A new Prdm1-Cre line is suitable for studying the second heart field development.

The second heart field (SHF) plays a pivotal role in heart development, particularly in outflow tract (OFT) morphogenesis and septation, as well as in the expansion of the right ventricle (RV). Two mouse Cre lines, the Mef2c-AHF-Cre (Mef2c-Cre) and Isl1-Cre, have been widely used to study the SHF development. However, Cre activity is triggered not only in the SHF but also in the RV in the Mef2c-Cre mice, and in the Isl1-Cre mice, Cre activation is not SHF-specific. Therefore, a more suitable SHF-Cre line is desirable for better understanding SHF development. Here, we generated and characterized the Prdm1-Cre knock-in mice. In comparison with Mef2c-Cre mice, the Cre activity is similar in the pharyngeal and splanchnic mesoderm, and in the OFT of the Prdm1-Cre mice. Nonetheless, it was noticed that Cre expression is largely reduced in the RV of Prdm1-Cre mice compared to the Mef2c-Cre mice. Furthermore, we deleted Hand2, Nkx2-5, Pdk1 and Tbx20 using both Mef2c-Cre and Prdm1-Cre mice to study OFT morphogenesis and septation, making a comparison between these two Cre lines. New insights were obtained in understanding SHF development including differentiation into cardiomyocytes in the OFT using Prdm1-Cre mice. In conclusion, we found that Prdm1-Cre mouse line is a more appropriate tool to monitor SHF development, while the Mef2c-Cre mice are excellent in studying the role and function of the SHF in OFT morphogenesis and septation.

Single-Cell RNA-Seq Analysis of Hearts in Patients with Fetal Tetralogy of Fallot.

INTRODUCTION: To explore the cytological characteristics of tetralogy of Fallot (TOF), we collected samples and investigated the differences in the cytological classification between normal fetal hearts and fetal hearts with congenital defects. We then performed single-cell sequencing analysis to search for possible differential genes of disease markers. METHODS: Here, the right ventricles of a heart sample with TOF and a healthy human fetal heart sample were analyzed through single-cell sequencing. Data quality control filtering, comparison, quantification, and identification of recovered cells on the raw data were performed using Cell Ranger, thereby ultimately obtaining gene expression matrices for each cell. Subsequently, Seurat was used for cell filtration, standardization, cell subgroup classification, differential expression gene analysis of each subgroup, and marker gene screening. RESULTS: Bioinformatic analysis identified 9,979 and 15,224 cells from the healthy and diseased samples, respectively, with an average read depth of 25,000/cell. The cardiomyocyte cell populations, derived from the abnormal samples identified through the first-level graph-based analysis, were separated into six distinct cell clusters. CONCLUSION: Our study provides some information on TOF in a fetus, which can offer a new reference for the early detection and treatment of TOF by comparing defective heart cells with normal heart cells.

Comorbidities in congenital heart disease: different patterns in childhood and adulthood.

BACKGROUND: Existing studies were no exploration of the association between congenital heart disease (CHD) in children and comorbidities. This study was to assess the prevalence and number of comorbidities in CHD among children and adults, and to compare the comorbidity patterns by children and adults using association rule analysis. METHODS: Patients identified by the International Classification of Diseases, Ninth Revision (ICD-9) code in the Medical Information Mart for Intensive Care III (MIMIC-III) 2001-2012 and MIMIC-IV 2008-2018 were included in this cross-sectional study. Association rule analysis was used to explore associations between CHD and comorbidities in children and adults using values of support (%), confidence (%), and lift. RESULTS: Among 60,400 eligible patients, 1.54% of adults had CHD and 0.83% of adults had CHD with at least one comorbidity, 13.79% had CHD and 12.37% had CHD with at least one comorbidity in children. The most common comorbidities were circulatory system diseases (53.78%), endocrine diseases (35.76%), and respiratory system diseases (23.46%) in adults with CHD, and the most common comorbidities were perinatal diseases (87.50%) in children with CHD. The comorbidity rate was 90.19% and 56.68% in children and adults, respectively. In children, perinatal diseases, circulatory system diseases, and endocrine diseases had the highest prevalence. The incidence of circulatory system diseases, perinatal diseases and endocrine diseases in CHD adults was confidence = 31.56%, 36.11%, and 23.23%, respectively. Perinatal diseases were common comorbidities among all CHD severity groups in children and adults. CONCLUSION: The prevalence of comorbidities in children with CHD was higher than that in adults with CHD. The most common comorbidities were perinatal diseases and endocrine diseases among children and adults with CHD, respectively. Our study provided insights into comorbidity patterns in children and adults with CHD.

Transcriptomic analysis reveals the role of a peptide derived from CRYAB on the CoCl2-induced hypoxic HL-1 cardiomyocytes.

