Aberrant Retinal Pigment Epithelial Cells Derived from Induced Pluripotent Stem Cells of a Retinitis Pigmentosa Patient with the PRPF6 Mutation.

Pre-mRNA processing factors (PRPFs) are vital components of the spliceosome and are involved in the physiological process necessary for pre-mRNA splicing to mature mRNA. As an important member, PRPF6 mutation resulting in autosomal dominant retinitis pigmentosa (adRP) is not common. Recently, we reported the establishment of an induced pluripotent stem cells (iPSCs; CSUASOi004-A) model by reprogramming the peripheral blood mononuclear cells of a PRPF6-related adRP patient, which could recapitulate a consistent disease-specific genotype. In this study, a disease model of retinal pigment epithelial (RPE) cells was generated from the iPSCs of this patient to further investigate the underlying molecular and pathological mechanisms. The results showed the irregular morphology, disorganized apical microvilli and reduced expressions of RPE-specific genes in the patient's iPSC-derived RPE cells. In addition, RPE cells carrying the PRPF6 mutation displayed a decrease in the phagocytosis of fluorescein isothiocyanate-labeled photoreceptor outer segments and exhibited impaired cell polarity and barrier function. This study will benefit the understanding of PRPF6-related RPE cells and future cell therapy.

Effect of porcine corneal stromal extract on keratocytes from SMILE-derived lenticules.

Propagating large amounts of human corneal stromal cells (hCSCs) in vitro while maintaining the physiological quality of their phenotypes is necessary for their application in cell therapy. Here, a novel medium to propagate hCSCs obtained from small incision lenticule extraction (SMILE)-derived lenticules was investigated and the feasibility of intrastromal injection of these hCSCs was assessed. Primary hCSCs were cultured in porcine corneal stroma extract (pCSE) with RIFA medium including ROCK inhibitor Y27632, insulin-transferrin-selenium, fibroblast growth factor 2, L-ascorbate 2-phosphate and 0.5% FBS (RIFA medium + pCSE). Protein profiling of the pCSE was identified using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). After subculturing in RIFA medium + pCSE or 10% FBS normal medium (NM), hCSCs at P4 were transplanted into mouse corneal stroma. Compared with NM, ALDH3A1, keratocan and lumican were significantly more expressed in the RIFA medium + pCSE. ALDH3A1 was also more expressed in the RIFA medium + pCSE than in the RIFA medium. Fibronectin and α-SMA were less expressed in the RIFA medium + pCSE than in the NM. Using Metascape analysis, the pCSE with its anti-fibrosis, pro-proliferation and anti-apoptosis activities, was beneficial for hCSC cultivation. The intrastromally implanted hCSCs in the RIFA medium + pCSE had positive CD34 expression but negative CD45 expression 35 days after injection. We provide a valuable new medium that is advantageous for the proliferation of hCSCs with the properties of physiological keratocytes. Intrastromal injection of hCSCs in RIFA medium + pCSE has the potential for clinical cell therapy.

Detection and functional characterization of a novel MEF2A variation responsible for familial dilated cardiomyopathy.

OBJECTIVES: Dilated cardiomyopathy (DCM) represents the most frequent form of cardiomyopathy, leading to heart failure, cardiac arrhythmias and death. Accumulating evidence convincingly demonstrates the crucial role of genetic defects in the pathogenesis of DCM, and over 100 culprit genes have been implicated with DCM. However, DCM is of substantial genetic heterogeneity, and the genetic determinants underpinning DCM remain largely elusive. METHODS: Whole-exome sequencing and bioinformatical analyses were implemented in a consanguineous Chinese family with DCM. A total of 380 clinically annotated control individuals and 166 more DCM index cases then underwent Sanger sequencing analysis for the identified genetic variation. The functional characteristics of the variant were delineated by utilizing a dual-luciferase assay system. RESULTS: A heterozygous variation in the MEF2A gene (encoding myocyte enhancer factor 2A, a transcription factor pivotal for embryonic cardiogenesis and postnatal cardiac adaptation), NM_001365204.1: c.718G>T; p. (Gly240*), was identified, and verified by Sanger sequencing to segregate with autosome-dominant DCM in the family with complete penetrance. The nonsense variation was neither detected in 760 control chromosomes nor found in 166 more DCM probands. Functional analyses revealed that the variant lost transactivation on the validated target genes MYH6 and FHL2, both causally linked to DCM. Furthermore, the variation nullified the synergistic activation between MEF2A and GATA4, another key transcription factor involved in DCM. CONCLUSIONS: The findings firstly indicate that MEF2A loss-of-function variation predisposes to DCM in humans, providing novel insight into the molecular mechanisms of DCM and suggesting potential implications for genetic testing and prognostic evaluation of DCM patients.

