In Vivo Base Editing of Scn5a Rescues Type 3 Long QT Syndrome in Mice.

BACKGROUND: Pathogenic variants in SCN5A can result in long QT syndrome type 3, a life-threatening genetic disease. Adenine base editors can convert targeted A T base pairs to G C base pairs, offering a promising tool to correct pathogenic variants. METHODS: We generated a long QT syndrome type 3 mouse model by introducing the T1307M pathogenic variant into the Scn5a gene. The adenine base editor was split into 2 smaller parts and delivered into the heart by adeno-associated virus serotype 9 (AAV9-ABEmax) to correct the T1307M pathogenic variant. RESULTS: Both homozygous and heterozygous T1307M mice showed significant QT prolongation. Carbachol administration induced Torsades de Pointes or ventricular tachycardia for homozygous T1307M mice (20%) but not for heterozygous or wild-type mice. A single intraperitoneal injection of AAV9-ABEmax at postnatal day 14 resulted in up to 99.20% Scn5a transcripts corrected in T1307M mice. Scn5a mRNA correction rate >60% eliminated QT prolongation; Scn5a mRNA correction rate <60% alleviated QT prolongation. Partial Scn5a correction resulted in cardiomyocytes heterogeneity, which did not induce severe arrhythmias. We did not detect off-target DNA or RNA editing events in ABEmax-treated mouse hearts. CONCLUSIONS: These findings show that in vivo AAV9-ABEmax editing can correct the variant Scn5a allele, effectively ameliorating arrhythmia phenotypes. Our results offer a proof of concept for the treatment of hereditary arrhythmias.

Intradermally delivered mRNA-encapsulating extracellular vesicles for collagen-replacement therapy.

The success of messenger RNA therapeutics largely depends on the availability of delivery systems that enable the safe, effective and stable translation of genetic material into functional proteins. Here we show that extracellular vesicles (EVs) produced via cellular nanoporation from human dermal fibroblasts, and encapsulating mRNA encoding for extracellular-matrix α1 type-I collagen (COL1A1) induced the formation of collagen-protein grafts and reduced wrinkle formation in the collagen-depleted dermal tissue of mice with photoaged skin. We also show that the intradermal delivery of the mRNA-loaded EVs via a microneedle array led to the prolonged and more uniform synthesis and replacement of collagen in the dermis of the animals. The intradermal delivery of EV-based COL1A1 mRNA may make for an effective protein-replacement therapy for the treatment of photoaged skin.

Losartan protects human stem cell-derived cardiomyocytes from angiotensin II-induced alcoholic cardiotoxicity.

Alcoholic cardiomyopathy (ACM) is a myocardial injury caused by long-term heavy drinking. Existing evidence indicates that high levels of oxidative stress are the key to pathological cardiomyopathy caused by long-term exposure to high concentrations of alcohol, while angiotensin II (AngII) and its type 1 receptor (AT1R) play an important role in excessive drinking. Whether oxidative stress-induced damage in ACM is related to AngII and AT1R is unclear, and the effects of alcohol on the electrophysiology of myocardial cells have not been reported. Most existing studies have used animal models. This study established an in vitro model of ACM based on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The transcriptional profiling of alcohol treatment was performed by RNA-seq analysis. The role of oxidative stress, the expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX), and the role of AngII and AT1R in the overactivation of oxidative stress were studied using fluorescent labeling, Western blotting, and high-content quantitative analysis. Real-time cell analysis(RTCA) and microelectrode array (MEA) were used to continuously monitor myocardial beating, observe the effects of alcohol on myocardial electrophysiological activity, and clarify the protective effects of the AT1R blocker losartan on ACM. We found that AngII and AT1R contribute to the effects of alcohol on the myocardium through oxidative stress damage, the mechanism of which may be achieved by regulating NOX.

Ranolazine rescues the heart failure phenotype of PLN-deficient human pluripotent stem cell-derived cardiomyocytes.

