significantly promoted more myotube formation and a higher fusion index for L6 cells. In cellular signaling, MSTN repression enhanced the expression of MyoG and MyoD, phosphorylation of Smad2, and the activity of Wnt1/GSK-3β/β-catenin pathway. Moreover, MSTN repression accelerated L6 cell growth with a higher cell proliferation index as well as a higher expression of cyclin D1 and cyclin E. Nonviral delivery of the dCas9 RNP complex significantly promoted myoblast differentiation and proliferation, providing a promising approach to improve muscle regeneration for SUI and PFD. Further characterization and validation of this approach in vivo are needed.

Yuming Sun,1– 6,&ast; Peilin Liu,7,&ast; Lifang Zhang,8 Shaorong Lei,1 Qian Gao7 1Department of Plastic and Cosmetic Surgery, Xiangya Hospital, Central South University, Changsha, People’s Republic of China; 2National Engineering Research Centre of Personalized Diagnostic and Therapeutic Technology, Xiangya Hospital, Central South University, Changsha, People’s Republic of China; 3Furong Laboratory, Central South University, Changsha, People’s Republic of China; 4Hunan Key Laboratory of Skin Cancer and Psoriasis, Hunan Engineering Research Centre of Skin Health and Disease, Xiangya Hospital, Central South University, Changsha, People’s Republic of China; 5National Clinical Research Centre for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, People’s Republic of China; 6Department of Dermatology, Xiangya Hospital, Central South University, Changsha, People’s Republic of China; 7Clinical Laboratory Department, Xiangya Hospital, Central South University, Changsha, People’s Republic of China; 8Department of Plastic and Cosmetic Surgery, Changsha Mylike Cosmetic Hospital, Changsha, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Qian Gao; Shaorong Lei, Email gaoqian@csu.edu.cn; leishaorong@csu.edu.cnBackground: The status of T lymphocyte subset counts in patients with COVID-19 remains uncertain. This study aimed to assess alterations in peripheral blood CD3+CD8+T (CD8+T) cells among hospitalized COVID-19 patients who have not received antiviral treatment and to evaluate their prognostic value within this patient population.Methods: A single-center, retrospective cohort study and a meta-analysis were conducted. The cohort study was performed at Xiangya Hospital from December 5, 2022, to January 31, 2023. We conducted a meta-analysis to explore the association between peripheral blood CD3+CD8+T cells and mortality in COVID-19 patients who did not receive antiviral therapy. All relevant studies in Embase, PubMed, Web of Science databases were systematically searched for meta-analysis.Results: The retrospective cohort study included 201 patients. A significant decrease in peripheral blood CD8+ T cell count was found to be associated with an increased risk of mortality (adjusted odds ratio [aOR]: 13.88; 95% confidence interval [CI]: 3.15– 61.23), after adjusting for gender, age, comorbidities, severity at admission, steroid therapy, and antibiotic therapy. The threshold value for CD8+T cell counts, determined by the receiver operating characteristic (ROC) curve analysis, was 145.5 (area under the curve [AUC]: 0.828, specificity: 90.3%, sensitivity: 72.9%, P< 0.001). Additionally, A total of 7 studies with 2765 participants were included in the meta-analysis. The meta-analysis reveals a significant association between lower CD8+ T cell counts and mortality (odds ratio [OR] = 3.543, 95% CI: 1.726 to 7.272; I2=93%).Conclusion: Peripheral blood CD8+ T cell can serve as a valuable prognostic biomarker for hospitalized patients who do not receive antiviral treatment.Keywords: peripheral blood CD3+CD8+T cell, prognostic biomarker, hospitalised COVID-19 patients