A New TBX5 Loss-of-Function Mutation Contributes to Congenital Heart Defect and Atrioventricular Block.

Congenital heart defect (CHD) represents the most common birth deformity, afflicting 1% of all births worldwide, and accounts for substantial morbidity and mortality. Increasing evidence highlights the pivotal roles of genetic etiologies in the pathogenesis of CHD, and pathogenic mutations in multiple genes, including TBX5 encoding a cardiac core transcription factor key to cardiovascular morphogenesis, have been involved in CHD. However, due to pronounced genetic heterogeneity of CHD, the genetic determinants underlying CHD in most cases remain obscure. In this investigation, by sequencing analysis of the coding exons and flanking introns of the TBX5 gene in 198 unrelated patients affected with CHD, a novel heterozygous mutation, NM_000192.3: c.692C>T; p. (Pro231Leu), was identified in an index patient with familial double outlet right ventricle (DORV), ventricular septal defect (VSD), and atrioventricular block (AVB). Genetic analysis of the proband's pedigree showed that the mutation co-segregated with the diseases. The missense mutation, which altered the amino acid conserved evolutionarily, was absent from 266 unrelated healthy subjects. Functional analyses with a dual-luciferase reporter assay system unveiled that the Pro231Leu-mutant TBX5 was associated with significantly reduced transcriptional activity on its target genes MYH6 and NPPA. Furthermore, the mutation disrupted the synergistic transactivation between TBX5 and NKX2-5 as well as GATA4, two other transcription factors causally linked to CHD. This study firstly links TBX5 loss-of-function mutation to familial DORV, VSD, and AVB, which provides novel insight into the mechanism underpinning CHD and AVB, suggesting potential implications for genetic evaluation and individualized treatment of patients affected by CHD and AVB.

[Qualitative Study on Survival Stressors of 15 Community-dwelling People with Mental Illness].

Objective To understand the survival stress of community-dwelling people with mental disorder. Methods Fifteen cases were selected by purposive sampling and received semi-structured individualized interviews.The data were analyzed by Colaizzi framework and themes were extracted. Results Four themes were extracted:physiological stress due to psychiatric symptoms and side effects of drugs;psychological stress due to the outcome of mental illness and to conflict of roles in daily life;social and environmental stress such as social discrimination,lack of job opportunities,and difficulty in obtaining social welfare resources;and interpersonal stress caused by discrimination and deteriorating family relations. Conclusions Community-dwelling people with mental illness have a higher level survival stress after returning to their families and society,with the stressors including symptoms of illness,social discrimination,and interpersonal relationship.Eliminating self-discrimination of the patients,improving social support and social welfare system,and increasing individualized community mental rehabilitation activities may reduce the survival stress of these patients and promote their rehabilitation.

First-in-man study of Heartech® percutaneous left ventricular partitioning device for treatment of heart failure postmyocardial infarction.