Phospholamban (PLN) is a key regulator that controls the function of the sarcoplasmic reticulum (SR) and is required for the regulation of cardiac contractile function. Although PLN-deficient mice demonstrated improved cardiac function, PLN loss in humans can result in dilated cardiomyopathy (DCM) or heart failure (HF). The CRISPR-Cas9 technology was used to create a PLN knockout human induced pluripotent stem cell (hiPSC) line in this study. PLN deletion hiPSCs-CMs had enhanced contractility at day 30, but proceeded to a cardiac failure phenotype at day 60, with decreased contractility, mitochondrial damage, increased ROS production, cellular energy metabolism imbalance, and poor Ca2+ handling. Furthermore, adding ranolazine to PLN knockout hiPSCs-CMs at day 60 can partially restore Ca2+ handling disorders and cellular energy metabolism, alleviating the PLN knockout phenotype of HF, implying that the disorder of intracellular Ca2+ transport and the imbalance of cellular energy metabolism are the primary mechanisms for PLN deficiency pathogenesis.

Rapid, Label-Free Prediction of Antibiotic Resistance in Salmonella typhimurium by Surface-Enhanced Raman Spectroscopy.

The rapid identification of bacterial antibiotic susceptibility is pivotal to the rational administration of antibacterial drugs. In this study, cefotaxime (CTX)-derived resistance in Salmonella typhimurium (abbr. CTXr-S. typhimurium) during 3 months of exposure was rapidly recorded using a portable Raman spectrometer. The molecular changes that occurred in the drug-resistant strains were sensitively monitored in whole cells by label-free surface-enhanced Raman scattering (SERS). Various degrees of resistant strains could be accurately discriminated by applying multivariate statistical analyses to bacterial SERS profiles. Minimum inhibitory concentration (MIC) values showed a positive linear correlation with the relative Raman intensities of I990/I1348, and the R2 reached 0.9962. The SERS results were consistent with the data obtained by MIC assays, mutant prevention concentration (MPC) determinations, and Kirby-Bauer antibiotic susceptibility tests (K-B tests). This preliminary proof-of-concept study indicates the high potential of the SERS method to supplement the time-consuming conventional method and help alleviate the challenges of antibiotic resistance in clinical therapy.

Generation of a COL1A2 homozygous knockout stem cell line via CRISPR/Cas9 system.

The loss of function of the COL1A2 gene can result in osteogenesis imperfecta (OI) types I, II, III, and IV and Ehlers-Danlos syndrome (cardiac valvular and arthrochalasia type).To further investigate the significance of COL1A2 in osteogenesis imperfecta and cardiac valve disease, we created a homozygous COL1A2-/- human embryonic stem cell line (WAe009-A-72) using CRISPR/Cas9. In vivo, the WAe009-A-72 cell line retained typical colony form, a normal karyotype, and robustly expressed pluripotency markers while differentiating into all three germ layers. This cell line is a potential tool for investigating the role of the COL1A2 gene in associated disorders.

Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG.

[Figure: see text].

hERG-deficient human embryonic stem cell-derived cardiomyocytes for modelling QT prolongation.

BACKGROUND: Long-QT syndrome type 2 (LQT2) is a common malignant hereditary arrhythmia. Due to the lack of suitable animal and human models, the pathogenesis of LQT2 caused by human ether-a-go-go-related gene (hERG) deficiency is still unclear. In this study, we generated an hERG-deficient human cardiomyocyte (CM) model that simulates 'human homozygous hERG mutations' to explore the underlying impact of hERG dysfunction and the genotype-phenotype relationship of hERG deficiency. METHODS: The KCNH2 was knocked out in the human embryonic stem cell (hESC) H9 line using the CRISPR/Cas9 system. Using a chemically defined differentiation protocol, we obtained and verified hERG-deficient CMs. Subsequently, high-throughput microelectrode array (MEA) assays and drug interventions were performed to characterise the electrophysiological signatures of hERG-deficient cell lines. RESULTS: Our results showed that KCNH2 knockout did not affect the pluripotency or differentiation efficiency of H9 cells. Using high-throughput MEA assays, we found that the electric field potential duration and action potential duration of hERG-deficient CMs were significantly longer than those of normal CMs. The hERG-deficient lines also exhibited irregular rhythm and some early afterdepolarisations. Moreover, we used the hERG-deficient human CM model to evaluate the potency of agents (nifedipine and magnesium chloride) that may ameliorate the phenotype. CONCLUSIONS: We established an hERG-deficient human CM model that exhibited QT prolongation, irregular rhythm and sensitivity to other ion channel blockers. This model serves as an important tool that can aid in understanding the fundamental impact of hERG dysfunction, elucidate the genotype-phenotype relationship of hERG deficiency and facilitate drug development.

