Prime editing-mediated correction of the leptin receptor in muscle cells of db/db mice.

Genetic diseases can be caused by monogenic diseases, which result from a single gene mutation in the DNA sequence. Many innovative approaches have been developed to cure monogenic genetic diseases, namely by genome editing. A specific type of genomic editing, prime editing, has the potential advantage to edit the human genome without requiring double-strand breaks or donor DNA templates for editing. Additionally, prime editing does not require a precisely positioned protospacer adjacent motif (PAM) sequence, which offers flexible target and more precise genomic editing. Here we detail a novel construction of a prime editing extended guide RNA (pegRNA) to target mutated leptin receptors in B6.BKS(D)-Leprdb/J mice (db/db mice). The pegRNA was then injected into the flexor digitorum brevis (FDB) muscle of db/db mice to demonstrate in vivo efficacy, which resulted in pegRNA mediated base transversion at endogenous base transversion. Genomic DNA sequencing confirmed that prime editing could correct the mutation of leptin receptor gene in db/db mice. Furthermore, prime editing treated skeletal muscle exhibited enhanced leptin receptor signals. Thus, the current study showed in vivo efficacy of prime editing to correct mutant protein and rescue the physiology associated with functional protein.

FOXO1 regulates RUNX2 ubiquitination through SMURF2 in calcific aortic valve disease.

The prevalence of calcific aortic valve disease (CAVD) remains substantial while there is currently no medical therapy available. Forkhead box O1 (FOXO1) is known to be involved in the pathogenesis of cardiovascular diseases, including vascular calcification and atherosclerosis; however, its specific role in calcific aortic valve disease remains to be elucidated. In this study, we identified FOXO1 significantly down-regulated in the aortic valve interstitial cells (VICs) of calcified aortic valves by investigating clinical specimens and GEO database analysis. FOXO1 silencing or inhibition promoted VICs osteogenic differentiation in vitro and aortic valve calcification in Apoe-/- mice, respectively. We identified that FOXO1 facilitated the ubiquitination and degradation of RUNX2, which process was mainly mediated by SMAD-specific E3 ubiquitin ligase 2 (SMURF2). Our discoveries unveil a heretofore unacknowledged mechanism involving the FOXO1/SMURF2/RUNX2 axis in CAVD, thereby proposing the potential therapeutic utility of FOXO1 or SMURF2 as viable strategies to impede the progression of CAVD.

Is Time Scheduling Important? An Analysis of Donor Heart Cross-clamp Times During Heart Transplantation.

Background: Outcomes in heart transplantation are affected by a variety of variables and patient factors. However, the impact of circadian rhythms, gene expression, and transcription remain underexplored. We thus evaluated the potential role of donor heart cross-clamp times on short-term and long-term outcomes after heart transplantation. Methods: A total of 31 713 heart transplants were identified from the United Network for Organ Sharing Database. Patients were first stratified on the basis of time of donor procurement: 12 am to 12 pm or 12 pm to 12 am. To evaluate a possible effect of circadian rhythms, donor time was further divided into 5 groups based on preclinical data: 4 am to 8 am; 8 am to 11 am; 11 am to 5 pm; 5 pm to 10 pm; 10 pm to 4 am. Groups were assessed with comparative statistics. Long-term survival was evaluated using Kaplan-Meier methods and a multivariate Cox proportional hazard model. Results: Patients who received hearts recovered between 12 am and 12 pm had significantly higher survival than those who received hearts recovered between 12 pm and 12 am. This survival difference was observed in both unadjusted (P = 0.002) and adjusted analyses (hazard ratio [HR]: 0.93; 95% confidence interval [CI], 0.89-0.97; P < 0.001). On unadjusted analysis, the survival difference among the 5 groups was insignificant (P = 0.07). Following adjustment, the periods of 11 am to 5 pm (HR: 1.09, 95% CI, 1.02-1.17; P = 0.012), 5 pm to 10 pm (HR: 1.11; 95% CI, 1.04-1.19; P = 0.002), and 10 pm to 4 am (HR: 1.07; 95% CI, 1.01-1.15; P = 0.034), were all independently associated with increased long-term mortality. Notably, the time of 8 am to 11 am was not associated with a change in survival (HR: 1.04; 95% CI, 0.96-1.14; P = 0.3). Conclusions: Given the independent association of donor timing and survival after adjustment in a large national cohort, further investigation into the role of donor circadian rhythm and donor procurement time is warranted in preclinical and clinical studies. Understanding the underlying mechanisms of this observation could potentially lead to the development of effective treatments and donor procurement processes that prepare the organs for transplantation in a better condition.