Acute myocardial infarction (AMI) is a life-threatening disease that often results in heart failure. CRYAB, a small heat shock protein, has been shown to have cardioprotective effects against oxidative stress-induced apoptosis in AMI. Previously, we purified a peptide derived from CRYAB (LEDQFFGEH), which we named PDFC. In this study, we determined the function of PDFC on HL-1 cardiomyocytes and explored the mechanism underlying its function. A hypoxic myocardiocyte cell line was generated by stimulation of HL-1 mouse cardiac muscle cells with different concentrations of CoCl2. Then, the hypoxic HL-1 cells were treated with the synthetic PDFC peptide, and cell proliferation, migration, and apoptosis were assessed to examine the effects of PDFC on HL-1 and hypoxic HL-1 cells. To examine the mechanism underlying the effects of PDFC on hypoxic cells, PDFC-treated hypoxic HL-1 cells were submitted for deep RNA sequencing. Finally, several differentially expressed genes in different pathways were selected for confirmation by RT-qPCR. Hypoxic myocardiocytes were generated by stimulating HL-1 cells with 800 µM CoCl2 for 24 h, which significantly upregulated HIF-1α. PDFC at 200 µg/ml showed the most positive effects on cell viability. Although hypoxic HL-1 cells and PDFC-treated hypoxic HL-1 cells both showed lower viability and migration and higher levels of apoptosis than untreated HL-1 cells, compared to hypoxic HL-1 cells, PDFC-treated hypoxic HL-1 cells showed higher viability and migration and lower apoptosis. The deep sequencing showed that 812 genes were upregulated and 1946 genes were downregulated. Among these differentially expressed genes, 699 of the upregulated genes and 1488 of the downregulated genes were protein-coding genes. Gene ontology and pathway enrichment analysis showed that the downregulated genes were dominant and that the PI3K-Akt pathway was located in the center of the network. A protein-protein interaction network was constructed, and 892 nodes were determined. In PDFC-treated hypoxic HL-1 cells, Fn1, Pik3r5, and Creb5 were downregulated, while Insr, Bcl2, Mapk14, and Pten were upregulated when compared to the levels in hypoxic HL-1 cells. In conclusion, this study reveals the significant bioactive effect of the CRYAB-derived peptide, PDFC on cardiomyocytes and the underlying mechanism.

[Influence of twin pregnancy by assisted reproductive technology on neonatal outcomes].

OBJECTIVE: To study the influence of twin pregnancy by assisted reproductive technology (ART) versus twin pregnancy by spontaneous conception (SC) on neonatal outcomes. METHODS: A retrospective analysis was performed for the clinical data of 3 356 live twins with a gestational age of ≥24 weeks who were born in Nanjing Maternal and Child Health Hospital from 2017 to 2019, with 2 006 twins (1 003 pairs) in the ART group and 1 350 (675 pairs) in the SC group. The two groups were compared in terms of the mother's general information and pregnancy comorbidities and the general information, diseases, and outcomes of neonates. RESULTS: Compared with the SC group, the ART group had a significantly higher maternal age (P < 0.05) and significantly higher rates of primiparity, cesarean section, and cervical cerclage (P < 0.05). Compared with the SC group, the ART group had significantly higher incidence rates of maternal pregnancy comorbidities including hypertension, gestational diabetes, and postpartum hemorrhage (P < 0.05). Compared with the SC group, the ART group had a significantly lower mean gestational age of neonates (P < 0.05) and a significantly higher proportion of very-low-birth-weight infants (6.8% vs 5.8%, P < 0.05), while ART did not increase the risks of preterm birth and low Apgar score. There were no significant differences between the two groups in the mortality rate of neonates and the incidence rates of neonatal diseases including respiratory distress syndrome, stage II/III necrotizing enterocolitis, bronchopulmonary dysplasia, and grade III-IV intracranial hemorrhage (P > 0.05). CONCLUSIONS: Compared with twin pregnancy by SC, twin pregnancy by ART does not increase the neonatal mortality rate and risk of adverse outcomes.

[A clinical analysis of sepsis in very low birth weight infants].

OBJECTIVE: To study the incidence and clinical features of sepsis in very low birth weight (VLBW) infants. METHODS: The clinical data were collected from VLBW infants, with a birth weight of < 1 500 g, who were admitted to the Department of Neonatology, Maternity Hospital Affiliated to Nanjing Medical University, from January 2019 to June 2020. The incidence of sepsis, distribution of pathogenic bacteria, and risk factors for sepsis were analyzed. RESULTS: A total of 369 infants were enrolled, and 138 infants had sepsis, among whom 84 had early-onset sepsis (EOS) and 54 had late-onset sepsis (LOS). Enterococcus faecalis (24%) and Streptococcus (21%) were the main pathogenic bacteria in infants with EOS, and Staphylococcus (41%) and Enterobacter (29%) were the main pathogenic bacteria in infants with LOS. The incidence of EOS and LOS decreased with the increase of gestational age and birth weight (P < 0.05). The multivariate logistic regression analysis showed that a high birth weight was a protective factor against EOS (OR=0.996, 95%CI:0.993-0.998, P < 0.05), while vaginal delivery (OR=2.781, 95%CI:1.190-6.500, P < 0.05) was a risk factor for EOS, and long duration of parenteral nutrition was a risk factor for LOS (OR=1.129, 95%CI:1.067-1.194, P < 0.05). CONCLUSIONS: Enterococcus faecalis is the most common pathogenic bacteria for EOS, and Staphylococcus is the most common pathogenic bacterium for LOS in VLBW infants. A high birth weight may reduce the risk of EOS in VLBW infants, while vaginal delivery may increase the risk of EOS. Prolonged parenteral nutrition may increase the risk of LOS.