The establishment of a homozygous SNTA1 knockout human embryonic stem cell line (WAe009-A-50) using the CRISPR/Cas9 system.

SNTA1 encodes α1-syntrophin, a scaffold protein, which is a component of the dystrophin-associated protein complex. Additionally, α1-syntrophin interacts with SCN5A and nNOS-PMCA4b complex in cardiomyocytes. SNTA1 is a susceptibility locus for arrhythmia and cardiomyopathy. We generated a homozygous SNTA1 knockout human embryonic stem cell (H9SNTA1KO) using the CRISPR/Cas9 system. H9SNTA1KO maintained pluripotency and a normal karyotype and differentiated into three germ layers in vivo.

Ascorbic acid can promote the generation and expansion of neuroepithelial-like stem cells derived from hiPS/ES cells under chemically defined conditions through promoting collagen synthesis.

INTRODUCTION: Spinal cord injury (SCI) is a neurological, medically incurable disorder. Human pluripotent stem cells (hPSCs) have the potential to generate neural stem/progenitor cells (NS/PCs), which hold promise in the treatment of SCI by transplantation. In our study, we aimed to establish a chemically defined culture system using serum-free medium and ascorbic acid (AA) to generate and expand long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably. METHODS: We induced human embryonic stem cells (hESCs)/induced PSCs (iPSCs) to neurospheres using a newly established in vitro induction system. Moreover, lt-NES cells were derived from hESC/iPSC-neurospheres using two induction systems, i.e., conventional N2 medium with gelatin-coated plates (coated) and N2+AA medium without pre-coated plates (AA), and were characterized by reverse transcription polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons. A microelectrode array (MEA) recording system was used to evaluate the functionality of the neurons differentiated from lt-NES cells. Finally, the mechanism underlying the induction of lt-NES cells by AA was explored through RNA-seq and the use of inhibitors. RESULTS: HESCs/iPSCs were efficiently induced to neurospheres using a newly established induction system in vitro. lt-NES cells derived from hESC/iPSC-neurospheres using the two induction systems (coated vs. AA) both expressed the neural pluripotency-associated genes PAX6, NESTIN, SOX1, and SOX2. After long-term cultivation, we found that they both exhibited long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retained the ability to differentiate into general functional neurons that express ß-tubulin at high levels. We also demonstrated that AA promotes the generation and long-term expansion of lt-NES cells by promoting collagen synthesis via the MEK-ERK1/2 pathway. CONCLUSIONS: This new chemically defined culture system was stable and effective regarding the generation and culture of lt-NES cells induced from hESCs/iPSCs using serum-free medium combined with AA. The lt-NES cells induced under this culture system maintained their long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons.

Generation of a TBX5 homozygous knockout embryonic stem cell line (WAe009-A-45) by CRISPR/Cas9 genome editing.

Holt-Oram syndrome (HOS), which is caused by genetic changes in the TBX5 gene, affects the hands and heart. HOS patients have heart defects, including atrial septal defects (ASD), ventricular septal defects (VSD) and heart conduction disease. Here, we generated a homozygous TBX5 knockout human embryonic stem cell (hESC) line (TBX5-KO) using a CRISPR/Cas9 system. The TBX5-KO maintained stem cell like morphology, pluripotency markers, normal karyotype, and could differentiate into all three germ layers in vivo. This cell line can provide an in vitro platform for studying the pathogenic mechanisms and biological function of TBX5 in the heart development.

RAD-Deficient Human Cardiomyocytes Develop Hypertrophic Cardiomyopathy Phenotypes Due to Calcium Dysregulation.