Global, regional, and national incidence, mortality, and disability-adjusted life years of non-rheumatic valvular heart disease and trend analysis from 1990 to 2019: Results from the Global Burden of Disease study 2019.

BACKGROUND: In the context of the population growing and aging worldwide, the incidence of non-rheumatic valvular heart disease increased rapidly. This study aimed to describe the burden of non-rheumatic valvular heart disease, providing an up-to-date and comprehensive analysis on the global and regional levels and time trends from 1900 to 2019. METHODS: The Global Burden of Disease 2019 was used to obtain data for this analysis. Non-rheumatic valvular heart disease in the Global Burden of Disease study includes both non-rheumatic calcific aortic valve disease and non-rheumatic degenerative mitral valve disease. The incidence, mortality, and disability-adjusted life year in 204 countries from 1990 to 2019 were analyzed by location, year, sex, age, and socio-demographic index. Estimated annual percentage change was calculated to represent the temporal trends from 1990 to 2019. Spearman's rank order correlation was used to determine the correlation between socio-demographic index and the incidence and burden of non-rheumatic valvular heart disease. RESULTS: Globally, there were 1.65 million (95% uncertainty interval, 1.56-1.76 million) incident cases, 0.16 million (95% uncertainty interval, 0.14-0.18 million) death cases, and 2.79 million (95% uncertainty interval, 2.52-3.31 million) disability-adjusted life years of non-rheumatic valvular heart disease. Compared with 1990, the number of incident cases, death cases, and disability-adjusted life years in 2019 increased by 104.58%, 210.60%, and 167.62%, respectively, the age-standardized incidence rate (estimated annual percentage change, 0.39; 95% confidence interval, 0.29 to 0.49) increased due to population growth, and the age-standardized death rates (estimated annual percentage change, -0.32; 95% confidence interval, -0.39 to -0.25) and age-standardized disability-adjusted life year rate (estimated annual percentage change, -0.81; 95% confidence interval, -0.87 to -0.74) decreased during this period. Regarding the socio-demographic index, the highest age-standardized incidence, death, and disability-adjusted life year rates of non-rheumatic valvular heart disease were found in high-socio-demographic index countries in 2019. Meantime, the age-standardized incidence rate remained increased from 1990 to 2019, while significant decreases were found in the age-standardized death rate and age-standardized disability-adjusted life year rate. Females have higher age-standardized incidence rate, while higher age-standardized death rate and age-standardized disability-adjusted life year rate belong to males globally during the period of 1990-2019. Increasing trends were observed for both incidence, death, and disability-adjusted life year rates with age. High systolic blood pressure was the leading cause for non-rheumatic valvular heart disease across all ages. CONCLUSIONS: From 1990 to 2019, the age-standardized incidence rate of non-rheumatic valvular heart disease remained increased, while age-standardized death rate and age-standardized disability-adjusted life year rate decreased, resulting from the growing population worldwide and improving medical resources. The aged, who has high systolic blood pressure and diet high in sodium, should pay more attention to, especially in high-socio-demographic index regions. With the population aging, the number of patients who require heart valve replacement is estimated to increase significantly in the future. Effective measures are warranted to control and treat the incidence and burden of non-rheumatic valvular heart disease.

Donor circadian clock influences the long-term survival of heart transplantation by immunoregulation.