Jingmei Li,1,&ast; Jin Zhang,1,&ast; Yan Gao,1 Sibei Lei,1 Jieping Wu,1 Xiaohua Chen,1 Kaiyu Wang,1 Xingmei Duan,2 Ke Men1 1Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, People’s Republic of China; 2Department of Pharmacy, Personalized Drug Therapy Key Laboratory of Sichuan Province Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Ke Men, State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital of Sichuan University, Chengdu, 610041, People’s Republic of China, Email mendingbob@hotmail.com Xingmei Duan, Department of Pharmacy, Personalized Drug Therapy Key Laboratory of Sichuan Province, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China, Email duanxingmei2003@163.comIntroduction: Cell-membrane nanocarriers are usually constructed by modifying the nanoparticle surface with cell membrane extracts, which has a direct benefit in endowing targeting capacity to nanocarriers based on their original cell types. However, delivering nucleic acid cargos by cell membrane–based nanoparticles is difficult owing to the strong negative charge of the cell membrane fraction. In this study, we developed a cancer cell membrane–based drug delivery system, the cMDS, for efficient siRNA delivery. Meanwhile, the cancer-specific immune response stimulated by the gene vector itself could offer synergistic anti-cancer ability.Methods: The cMDS was prepared by ultrasound, and its transfection efficiency and anti-cancer ability were examined using cultures of CT26 cells. MTT and red blood cell hemolysis tests were performed to assess the safety of cMDS, while its targeted gene delivery and strong immune stimulation were investigated in a subcutaneous tumor model. Moreover, the detailed anti-cancer immune stimulation mechanisms of cMDS are uncovered by protein chip analysis.Results: The cMDS was spherical core-shell structure. It showed high transfection efficiency and anti-cancer ability in vitro. In animal experiments, intravenously administered cMDS/siStat3 complex efficiently suppress the growth of colon cancer. Moreover, the result of protein chip analysis suggested that cMDS affect the migration and chemotaxis of immune cells.Conclusion: The cMDS shows obvious tumor tissue-specific accumulation properties and strong immune stimulation ability. It is an advanced targeted gene delivery system with potent immunotherapeutic properties.Keywords: bioinspired material, cancer cell membrane, targeted siRNA delivery, immunotherapeutic, colon cancer

Abstract Manipulating gene expression in the host genome with high precision is crucial for controlling cellular function and behavior. Here, we present a precise, non-invasive, and tunable strategy for controlling the expression of multiple endogenous genes both in vitro and in vivo, utilizing ultrasound as the stimulus. By engineering a hyper-efficient dCas12a and effector under a heat shock promoter, we demonstrate a system that can be inducibly activated through thermal energy produced by ultrasound absorption. This system allows versatile thermal induction of gene activation or base editing across cell types, including primary T cells, and enables multiplexed gene activation using a single guide RNA array. In mouse models, localized temperature elevation guided by high-intensity focused ultrasound effectively triggers reporter gene expression in implanted cells. Our work underscores the potential of ultrasound as a clinically viable approach to enhance cell and gene-based therapies via precision genome and epigenome engineering.

Xin Zhao,1,&ast; Pingyuan Ge,1,&ast; Shaohua Lei,1 Siqi Guo,1 Peng Zhou,1 Li Zhao,1 Yiyu Qi,1 Xiaotong Wei,2 Weizhen Wu,2 Ningjing Wang,2 Rui Guo,3 Nianyun Yang,1 Qingqing Xiao,1 Qichun Zhang,1,2 Huaxu Zhu1 1Department of Traditional Chinese Medicine Processing and Preparation, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of China; 2Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, People’s Republic of China; 3School of Medicine and Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Qichun Zhang; Huaxu Zhu, Email zhangqichun@njucm.edu.cn; zhuhx@njucm.edu.cnIntroduction: Neuroinflammation is one of the major pathogeneses in Alzheimer’s disease (AD) and mainly involves abnormal inflammatory activation of microglia by multiple pathological stimuli. The treatment of AD remains a major challenge due to the multifactorial characterization of AD and the inefficient ability of therapeutic drugs to permeate through the blood‒brain barrier (BBB). Accordingly, drug combination treatment and drug carrier delivery have become important therapeutic tools for the treatment of multifactorial diseases, especially AD.Methods: Inflammatory cytokine levels in microglia, including NO, TNF-α, IL-1β, IL-4, and IL-10, were detected. The Morris water maze and object location task were used to investigate the learning and memory functions of APP/PS1 mice in different treatment groups. The number of neurons and plasticity of synapses were evaluated by immunofluorescence double labelling. Additionally, the ratio of β-amyloid plaques and the number of activated microglia were evaluated by immunofluorescence staining. The concentrations of β-amyloid plaques and inflammatory factors in the hippocampus were determined by ELISA. Microglia-derived exosomes (Exos) were extracted and purified by size exclusion chromatography. The distribution of exosomes and drugs was investigated in vitro and in vivo.Results: Compared to single drug interventions, the combination of Ber and Pal (Ber/Pal) modulated microglial inflammatory cytokine levels. Ber/Pal promoted the recovery of learning and memory impairment in APP/PS1 mice. Immunofluorescence staining indicated that Ber/Pal restored neurons, inhibited Aβ plaque formation and microglial activation, and regulated the secretion of inflammatory factors. Exos promoted the accumulation of drugs in cells and tissues and improved the targeting of drugs across the BBB.Conclusion: Ber/Pal could offer a synergistic and more comprehensive therapeutic effect in AD. Additionally, the microglia-derived Exos-Ber/Pal delivery system promoted the targeting and permeation of drugs into the brain, suggesting a creative strategy for targeting AD therapy by regulating neuroinflammation in microglial cells.Keywords: Alzheimer’s disease, neuroinflammation, phytoconstituents, drug combination, BBB permeability, exosomes