Ras associated with diabetes (RAD) is a membrane protein that acts as a calcium channel regulator by interacting with cardiac L-type Ca2 + channels (LTCC). RAD defects can disrupt intracellular calcium dynamics and lead to cardiac hypertrophy. However, due to the lack of reliable human disease models, the pathological mechanism of RAD deficiency leading to cardiac hypertrophy is not well understood. In this study, we created a RRAD -/- H9 cell line using CRISPR/Cas9 technology. RAD disruption did not affect the ability and efficiency of cardiomyocytes differentiation. However, RAD deficient hESC-CMs recapitulate hypertrophic phenotype in vitro. Further studies have shown that elevated intracellular calcium level and abnormal calcium regulation are the core mechanisms by which RAD deficiency leads to cardiac hypertrophy. More importantly, management of calcium dysregulation has been found to be an effective way to prevent the development of cardiac hypertrophy in vitro.

LncRNA MEG3 influences the proliferation and apoptosis of psoriasis epidermal cells by targeting miR-21/caspase-8.

BACKGROUND: It was reported that microRNA-21(miR-21) was differentially expressed in the keratinocytes of psoriasis patients, and it may influence the apoptosis and proliferation of cells. The role of lncRNA maternally expressed gene3 (MEG3), a competing endogenous RNAs of miR-21, in the progression of psoriasis remains unclear. We aimed to unfold the influence of MEG3 and miR-21 on the proliferation and apoptosis of psoriasis epidermal cells. METHODS: 50µg/L TNF-α was used to treat HaCaTs and NHEKs cells for 24 h, and then different experiments were conducted. qRT-PCR were applied for measuring the mRNA level of MEG3, miR-2, and caspase-8, and the protein expression of caspase-8 was measured with western blotting. Flow cytometry was used for assessing apoptosis. Cell proliferation was detected using MTT and colony formation assays. Dual luciferase reporter assay was applied for confirming the binding site between MEG3 and miR-21, miR-21 and Caspase-8. RESULTS: A cell model for in vitro studying the role of MEG3 in psoriasis pathophysiology was established using HaCaT and HHEKs. MEG3 was significantly down-regulated in HaCaT, HHEKs, and psoriatic skin samples. MEG3 inhibits proliferation and promotes apoptosis of Activated-HaCaT (Act-HaCaT) and Activated-HHEKs (Act- HHEK) by regulating miR-21, and the binding site between MEG3 and miR-21 was identified. We also found that miR-21 could inhibit the level of caspase-8 and identified the binding site between caspase-8 and miR-21. Some down-stream proteins of caspase-8, Cleaved caspase-8, cytc, and apaf-1 were regulated by miR-21 and MEG3. CONCLUSION: MEG3/miR-21 axis may regulate the expression of caspase-8, and further influence the proliferation and apoptosis of psoriasis keratinocyte, Act-HaCaT and Act- HHEK. Therefore, our findings may provide a new thought for the study of pathogenesis and treatment of psoriasis.

MLP-deficient human pluripotent stem cell derived cardiomyocytes develop hypertrophic cardiomyopathy and heart failure phenotypes due to abnormal calcium handling.

Muscle LIM protein (MLP, CSRP3) is a key regulator of striated muscle function, and its mutations can lead to both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, due to lack of human models, mechanisms underlining the pathogenesis of MLP defects remain unclear. In this study, we generated a knockout MLP/CSRP3 human embryonic stem cell (hESC) H9 cell line using CRISPR/Cas9 mediated gene disruption. CSRP3 disruption had no impact on the cardiac differentiation of H9 cells and led to confirmed MLP deficiency in hESC-derived cardiomyocytes (ESC-CMs). MLP-deficient hESC-CMs were found to develop phenotypic features of HCM early after differentiation, such as enlarged cell size, multinucleation, and disorganized sarcomeric ultrastructure. Cellular phenotypes of MLP-deficient hESC-CMs subsequently progressed to mimic heart failure (HF) by 30 days post differentiation, including exhibiting mitochondrial damage, increased ROS generation, and impaired Ca2+ handling. Pharmaceutical treatment with beta agonist, such as isoproterenol, was found to accelerate the manifestation of HCM and HF, consistent with transgenic animal models of MLP deficiency. Furthermore, restoration of Ca2+ homeostasis by verapamil prevented the development of HCM and HF phenotypes, suggesting that elevated intracellular Ca2+ concentration is a central mechanism for pathogenesis of MLP deficiency. In summary, MLP-deficient hESC-CMs recapitulate the pathogenesis of HCM and its progression toward HF, providing an important human model for investigation of CSRP3/MLP-associated disease pathogenesis. More importantly, correction of the autonomous dysfunction of Ca2+ handling was found to be an effective method for treating the in vitro development of cardiomyopathy disease phenotype.