AIMS: Circadian clocks play important role in immunoregulation. We aimed to investigate cardiac circadian clock specific pathways and compare cardiac grafts procured at different timing on survival after transplantation to explore novel criteria for donor selection. METHODS AND RESULTS: In primate heart, phase set enrichment analysis (PSEA) showed rhythmic transcripts were enriched in antigen processing and presentation during activation of circadian rhythm. Digital sorting of immune cell composition and single-sample gene set enrichment analysis (ssGSEA) in unused donor transcriptomes showed the pathway, positive regulation of circadian rhythm significantly correlates with allograft rejection and antigen presentation pathways as well as with increased compositions of matured dendritic cell, CD4+ T cell, and naive B cell. Single-centre retrospective cohort of 390 adult heart transplants between 1 January 2015 and 31 December 2020 was used to generate a propensity score matching (PSM) cohort. Survival curve differed significantly showing inferior long-term survival when donor hearts were procured at activation group (12 pm to 12 am) compared to repression group (12 am to 12 pm) (6-year survival: 64.2% vs. 75.8%, P = 0.0065). Activation group was also associated with significantly higher rates of in-hospital death, cardiopulmonary resuscitation, and usage of mechanical circulatory support after heart transplantation compared to repression group. Furthermore, tendency for post-transplant free of rejection rates was higher in repression group compared to activation group (acute rejection, Gehan-Breslow P = 0.11 and 0.04; chronic rejection, Log rank P = 0.077 and 0.15, in full and PSM cohorts, respectively). Adjusted Cox regression analysis showed that activation group was associated with 2.20 times increased hazard of death (hazard ratio: 2.20; 95% confidence interval: 1.23-3.95; P = 0.008) compared to repression group. CONCLUSIONS: Circadian immunity may represent donor-related risk factors for cardiac allograft rejection through activating genes related to antigen presentation pathway and immune cells oscillation at specific time of day. Molecular circadian clock should be considered during retrieval of cardiac allografts in order to maximize graft durability.

Platelet membrane-coated alterbrassicene A nanoparticle inhibits calcification of the aortic valve by suppressing phosphorylation P65 NF-κB.

Rationale: Calcific aortic valve disease (CAVD) is a leading cause of cardiovascular mortality and morbidity with increasing prevalence and incidence. The pathobiology of CAVD involves valvular fibrocalcification, and osteogenic and fibrogenic activities are elevated in aortic valve interstitial cells (VICs) from diseased valves. It has been demonstrated that activated NF-κB pathway was present in the early stage of CAVD process. There is currently no effective clinical drugs targeting NF-κB pathway for CAVD treatment. Therefore, it is of great clinical significance to seek effective treatments for valve calcification. Methods: In this study, we established immortal human valve interstitial cells (im-hVICs) with pGMLV-SV40T-puro lentivirus. Alizarin red staining and western blotting were performed to evaluate the calcification of immortal VICs supplemented with different compounds. The natural fusicoccane diterpenoid alterbrassicene A (ABA) was found to have potential therapeutic functions. Ribonucleic acid sequencing was used to identify the potential target of ABA. Platelet membrane-coated nanoparticle of ABA (PNP-ABA) was fabricated and the IBIDI pump was used to evaluate the adhesion ability of PNP-ABA. Murine wire-induced aortic valve stenosis model was conducted for in vivo study of PNP-ABA. Results: The natural fusicoccane diterpenoid ABA was found to significantly reduce the calcification of human VICs during osteogenic induction via inhibiting the phosphorylation P65. Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein-2 (BMP2) were down regulated with the treatment of ABA in human VICs. Additionally, molecular docking results revealed that ABA bound to RelA (P65) protein. Phosphorylation of P65 (Ser536) was alleviated by ABA treatment, as well as the nuclear translocation of P65 during osteogenic induction in human VICs. Alizarin red staining showed that ABA inhibited osteogenic differentiation of VICs in a dose-dependent manner. PNP-ABA attenuated aortic valve calcification in murine wire-induced aortic valve stenosis model in vivo. Conclusions: The establishment of im-hVICs provides a convenient cell line for the study of CAVD. Moreover, our current research highlights a novel natural compound, ABA, as a promising candidate to prevent the progression of CAVD.

Identifying hub genes of calcific aortic valve disease and revealing the immune infiltration landscape based on multiple WGCNA and single-cell sequence analysis.