OBJECTIVE: This first-in-man (FIM) study aimed to determine the safety and efficacy of the Heartech® left ventricular partitioning device (LVPD) in patients with chronic heart failure (HF) postmyocardial infarction. METHODS: Sixteen patients were enrolled from three cardiac intervention centers in China. All patients underwent percutaneous ventricular restoration (PVR) procedures with implantation of the Heartech® LVPD. Safety and immediate success rates were recorded. Major adverse cardiovascular and cerebrovascular events (MACCEs) including all-cause mortality, myocardial infarction, stroke, emergent or selective surgery or interventional therapy, renal failure requiring hemodialysis, and major bleeding were recorded. Efficacy features included functional status, echocardiographic characteristics, life quality characteristics including peak oxygen consumption of cardiopulmonary exercise testing (CPET), European five-dimensional health scale (EQ-5D), 6-min walk test (6MWT) at baseline and during follow-up (NCT02938637). RESULTS: The device success rate was 93.75% (15 successes in 16 patients) with 100% safety. During follow-up of 36 ± 4.5 days, no MACCEs were found. The left ventricular end-systolic volume index decreased significantly (LVESVi, 72.47 ± 22.77 mL/m2 vs. 50.13 ± 13.36 mL/m2 , p < .001) as did left ventricular end diastolic volume index (LVEDVi, 106.27 ± 28.01 mL/m2 vs. 83.20 ± 16.87 mL/m2 , p = .001). Left ventricular ejection fraction (LVEF, 32.47 ± 6.98% vs. 40.41 ± 6.15, p < .001), 6MWT (383.13 ± 108.70 vs. 453.47 ± 88.24, p < 0.001) and EQ-5D (65.93 ± 11.25 vs. 78.67 ± 8.35, p < .001) improved significantly. CONCLUSIONS: Heartech® LVPD appeared to be safe and effective for treatment of HF postmyocardial infarction.

Inhibition of gasdermin D palmitoylation by disulfiram is crucial for the treatment of myocardial infarction.

To investigate the role of S-palmitoylation in pyroptosis following acute myocardial infarction (AMI). Myocardial ischemic injury is mainly related to the death of terminally differentiated cardiomyocytes. Pyroptosis is a new form of programmed cell death and recently is identified a potential mechanism of cardiomyocyte loss. However, the role of S-palmitoylation in pyroptosis following MI remains elusive. AMI was mimicked by permanent left anterior descending artery ligation. The palmitoylated proteins labeled by Click-iT palmitic acid were precipitated using streptavidin magnetic bead conjugate. The short-term palmitic acid dietary intake by modified western diet with palm oil for 7 days is compared with modified western diet with olive oil. Palmitoylation is increased in myocardial infarction and anoxic cardiomyocytes. Pyroptosis, but not apoptosis and necrosis, is more relevant with palmitoylation in the process of myocardial ischemia injury. The gasdermin D (GSDMD) Cys192 palmitoylation promotes its cytomembrane localization by ZDHHC14. GSDMD Cys192 palmitoylation aggravates in vitro cardiomyocyte pyroptosis. The short-term palmitic acid dietary intake or ML348 deteriorates myocardial pyroptosis, infarct size and cardiac function in AMI mice by GSDMD palmitoylation. Disulfiram antagonizes Cys192 palmitoylation of GSDMD-N-terminal and reduces myocardial pyroptosis and injury in AMI mice. We identifies ZHDDC14 induced palmitoylation as a crucial node for modulating GSDMD-N-terminal cytomembrane localization and establishes Disulfiram targeting GSDMD Cys192 palmitoylation as a potential clinical intervention for myocardial pyroptosis.

Identification, Expression, Characteristic Analysis, and Immune Function of Two Akirin Genes in Grass Carp (Ctenopharyngodon idella).

Intensive aquaculture of grass carp often leads to decreased immunity and increased disease prevalence, resulting in economic losses. Improving grass carp immunity is therefore a critical strategy for addressing these challenges. Akirin reportedly participates in myogenesis, growth, and immune responses. However, its role in grass carp remains unclear. Herein, we isolated akirins from the spleen of grass carp and analyzed their tissue-specific expression. Akirin expression was detected following treatment with poly (I:C), LPS, and Aeromonas hydrophila (A. hydrophila). The immunological function of the akirin protein was evaluated in head kidney leukocytes (HKLs). The results revealed that the coding sequence (CDS) of akirin1 is 570 bp, encoding 189 amino acids. There was one predicted nuclear localization signal (NLS) and two predicted α- helix domains. The CDS of akirin2 is 558 bp, encoding 185 amino acids. There were two predicted NLSs and two predicted α-helix domains. Tissue-specific expression analysis showed that akirins are widely detected in grass carp tissues. akirin1 was highly detected in the brain, kidneys, heart, spleen, and gonads, while akirin2 was highly detected in the brain, liver, gonads, kidneys, spleen, and heart. The mRNA levels of akirins were promoted after treatment with poly (I:C), LPS, and A. hydrophila. Recombinant akirin proteins were produced in Escherichia coli (E. coli). il-1ß, ifnγ, il-6, tnfα, il-4, iκbα, and nfκb were markedly increased in grass carp HKLs by treatment with the akirin protein. These results suggest that akirins play a role in the immunological regulation of grass carp.