MicroRNA-210-3p Targets RGMA to Enhance the Angiogenic Functions of Endothelial Progenitor Cells Under Hypoxic Conditions.

Endothelial progenitor cells (EPCs) are multipotential stem cells considered to have immense clinical value for revascularization. However, the clinical application of EPCs has been hampered by their clinical potency in ischemic anoxic environments. This study aimed to explore the effect of microRNA-210 (miR-210) on EPCs under oxygen-glucose deprivation (OGD) conditions. We generated a model of EPCs cultured under OGD conditions to simulate ischemia and explore the expression of miR-210 in vitro. With longer exposure to hypoxia, we found that miR-210-3p expression was highly upregulated in OGD groups compared to that in controls from 4 to 24 h, but not miR-210-5p. We then transfected a miR-210-3p mimic and inhibitor into EPCs, and after 24 h, we exposed them to OGD conditions for 4 h to simulate ischemia. We detected miR-210 by real-time polymerase chain reaction (RT-PCR) and tested the proliferation, migration, and tube formation of normal EPCs and OGD-treated EPCs by CCK-8, transwell chamber, and Matrigel assays, respectively. The direct targets of miR-210-3p were predicted using miRWalk. Compared to that in normal EPCs, higher miR-210-3p expression was found in OGD-treated EPCs (p < 0.05). Moreover, upregulation of miR-210-3p was found to promote proliferation, migration, and tube formation in EPCs under normal and OGD conditions (p < 0.05), whereas down-regulation inhibited these abilities in OGD-treated EPCs (p < 0.05). Repulsive guidance molecule A (RGMA), a negative regulator of angiogenesis, was predicted to be a target of miR-210-3p. Accordingly, upregulation of miR-210-3p was found to inhibit its expression at the protein level in OGD-treated EPCs, whereas downregulation of miR-210-3p inhibited its expression (p < 0.05). A dual-luciferase reporter system confirmed that RGMA is a direct target of miR-210-3p. MicroRNA-210-3p overexpression enhances the angiogenic properties of OGD-treated EPCs by inhibiting RGMA.

[Characteristics and Heavy Metal Adsorption Performance of Sewage Sludge-derived Biochar from Co-pyrolysis with Transition Metals].

To enhance the heavy metal cation adsorption capacity of sewage sludge-derived biochar in an aqueous medium with a high concentration of Ca2+, modified biochars were obtained from co-pyrolysis of sewage sludge and transition metal oxides (with a sewage sludge:transition metal mass ratio of 10:1), such as Fe2O3, MnO2, and ZnO. The properties of the modified biochars were characterized, and the Cd2+ adsorption effect of the modified biochars was determined as well. The H/C atom ratios of the modified biochars were all lower than 0.31, indicating that the transition metal oxides catalyzed the decomposition and volatilization of organic matter in sewage sludge. The majority of the added Fe and Mn remained in the modified biochars, and existed as a simple substance and oxide, respectively; while significant loss of Zn occurred. The pores of the modified biochars were mainly mesopores with an average pore size of approximately 3.8 nm, and the specific surface area of the modified biochars was larger than 50 m2·g-1. When the initial Cd2+ concentration was increased from 0 mg·L-1 to approximately 200 mg·L-1, the Cd2+ adsorption capacity of the Fe-modified biochar declined from 43.17 mg·g-1 to 27.88 mg·g-1, which was still higher than that of the unmodified biochar by at least 10 mg·g-1. In aqueous media with a high concentration of Ca2+, the Fe-modified biochar showed better Cd2+ adsorption performance; thus, compared to MnO2 and ZnO, Fe2O3 was the best choice to enhance the heavy metal adsorption performance of the sewage sludge-derived biochar.

Doxorubicin-induced cardiotoxicity is maturation dependent due to the shift from topoisomerase IIα to IIß in human stem cell derived cardiomyocytes.