Background: Calcific aortic valve disease (CAVD) is a progressive fibrocalcific disease that can be treated only through valve replacement. This study aimed to determine the role of hub genes and immune cell infiltration in CAVD progression. Methods: In this study, bioinformatics analysis was used to identify hub genes involved in CAVD. The datasets were downloaded from the Gene Expression Omnibus (GEO) database. Gene expression differences were evaluated via pathway and Gene Ontology analyses. Weighted gene co-expression network analysis (WGCNA) and differentially expressed genes were used to screen hub genes. The CIBERSORT algorithm was used to compare immune infiltration into the calcified aortic valve based on the hub genes between high- and low-expression groups. We also performed single-cell RNA sequencing based on six different human aortic valve leaflets. The expression of hub genes was identified in human and mouse samples through quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry, immunofluorescence, and ELISA, and clinical features of the patients were investigated. Results: In total, 454 differentially expressed genes were obtained from the GEO database. WGCNA was used to find 12 co-expression modules in the Array Express database, of which one hub module (brown module) was most correlated with CAVD. Two hub genes were identified after combining the differentially expressed genes S100A8 and S100A9. Regarding these genes, the immune infiltration profiles varied between high- and low-expression groups. Compared with that in the low hub gene expression group, the high hub gene expression group had a higher proportion of activated NK cells (p < 0.01) and M1 macrophages (p < 0.05). The expression of S100A8 and S100A9 was consistent with single-gene RNA sequencing results, confirming that the expression levels of these two hub genes are significantly upregulated in patients with CAVD (p < 0.01). Furthermore, these results were verified using mouse and human samples by performing immunofluorescence, immunohistochemistry, qPCR, and ELISA analyses. Finally, the localization of S100A8 and S100A9 in monocytes and macrophages was confirmed via immunofluorescence using human aortic valves. Conclusion: These results demonstrate that S100A8 and S100A9 are two hub genes involved in CAVD, which might play an important role in its development through immune-related signaling pathways.

MG53 Inhibits Necroptosis Through Ubiquitination-Dependent RIPK1 Degradation for Cardiac Protection Following Ischemia/Reperfusion Injury.

Rationale: While reactive oxygen species (ROS) has been recognized as one of the main causes of cardiac injury following myocardial infarction, the clinical application of antioxidants has shown limited effects on protecting hearts against ischemia-reperfusion (I/R) injury. Thus, the precise role of ROS following cardiac injury remains to be fully elucidated. Objective: We investigated the role of mitsugumin 53 (MG53) in regulating necroptosis following I/R injury to the hearts and the involvement of ROS in MG53-mediated cardioprotection. Methods and Results: Antioxidants were used to test the role of ROS in MG53-mediated cardioprotection in the mouse model of I/R injury and induced human pluripotent stem cells (hiPSCs)-derived cardiomyocytes subjected to hypoxia or re-oxygenation (H/R) injury. Western blotting and co-immunoprecipitation were used to identify potential cell death pathways that MG53 was involved in. CRISPR/Cas 9-mediated genome editing and mutagenesis assays were performed to further identify specific interaction amino acids between MG53 and its ubiquitin E3 ligase substrate. We found that MG53 could protect myocardial injury via inhibiting the necroptosis pathway. Upon injury, the generation of ROS in the infarct zone of the hearts promoted interaction between MG53 and receptor-interacting protein kinase 1 (RIPK1). As an E3 ubiquitin ligase, MG53 added multiple ubiquitin chains to RIPK1 at the sites of K316, K604, and K627 for proteasome-mediated RIPK1 degradation and inhibited necroptosis. The application of N-acetyl cysteine (NAC) disrupted the interaction between MG53 and RIPK1 and abolished MG53-mediated cardioprotective effects. Conclusions: Taken together, this study provided a molecular mechanism of a potential beneficial role of ROS following acute myocardial infarction. Thus, fine-tuning ROS levels might be critical for cardioprotection.

Ubiquitin Carboxyl-Terminal Hydrolase L1 of Cardiomyocytes Promotes Macroautophagy and Proteostasis and Protects Against Post-myocardial Infarction Cardiac Remodeling and Heart Failure.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinase known to play essential roles in the nervous tissue. Myocardial upregulation of UCHL1 was observed in human dilated cardiomyopathy and several animal models of heart disease, but the (patho)physiological significance of UCHL1 in cardiomyocytes remains undefined. Hence, we conducted this study to fill this critical gap. We produced cardiomyocyte-restricted Uchl1 knockout (CKO) by coupling the Uchl1-floxed allele with transgenic Myh6-Cre in C57B/6J inbred mice. Mice transgenic for Myh6-Cre were used as controls (CTL). Myocardial Uchl1 proteins were markedly reduced in CKO mice but they did not display discernible abnormal phenotype. Ten-week old CTL or CKO mice were subjected to left anterior descending artery ligation (myocardial infarction, MI) or sham surgery (Sham) and characterized at 7- and 28-day after surgery. Compared with Sham mice, significant increases in myocardial UCHL1 proteins were detected in CTL MI but not in CKO MI mice. MI-induced left ventricular (LV) chamber dilation, reduction of ejection fraction (EF) and fractional shortening (FS), and LV anterior wall thinning detected by echocardiography were comparable between the CTL MI and CKO MI groups 7-day post-MI. However, by 28-day post-MI, MI-induced LV chamber dilatation, EF and FS reduction, increases of myocardial ubiquitin conjugates, and increases in the heart weight to body weight ratio and the ventricular weight to body weight ratio were significantly more pronounced in CKO MI than CTL MI mice. As further revealed by LV pressure-volume relationship analyses, CKO MI mice but not CTL MI mice displayed significant decreases in stroke volume, cardiac output, and the maximum rates of LV pressure rising or declining and of LV volume declining, as well as significant increases in LV end-diastolic pressure and Tau, compared with their respective Sham controls. LC3-II flux assays reveal that autophagic flux is decreased in CKO mouse myocardium as well as in cultured Uchl1-deficient cardiomyocytes. In conclusion, UCHL1 of cardiomyocytes is dispensable for development but promotes macroautophagy in cardiomyocytes. Upregulation of UCHL1 in post-MI hearts occurs primarily in the cardiomyocytes and protects against post-MI cardiac remodeling and malfunction likely through supporting autophagic flux and proteostasis during a stress condition.