Somatic GATA4 mutation contributes to tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most prevalent cyanotic congenital heart pathology and causes infant morbidity and mortality worldwide. GATA-binding protein 4 (GATA4) serves as a pivotal transcriptional factor for embryonic cardiogenesis and germline GATA4 mutations are causally linked to TOF. However, the effects of somatic GATA4 mutations on the pathogenesis of TOF remain to be ascertained. In the present study, sequencing assay of GATA4 was performed utilizing genomic DNA derived from resected heart tissue specimens as well as matched peripheral blood specimens of 62 patients with non-familial TOF who underwent surgical treatment for TOF. Sequencing of GATA4 was also performed using the heart tissue specimens as well as matched peripheral venous blood samples of 68 sporadic cases who underwent heart valve displacement because of rheumatic heart disorder and the peripheral venous whole blood samples of 216 healthy subjects. The function of the mutant was explored by dual-luciferase activity analysis. Consequently, a new GATA4 mutation, NM_002052.5:c.708T>G;p.(Tyr236*), was found in the heart tissue of one patient with TOF. No mutation was detected in the heart tissue of the 68 cases suffering from rheumatic heart disorder or in the venous blood samples of all 346 individuals. GATA4 mutant failed to transactivate its target gene, myosin heavy chain 6. Additionally, this mutation nullified the synergistic transactivation between GATA4 and T-box transcription factor 5 or NK2 homeobox 5, two genes causative for TOF. Somatic GATA4 mutation predisposes TOF, highlighting the significant contribution of somatic variations to the molecular pathogenesis underpinning TOF.

Introduction of an RS1 mutation causative variant consistent with identified XLRS patient using CRISPR/Cas9 technology in normal iPSC.

X-linked retinoschisis (XLRS) is a common retinal genetic disease that occurs in juvenile males and causes progressive visual impairment. This presents a schisis in the macula or peripheral retina of bilateral eyes, which has no effective treatment. Here, we introduced the RS1 (c.C304T, p.R102W) mutation into a normal induced pluripotent stem (iPS) cell line using CRISPR/Cas9 technology. This missense mutation was consistent with that observed in the XLRS patient-derived iPS cell line (CSUASOi001-A). Conclusively, establishing a directed gene mutation cell line (CSUi007-A) provides a useful cell resource to investigate XLRS pathogenesis.

Molecular dynamics-guided optimization of BGM0504 enhances dual-target agonism for combating diabetes and obesity.

The dual activation of glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) has emerged as a promising therapeutic strategy for managing type 2 diabetes and obesity. Tirzepatide, a dual agonist peptide, has exhibited superior clinical efficacy in glycemic and weight control compared to selective GLP-1R agonists. Nevertheless, the structural basis of Tirzepatide's extended half-life, attributed to an acylation side chain on the parent peptide, raises questions regarding its partial agonistic activity. Employing molecular dynamics simulations, we explored the dynamic processes of peptide-receptor interactions. We uncovered a crucial salt bridge between parent peptide and GLP-1R/GIPR at K20, a feature not discernible in cryo-electron microscopy structures. Building upon these insights, we developed an optimization strategy based on the parent peptide which involved repositioning the acylation side chain. The results of both in vitro and in vivo experiments demonstrated that the optimized peptide has twofold to threefold increase in agonistic activity compared to Tirzepatide while maintaining its extended half-life in plasma. This led to the design of BGM0504, which proved to be more effective than its predecessor, Tirzepatide, in both laboratory and animal studies.