Abstract Chronic wounds impose a significant healthcare burden to a broad patient population. Cell-based therapies, while having shown benefits for the treatment of chronic wounds, have not yet achieved widespread adoption into clinical practice. We developed a CRISPR/Cas9 approach to precisely edit murine dendritic cells to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing of tolerogenic dendritic cells, we identified N-myc downregulated gene 2 (Ndrg2), which marks a specific population of dendritic cell progenitors, as a promising target for CRISPR knockout. Ndrg2-knockout alters the transcriptomic profile of dendritic cells and preserves an immature cell state with a strong pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a therapeutic hydrogel for in vivo cell delivery and developed an effective translational approach for dendritic cell-based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in dendritic cells for treating various types of chronic wounds.

A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.

Abstract Background Chimeric antigen receptor (CAR)-T cell therapies for the treatment of hematological malignancies experienced tremendous progress in the last decade. However, essential limitations need to be addressed to further improve efficacy and reduce toxicity to assure CAR-T cell persistence, trafficking to the tumor site, resistance to an hostile tumor microenvironment (TME), and containment of toxicity restricting production of powerful but potentially toxic bioproducts to the TME; the last could be achieved through contextual release upon tumor antigen encounter of factors capable of converting an immune suppressive TME into one conducive to immune rejection. Methods We created an HER2-targeting CAR-T (RB-312) using a clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system, which induces the expression of the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. This circuit includes two lentiviral constructs. The first one (HER2-TEV) expresses an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3z co-stimulatory domains linked to the tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the interleukin (IL)-12A and IL12B transcription start site (TSS), respectively. The second construct (LdCV) encodes linker for activation of T cells (LAT) fused to nuclease-deactivated Streptococcus Pyogenes Cas9 (dCas9)-VP64-p65-Rta (VPR) via a TEV-cleavable sequence (TCS). Activation of the CAR brings HER2-TEV in close proximity to LdCV releasing dCas9 for nuclear localization. This conditional circuit leads to conditional and reversible induction of the IL-12/p70 heterodimer. RB-312 was compared in vitro to controls (cRB-312), lacking the IL-12 sgRNAs and conventional HER2 CAR (convCAR). Results The inducible CRISPRa system activated endogenous IL-12 expression resulting in enhanced secondary interferon (FN)-γ production, cytotoxicity, and CAR-T proliferation in vitro, prolonged in vivo persistence and greater suppression of HER2+ FaDu oropharyngeal cancer cell growth compared to the conventional CAR-T cell product. No systemic IL-12 was detected in the peripheral circulation. Moreover, the combination with programmed death ligand (PD-L1) blockade demonstrated robust synergistic effects. Conclusions RB-312, the first clinically relevant product incorporating a CRISPRa system with non-gene editing and reversible upregulation of endogenous gene expression that promotes CAR-T cells persistence and effectiveness against HER2-expressing tumors. The autocrine effects of reversible, nanoscale IL-12 production limits the risk of off-tumor leakage and systemic toxicity.

The control of the in-plane domain evolution in ferroelectric thin films is not only critical to understanding ferroelectric phenomena but also to enabling functional device fabrication. However, in-plane polarized ferroelectric thin films typically exhibit complicated multi-domain states, not desirable for optoelectronic device performance. Here we report a strategy combining interfacial symmetry engineering and anisotropic strain to design single-domain, in-plane polarized ferroelectric BaTiO3 thin films. Theoretical calculations predict the key role of the BaTiO3/PrScO3 [Formula: see text] substrate interfacial environment, where anisotropic strain, monoclinic distortions, and interfacial electrostatic potential stabilize a single-variant spontaneous polarization. A combination of scanning transmission electron microscopy, piezoresponse force microscopy, ferroelectric hysteresis loop measurements, and second harmonic generation measurements directly reveals the stabilization of the in-plane quasi-single-domain polarization state. This work offers design principles for engineering in-plane domains of ferroelectric oxide thin films, which is a prerequisite for high performance optoelectronic devices.

Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and ∼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1+/CD45- tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-β, producing 150 pg/106 cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-β). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-β plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.

Aging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue and organismal levels. At the chromatin level, aging associates with progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis. Nuclear reprogramming to pluripotency can revert both the age and the identity of any cell to that of an embryonic cell. Recent evidence shows that transient reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice. However, it is unknown how this form of rejuvenation would apply to naturally aged human cells. Here we show that transient expression of nuclear reprogramming factors, mediated by expression of mRNAs, promotes a rapid and broad amelioration of cellular aging, including resetting of epigenetic clock, reduction of the inflammatory profile in chondrocytes, and restoration of youthful regenerative response to aged, human muscle stem cells, in each case without abolishing cellular identity.