UCHL1 protects against ischemic heart injury via activating HIF-1α signal pathway.

Ubiquitin carboxyl-terminal esterase L1 (UCHL1) has been thought to be a neuron specific protein and shown to play critical roles in Parkinson's Disease and stroke via de-ubiquiting and stabilizing key pathological proteins, such as α-synuclein. In the present study, we found that UCHL1 was significantly increased in both mouse and human cardiomyocytes following myocardial infarction (MI). When LDN-57444, a pharmacological inhibitor of UCHL1, was used to treat mice subjected to MI surgery, we found that administration of LDN-57444 compromised cardiac function when compared with vehicle treated hearts, suggesting a potential protective role of UCHL1 in response to MI. When UCHL1 was knockout by CRISPR/Cas 9 gene editing technique in human induced pluripotent stem cells (hiPSCs), we found that cardiomyocytes derived from UCHL1-/- hiPSCs were more susceptible to hypoxia/re-oxygenation induced injury as compared to wild type cardiomyocytes. To study the potential targets of UCHL1, a BioID based proximity labeling approach followed by mass spectrum analysis was performed. The result suggested that UCHL1 could bind to and stabilize HIF-1α following MI. Indeed, expression of HIF-1α was lower in UCHL1-/- cells as determined by Western blotting and HIF-1α target genes were also suppressed in UCHL1-/- cells as quantified by real time RT-PCR. Recombinant UCHL1 (rUCHL1) protein was purified by E. Coli fermentation and intraperitoneally (I.P.) delivered to mice. We found that administration of rUCHL1 could significantly preserve cardiac function following MI as compared to control group. Finally, adeno associated virus mediated cardiac specific UCHL1 delivery (AAV9-cTNT-m-UCHL1) was performed in neonatal mice. UCHL1 overexpressing hearts were more resistant to MI injury as compare to the hearts infected with control virus. In summary, our data revealed a novel protective role of UCHL1 on MI via stabilizing HIF-1α and promoting HIF-1α signaling.

Serp-1 Promotes Corneal Wound Healing by Facilitating Re-epithelialization and Inhibiting Fibrosis and Angiogenesis.

Purpose: Chemical corneal injuries carry a high morbidity and commonly lead to visual impairment. Here, we investigate the role of Serp-1, a serine protease inhibitor, in corneal wound healing. Methods: An alkaline-induced corneal injury was induced in 14 mice. Following injury, five mice received daily topical saline application while nine mice received Serp-1 100 µL topically combined with a daily subcutaneous injection of 100 ng/gram body weight of Serp-1. Corneal damage was monitored daily through fluorescein staining and imaging. Cross sectional corneal H&E staining were obtained. CD31 was used as marker for neovascularization. Results: Serp-1 facilitates corneal wound healing by reducing fibrosis and neovascularization while mitigating inflammatory cell infiltration with no noticeable harm related to its application. Conclusions: Serp-1 effectively mitigates inflammation, decreases fibrosis, and reduce neovascularization in a murine model of corneal injury without affecting other organs. Translational Relavence: Our study provides preclinical data for topical application of Serp-1 to treat corneal wounds.

Recombinant Human MG53 Protein Protects Against Alkaline-Induced Corneal Injuries in Mice.