Impact of chronic obstructive pulmonary disease on procedural outcomes and quality of life in patients with atrial fibrillation undergoing catheter ablation.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a risk factor for atrial fibrillation (AF). The aim of this study was to investigate the impact of COPD on outcomes of catheter ablation in patients with AF in terms of recurrence and quality of life (QoL). METHODS: In this prospective study, 550 consecutive patients with symptomatic, medication-refractory AF underwent first catheter ablation. Patients were classified into those with COPD (group 1, n = 54) and those without COPD (group 2, n = 496). Patients were followed up for atrial tachyarrhythmia (ATa) recurrence for at least 24 months. The Medical Outcomes Study SF-36 Health Survey was used to assess QoL at baseline and 24 months after ablation. RESULTS: After a single ablation, 24 patients in group 1 (44.4%) and 142 in group 2 (28.6%) had ATa recurrence during a mean follow-up of 31.4 ± 4.8 months (P = 0.016). The second ablation was performed in 19 patients (35.2%) from group 1 and in 109 patients (22.0%) from group 2 (P = 0.029). Multivariate logistic analysis showed that nonparoxysmal AF (P = 0.013, OR = 1.767, 95% CI: 1.129-2.765) as well as the presence of COPD (P = 0.029, OR = 1.951, 95% CI: 1.070-3.557) was the independent predictor for higher ATa recurrence. Moreover, patients in group 1 had significantly lower baseline scores on all SF-36 Health Survey subscales. At 24-month follow-up, both mental component summary and physical component summary scores improved markedly in group 1 and 2. CONCLUSIONS: Although the presence of COPD predicted higher recurrence after single-catheter ablation in AF patients, significant improvements in QoL were observed in the postablation COPD population.

Efficacy and safety of catheter ablation for long-standing persistent atrial fibrillation in women.

OBJECTIVES: It is uncertain whether gender affects the outcomes of catheter ablation (CA) for atrial fibrillation (AF). The objective of the study is to evaluate the efficacy and safety of CA for long-standing persistent AF in women. METHODS: Between January 2010 and May 2011, 220 consecutive patients (73 females, 33.2%), with long-standing persistent AF who underwent CA were prospectively recruited. Gender-related differences in clinical presentation, periprocedural complications, and outcomes were compared. RESULTS: Women were less likely to have lone AF than men (27.4% vs 47.6%; P = 0.004). The incidence of rheumatic heart disease was higher in women (19.2% in women vs 1.4% in men; P < 0.001). Women had a lower initial ablation success rate than men (35.6% vs 57.1%; P = 0.003). Hematomas occurred more often in women (6.8% in women vs 0.7% in men; P = 0.027). A Cox regression analysis demonstrated total duration of AF (per month, hazard ratio [HR] 1.003, confidence interval [CI] 1.001-1.006; P = 0.006) and gender (HR 1.663, CI 1.114-2.485; P = 0.013) as the independent predictors for recurrence after the first CA. CONCLUSIONS: Women and long AF duration were closely related to the recurrence of AF after the first ablation in patients with long-standing persistent AF. Women also had a higher risk of vascular complications.

ANK6, a mitochondrial ankyrin repeat protein, is required for male-female gamete recognition in Arabidopsis thaliana.

Double fertilization in angiosperms involves several successive steps, including guidance and reception of the pollen tube and male-female gamete recognition. Each step entails extensive communication and interaction between two different reproductive cell or tissue types. Extensive research has focused on the pollen tube, namely, its interaction with the stigma and reception by maternal cells. Little is known, however, about the mechanism by which the gametes recognize each other and interact to form a zygote. We report that an ankyrin repeat protein (ANK6) is essential for fertilization, specifically for gamete recognition. ANK6 (At5g61230) was highly expressed in the male and female gametophytes before and during but not after fertilization. Genetic analysis of a T-DNA insertional mutant suggested that loss of function of ANK6 results in embryonic lethality. Moreover, male-female gamete recognition was found to be impaired only when an ank6 male gamete reached an ank6 female gamete, thereby preventing formation of homozygous zygotes. ANK6 was localized to the mitochondria, where it interacted with SIG5, a transcription initiation factor previously found to be essential for fertility. These results show that ANK6 plays a central role in male-female gamete recognition, possibly by regulating mitochondrial gene expression.

Methods for RPE Sheet Construction Using iPS-CM in Conjunction with Natural Scaffold of Corneal Lenticule Derived from SMILE.