Doxorubicin (DOX) is widely used to treat various cancers affecting adults and children; however, its clinical application is limited by its cardiotoxicity. Previous studies have shown that children are more susceptible to the cardiotoxic effects of DOX than adults, which may be related to different maturity levels of cardiomyocyte, but the underlying mechanisms are not fully understood. Moreover, researchers investigating DOX-induced cardiotoxicity caused by human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have shown that dexrazoxane, the recognized cardioprotective drug for treating DOX-induced cardiotoxicity, does not alleviate the toxicity of DOX on hiPSC-CMs cultured for 30 days. We have suggested that this may be ascribed to the immaturity of the 30 days hiPSC-CMs. In this study, we investigated the mechanisms of DOX induced cardiotoxicity in cardiomyocytes of different maturity. We selected 30-day-old and 60-day-old hiPSC-CMs (day 30 and day 60 groups), which we term 'immature' and 'relatively mature' hiPSC-CMs, respectively. The day 30 CMs were found to be more susceptible to DOX than the day 60 CMs. DOX leads to more ROS (reactive oxygen species) production in the day 60 CMs than in the relatively immature group due to increased mitochondria number. Moreover, the day 60 CMs mainly expressed topoisomerase IIß presented less severe DNA damage, whereas the day 30 CMs dominantly expressed topoisomerase IIα exhibited much more severe DNA damage. These results suggest that immature cardiomyocytes are more sensitive to DOX as a result of a higher concentration of topoisomerase IIα, which leads to more DNA damage.

Chlorogenic acid: A potent molecule that protects cardiomyocytes from TNF-α-induced injury via inhibiting NF-κB and JNK signals.

The traditional Chinese herb Lonicerae Japonicae Flos has shown significant clinical benefits in the treatment of heart failure, but the mechanism remains unclear. As the main active ingredient found in the plasma after oral administration of Lonicerae Japonicae Flos, chlorogenic acid (CGA) has been reported to possess anti-inflammatory, anti-oxidant and anti-apoptosis function. We firstly confirmed the cardioprotective effects of CGA in transverse aortic constriction (TAC)-induced heart failure mouse model, through mitigating the TNF-α-induced toxicity. We further used TNF-α-induced cardiac injury in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to elucidate the underlying mechanisms. CGA pre-treatment could reverse TNF-α-induced cellular injuries, including improved cell viability, increased mitochondrial membrane potential and inhibited cardiomyocytes apoptosis. We then examined the NF-κB/p65 and major mitogen-activated protein kinases (MAPKs) signalling pathways involved in TNF-α-induced apoptosis of hiPSC-CMs. Importantly, CGA can directly inhibit NF-κB signal by suppressing the phosphorylation of NF-κB/p65. As for the MAPKs, CGA suppressed the activity of only c-Jun N-terminal kinase (JNK), but enhanced extracellular signal-regulated kinase1/2 (ERK1/2) and had no effect on p38. In summary, our study revealed that CGA has profound cardioprotective effects through inhibiting the activation of NF-κB and JNK pathway, providing a novel therapeutic alternative for prevention and treatment of heart failure.

Uric acid: a potent molecular contributor to pluripotent stem cell cardiac differentiation via mesoderm specification.

Congenital heart disease (CHD) is the most common cause of congenital anomaly and a leading cause of morbidity and mortality worldwide. Generation of cardiomyoctyes derived from pluripotent stem cells (PSCs) has opened new avenues for investigation of human cardiac development. Here we report that uric acid (UA), a physiologically abundant compound during embryonic development, can consistently and robustly enhance cardiac differentiation of human PSCs including hESCs and hiPSCs, in replacement of ascorbic acid (AA). We optimized treatment conditions and demonstrate that differentiation day 0-2, a period for specification of mesoderm cells, was a critical time for UA effects. This was further confirmed by UA-induced upregulation of mesodermal markers. Furthermore, we show that the developing mesoderm may be by directly promoted by SNAI pathway-mediated epithelial-mesenchymal transition (EMT) at 0-24 h and a lengthened G0/G1 phase by increasing the ubiquitination degradation in 24-48 h. These findings demonstrate that UA plays a critical role in mesoderm differentiation, and its level might be a useful indicator for CHD in early fetal ultrasound screening.