INTRODUCTION: The current study was designed to test the potential role of recombinant human MG53 (rhMG53) protein on protecting against alkaline-induced corneal injury in mice. MATERIALS AND METHODS: A round filter paper with 2-mm diameter was soaked in 1 mol/L of NaOH solution. The mouse alkaline injury was generated by placing the filter paper directly on the cornea for 30 seconds and washed with 30-mL saline; 10 µL of rhMG53 solution (20 µg/mL) or saline control was topically administrated on the mouse corneas (twice per day for 10 days). Re-epithelialization was measured by fluorescein staining and imaged by a slit lamp equipped with a digital camera. Clinical neovascularization and opacity scores were measured every day after injury. Ten days after injury, mice were sacrificed and corneas were dissected out for flat mount staining of CD31 for neovascularization. RESULTS: MG53 was present in both dog aqueous humor and human tears. mg53-/- corneas were more susceptible to alkaline-induced corneal injury. Topical treatment of rhMG53 improved re-epithelialization, suppressed neovascularization, and fibrosis induced by alkaline injury. CONCLUSIONS: rhMG53 may be an effective means to treat corneal wounding.

Cavin3 Suppresses Breast Cancer Metastasis via Inhibiting AKT Pathway.

OBJECTIVE: Cavin3 is a putative tumor suppressor protein. However, its molecular action on tumor regulation is largely unknown. The aim of the current study is to explore the implication of cavin3 alteration, its clinical significance, and any potential molecular mechanisms in the regulation of breast cancer (BC). METHODS: TCGA (The Cancer Genome Atlas) and GTEx (Genotype-Tissue Expression) data bases, and 17 freshly paired BC and adjacent normal tissues were analyzed for mRNA levels of Cavin3. Furthermore, cavin3 protein expression from 407 primary BC samples were assessed by immunohistochemistry (IHC) and measured by H-score. The clinical significance of cavin3 expression was explored by Kaplan-Meier analysis and the Cox regression method. In vitro biological assays were performed to elucidate the function and underlying mechanisms of cavin 3 in BC cell lines. RESULTS: Cavin3 mRNA was dramatically down-regulated in BC compared with the negative control. The median H-score of cavin3 protein by IHC was 50 (range 0-270). There were 232 (57%) and 175 (43%) cases scored as low (H-score≤50) and high (H-score >50) levels of cavin3, respectively. Low cavin3 was correlated with a higher T and N stage, and worse distant metastasis-free survival (DMFS) and overall survival (OS). Multivariate survival analysis revealed low cavin3 was an independent fact for worse DMFS. In BC cells, an overexpression of cavin3 could inhibit cell migration and invasion, and significantly decreased the level of p-Akt. Knockout of cavin3, meanwhile, promoted cell invasion ability and increased the level of p-AKT. CONCLUSION: Cavin3 expression is significantly lower in BC and is correlated with distant metastasis and worse survival. Cavin3 functions as a metastasis suppressor via inhibiting the AKT pathway, suggesting cavin3 as a potential prognostic biomarker and a target for BC treatment.

A simple, quick, and efficient CRISPR/Cas9 genome editing method for human induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) have become an essential research platform to study different human diseases once being discovered by Dr. Shinya Yamanaka in 2006. Another breakthrough in biomedical research is the application of CRISPR/Cas9 system for genome editing in mammalian cells. Although numerous studies have been done to develop methods for gene editing in iPSCs, the current approaches suffer from several limitations, including time and labor consuming, low editing efficiency, and potential off-target effects. In the current study, we report an electroporation-mediated plasmid CRISPR/Cas9 delivery approach for genome editing in iPSCs. With this approach, an edited iPSC cell line could be obtained within 2 weeks. In addition, the transit introducing of CRISPR/Cas9 machinery could minimize genomic integration of Cas9 gene, which avoided potential long-term side effects of Cas9 enzyme. We showed that CRISPR/Cas9-mediated genomic editing did not affect pluripotency and differentiation ability of iPSCs. With the quickly evolving of both iPSC and CRISPR/Cas9-mediated genome editing research fields, we believe that our method can significantly facilitate the application of genome editing in iPSCs research.

Diabetes inhibits corneal epithelial cell migration and tight junction formation in mice and human via increasing ROS and impairing Akt signaling.