In vitro generation of a functional retinal pigment epithelium (RPE) monolayer sheet is useful and promising for RPE cell therapy. Here, we outline a method to construct engineered RPE sheets treated by induced pluripotent stem cell-conditioned medium (iPS-CM) in conjunction with femtosecond laser intrastromal lenticule (FLI-lenticule) scaffold to aid in enhanced RPE characteristics and cilium assembly. Such a strategy to construct RPE sheets is a promising avenue for developing RPE cell therapy, disease models, and drug screening tools.

Carbodiimide crosslinked decellularized lenticules as a drug carrier for sustained antibacterial eye treatments.

In this study, the drug-loading and antibacterial activity of carbodiimide/N-hydroxysuccinimide (EDC/NHS) crosslinked decellularized lenticules (CDLs) were evaluated. Small incision lenticule extraction derived lenticules were decellularized and modified with crosslinking concentrations of 0.00 (E/L00, non-crosslinked), 0.01 (E/L01), 0.05 (E/L05) and 0.25 mmol (E/L25) EDC per mg lenticules at 5:1 EDC/NHS ratios with non-decellularized non-crosslinked lenticules (NDLs) as controls. NDLs and EDC/NHS CDLs had similar water contents. The light transmittance percentages (400-800 nm) were 91.55 ± 1.16%, 88.68 ± 1.19%, 80.86 ± 1.94%, 85.12 ± 2.42% and 85.62 ± 2.84% for NDLs, E/L00, E/L01, E/L05 and E/L25, respectively (P< 0.01). The EDC/NHS CDLs (diameter: 6.36 ± 0.18 mm; central thickness: 117.31 ± 3.46 µm) were soaked in 3% (wt./vol.) levofloxacin (LEV) solution for 3 h. The drug release concentrations of LEV-impregnated EDC/NHS CDLs were determined by high-performance liquid chromatography. Zone inhibition (ZOI) againstStaphylococcus aureusof E/L01, E/L05 and E/L25 were superior to E/L00 CDLs (P< 0.01) and among the different crosslinked groups, E/L05 lenticules produced the largest ZOIs and their drug concentration release over 21 d was the highest. EDC/NHS crosslinking can improve the drug-loading effect and antibacterial activity of decellularized lenticules. LEV-impregnated EDC/NHS CDLs are promising drug delivery carriers.

Identification of BMP10 as a Novel Gene Contributing to Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM), characterized by left ventricular or biventricular enlargement with systolic dysfunction, is the most common type of cardiac muscle disease. It is a major cause of congestive heart failure and the most frequent indication for heart transplantation. Aggregating evidence has convincingly demonstrated that DCM has an underlying genetic basis, though the genetic defects responsible for DCM in a larger proportion of cases remain elusive, motivating the ongoing research for new DCM-causative genes. In the current investigation, a multigenerational family affected with autosomal-dominant DCM was recruited from the Chinese Han population. By whole-exome sequencing and Sanger sequencing analyses of the DNAs from the family members, a new BMP10 variation, NM_014482.3:c.166C > T;p.(Gln56*), was discovered and verified to be in co-segregation with the DCM phenotype in the entire family. The heterozygous BMP10 variant was not detected in 268 healthy volunteers enrolled as control subjects. The functional measurement via dual-luciferase reporter assay revealed that Gln56*-mutant BMP10 lost the ability to transactivate its target genes NKX2.5 and TBX20, two genes that had been causally linked to DCM. The findings strongly indicate BMP10 as a new gene contributing to DCM in humans and support BMP10 haploinsufficiency as an alternative pathogenic mechanism underpinning DCM, implying potential implications for the early genetic diagnosis and precision prophylaxis of DCM.

VEZF1 loss-of-function mutation underlying familial dilated cardiomyopathy.

Dilated cardiomyopathy (DCM), characteristic of left ventricular or biventricular dilation with systolic dysfunction, is the most common form of cardiomyopathy, and a leading cause of heart failure and sudden cardiac death. Aggregating evidence highlights the underlying genetic basis of DCM, and mutations in over 100 genes have been causally linked to DCM. Nevertheless, due to pronounced genetic heterogeneity, the genetic defects underpinning DCM in most cases remain obscure. Hence, this study was sought to identify novel genetic determinants of DCM. In this investigation, whole-exome sequencing and bioinformatics analyses were conducted in a family suffering from DCM, and a novel heterozygous mutation in the VEZF1 gene (coding for a zinc finger-containing transcription factor critical for cardiovascular development and structural remodeling), NM_007146.3: c.490A > T; p.(Lys164*), was identified. The nonsense mutation was validated by Sanger sequencing and segregated with autosome-dominant DCM in the family with complete penetrance. The mutation was neither detected in another cohort of 200 unrelated DCM patients nor observed in 400 unrelated healthy individuals nor retrieved in the Single Nucleotide Polymorphism database, the Human Gene Mutation Database and the Genome Aggregation Database. Biological analyses by utilizing a dual-luciferase reporter assay system revealed that the mutant VEZF1 protein failed to transactivate the promoters of MYH7 and ET1, two genes that have been associated with DCM. The findings indicate VEZF1 as a new gene responsible for DCM, which provides novel insight into the molecular pathogenesis of DCM, implying potential implications for personalized precisive medical management of the patients affected with DCM.

One-stop assembly of adherent 3D retinal organoids from hiPSCs based on 3D-printed derived PDMS microwell platform.

The three-dimensional (3D) retinal organoids (ROs) derived from human induced pluripotent stem cells (hiPSCs), mimicking the growth and development of the human retina, is a promising model for investigating inherited retinal diseasesin vitro. However, the efficient generation of homogenous ROs remains a challenge. Here we introduce a novel polydimethylsiloxane (PDMS) microwell platform containing 62 V-bottom micro-cavities for the ROs differentiation from hiPSCs. The uniform adherent 3D ROs could spontaneously form using neural retina (NR) induction. Our results showed that the complex of NR (expressing VSX2), ciliary margin (CM) (expressing RDH10), and retinal pigment epithelium (RPE) (expressing ZO-1, MITF, and RPE65) developed in the PDMS microwell after the differentiation. It is important to note that ROs in PDMS microwell platforms not only enable one-stop assembly but also maintain homogeneity and mature differentiation over a period of more than 25 weeks without the use of BMP4 and Matrigel. Retinal ganglion cells (expressing BRN3a), amacrine cells (expressing AP2a), horizontal cells (expressing PROX1 and AP2α), photoreceptor cells for cone (expressing S-opsin and L/M-opsin) and rod (expressing Rod opsin), bipolar cells (expressing VSX2 and PKCα), and Müller glial cells (expressing GS and Sox9) gradually emerged. Furthermore, we replaced fetal bovine serum with human platelet lysate and established a xeno-free culture workflow that facilitates clinical application. Thus, our PDMS microwell platform for one-stop assembly and long-term culture of ROs using a xeno-free workflow is favorable for retinal disease modeling, drug screening, and manufacturing ROs for clinical translation.

Extensive antiarrhythmic drugs after catheter ablation of persistent atrial fibrillation.

OBJECTIVE: Whether early rhythm suppression with extensive antiarrhythmic drugs (AADs) in persistent atrial fibrillation (AF) after catheter ablation decreases arrhythmia recurrence is unknown. We now report the 12-month follow-up data in this prospective and randomized study. METHODS AND RESULTS: 123 consecutive patients with persistent AF undergoing catheter ablation were randomly divided into an extensive AADs therapy group (group 1:62 patients using both class Ic and III AADs) or one AAD therapy group (group 2:61 patients using class Ic or III AADs alone) for the initial 2 months after ablation. Recurrence of atrial tachyarrhythmias (ATa) was valuated at both 2 months and 12 months following ablation. During the first 2 months after ablation, less ATa were found in group 1 compared with group 2 (17/62 versus 29/61, P = 0.021). However, there was no difference with regard to ATa at 12 months between the groups (21/62 versus 22/61, P = 0.799). ATa at 2 months and left atrial diameter (LAD) were the statistically significant predictors of ATa during 12-month follow-up. CONCLUSIONS: While use of extensive AADs within the initial 2 months after persistent AF ablation decreases early ATa, it does not prevent late ATa. Moreover, LAD as well as early ATa is a strong predictor of ATa at 12 months.