Corneal wounds usually heal quickly; but diabetic patients have more fragile corneas and experience delayed and painful healing. In the present study, we compared the healing capacity of corneal epithelial cells (CECs) between normal and diabetic conditions and the potential mechanisms. Primary murine CEC derived from wild-type and diabetic (db/db) mice, as well as primary human CEC were prepared. Human CEC were exposed to high glucose (30 mM) to mimic diabetic conditions. Cell migration and proliferation were assessed using Scratch test and MTT assays, respectively. Reactive oxygen species (ROS) production in the cells was measured using dichlorofluorescein reagent. Western blot was used to evaluate the expression levels of Akt. Transepithelial electrical resistance (TEER) and zonula occludens-1 (ZO-1) expression were used to determine tight junction integrity. We found that the diabetic CEC displayed significantly slower cell proliferation and migration compared with the normal CEC from both mice and humans. Furthermore, ROS production was markedly increased in CEC grown under diabetic conditions. Treatment with an antioxidant N-acetyl cysteine (NAC, 100 µM) significantly decreased ROS production and increased wound healing in diabetic CEC. Barrier function was significantly reduced in both diabetic mouse and human CEC, while NAC treatment mitigated these effects. We further showed that Akt signaling was impaired in diabetic CEC, which was partially improved by NAC treatment. These results show that diabetic conditions lead to delayed wound-healing capacity of CEC and impaired tight junction formation in both mice and human. Increased ROS production and inhibited Akt signaling may contribute to this outcome, implicating these as potential targets for treating corneal wounds in diabetic patients.

MG53 promotes corneal wound healing and mitigates fibrotic remodeling in rodents.

The cornea plays an important role in transmitting light and providing protection to the eye, but is susceptible to injury and infection. Standard treatments for corneal wounds include topical lubricants, antibiotics, bandage contact lens, and surgery. However, these measures are often ineffective. Here we show that MG53, a protein with an essential role in cell membrane repair, contributes to the corneal injury-repair process. Native MG53 is present in the corneal epithelia, tear film, and aqueous humor, suggesting its potential function in corneal homeostasis. Knockout of MG53 in mice causes impaired healing and regenerative capacity following injury. Exogenous recombinant human MG53 (rhMG53) protein protects the corneal epithelia against mechanical injury and enhances healing by promoting migration of corneal fibroblasts. Using in vivo alkaline-induced injury to the rat cornea, we show that rhMG53 promotes re-epithelialization and reduces post-injury fibrosis and vascularization. Finally, we show that rhMG53 modulates TGF-ß-mediated fibrotic remodeling associated with corneal injury. Overall, our data support the bi-functional role of MG53 in facilitating corneal healing and maintaining corneal transparency by reducing fibrosis and vascularization associated with corneal injuries.

Limited prognostic value of myocardial viability assessment in patients with coronary artery diseases and severe left ventricular dysfunction.

BACKGROUND: Myocardial viability assessment is typically performed in patients with coronary artery disease (CAD) and severe left ventricular (LV) dysfunction to identify those who might benefit from revascularization and assist in decision making process. However, the prognostic value of myocardial viability testing remains a debating issue. METHODS: Positron Emission Tomography using 18F-fluorodeoxyglucose (18FDG-PET) was performed in 81 patients with ischemic LV dysfunction [ejection fraction (EF) ≤35%] for myocardial viability assessment prior to coronary artery bypass surgery. Fifty-three of them received finally coronary artery bypass grafting and were divided into two groups according to the extent of myocardial scar: one group with scar burden ≥10% (n=30) and the other with scar burden <10% (n=23). The remaining patients were contraindicated for CABG and received optimal medical treatment (OMT, n=28). All patients were followed up and the primary endpoint was all-cause mortality and the secondary endpoint was a composite of all-cause mortality and major adverse cardiovascular and cerebrovascular events (MACCE). RESULTS: 18FDG-PET revealed a different profile of myocardial viability among three groups with respect to the extent of myocardial scar, the hibernating myocardium (both P<0.01), some echocardiographic parameters such as left ventricular diastolic dimension (LVDD) and EF were also significantly different (both P<0.05). Nevertheless, the baseline prevalence of comorbidities and functional classifications were comparable. The per-procedural parameters were not significantly different between two CABG groups. In a median follow-up time of 32 months, Kaplan Meier analysis uncovered no significant difference in terms of overall survival (P=0.74) and MACCE-free survival (P=0.66) among three groups. CONCLUSIONS: Myocardial viability assessment using 18FDG-PET is of limited prognostic value in patients with CAD and severe LV dysfunction. In patients with substantial myocardial scar burden despite the existence of considerable hibernating myocardium, functional recovery following surgical revascularization is not necessarily translated to survival benefits.