Differential contribution of the two waves of cardiac progenitors and their derivatives to aorta and pulmonary artery.

During mouse development, part of the cells derived from the second heart field (SHF) progenitors contributes to the elongation and enlargement of the outflow tract (OFT) that subsequently septates into the trunks of aorta (Ao) and pulmonary artery (PA). Thus, the cardiac progenitor-originated cells are distributed to both Ao and PA. Here, we investigated that how these cells are assigned to the two great arteries during OFT septation through lineage tracing technology. By use of the inducible Mef2c-AHF-CreERT2; Rosa26-mTmG reporter system, two waves of SHF progenitors and their derivatives were identified, and they made differential contribution to the Ao and PA, respectively. While the early wave of cells (at E7.5) was preferentially destined to the Ao, the second wave of cells (from E8.5 till E11.5) made its favorite path to the PA. In addition, we unveiled PDK1 as a critical regulator of the second wave of cells as deletion of Pdk1 resulted in poorly developed PA leading to pulmonary stenosis. Thus, this study provides insights into the understanding of the pre-determined cell fate of the cardiac progenitor-derived cells with preferential contribution to the Ao and PA, as well as of the pathogenesis of pulmonary stenosis.

Profiles analysis reveals circular RNAs involving zebrafish physiological development.

Recent studies have found that known functions of circular RNAs (circRNAs) include sequestration of microRNAs (miRNAs) or proteins, modulation of transcription and interference with splicing, and even translation to produce polypeptides. The zebrafish model is also demonstrably similar to humans in many studies. To explore the changes in circRNAs during embryonic development and to further research the mechanism of action of circRNAs in development-related diseases, Zebrafish embryos at the blastula period, gastrula period, segmentation period, throat stage, and incubation period were collected. Illumina deep-sequencing technology and CircRNA Identifier (CIRI) algorithm were used to detect circRNAs. In total, we identified 1,028 circRNAs (junction reads ≥5 and p < 0.05). Considering that the function of circRNAs is related to host genes, a bioinformatics analysis revealed these differentially expressed host genes are involved in NOTCH signaling pathways, cardiovascular system development, retinal ganglion cell axon guidance, and so on. Moreover, circRNAs can participate in biological regulation through the function of miRNA sponges. TargetScan and miRanda were used to predict 73 miRNAs binding to circRNAs such as miR-19b, miR-124, and so on. Some miRNAs play important roles in embryogenesis. The peak expression of circRNAs is distributed at different time points, suggesting that it may be involved in embryogenesis at different stages. Our study provides a foundation for understanding the dynamic regulation of circRNA transcriptomes during embryogenesis and identifies novel key circRNAs that might control embryonic development in a zebrafish model.

Peptidomic Analysis of Maternal Serum to Identify Biomarker Candidates for Prenatal Diagnosis of Tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease. To identify endogenous peptides possibly involved in the progression of TOF, we performed comparative peptidomic profiling of maternal serum between normal fetuses and fetuses suffering from TOF. A total of 278 differentially expressed peptides, including 94 over-expressed peptides and 184 under-expressed peptides, originating from 227 protein precursors were identified by liquid chromatography/mass spectrometry (LC/MS) in maternal serum of fetuses with TOF compared to normal controls. Further, ingenuity pathway analysis (IPA) was used to identify putative roles for these peptides in cardiovascular development. Two peptides were derived from functional domains of proteins involved in heart development and associated with TOF; these may represent candidate bioactive peptides involved in TOF. These peptides may be related to the pathologic changes in the heart associated with TOF, and may be useful as novel biomarkers for prenatal diagnosis of TOF. J. Cell. Biochem. 119: 468-477, 2018. © 2017 Wiley Periodicals, Inc.

Akt1 signaling coordinates BMP signaling and ß-catenin activity to regulate second heart field progenitor development.

Second heart field (SHF) progenitors exhibit continued proliferation and delayed differentiation, which are modulated by FGF4/8/10, BMP and canonical Wnt/ß-catenin signaling. PTEN-Akt signaling regulates the stem cell/progenitor cell homeostasis in several systems, such as hematopoietic stem cells, intestinal stem cells and neural progenitor cells. To address whether PTEN-Akt signaling is involved in regulating cardiac progenitors, we deleted Pten in SHF progenitors. Deletion of Pten caused SHF expansion and increased the size of the SHF derivatives, the right ventricle and the outflow tract. Cell proliferation of cardiac progenitors was enhanced, whereas cardiac differentiation was unaffected by Pten deletion. Removal of Akt1 rescued the phenotype and early lethality of Pten deletion mice, suggesting that Akt1 was the key downstream target that was negatively regulated by PTEN in cardiac progenitors. Furthermore, we found that inhibition of FOXO by Akt1 suppressed the expression of the gene encoding the BMP ligand (BMP7), leading to dampened BMP signaling in the hearts of Pten deletion mice. Cardiac activation of Akt also increased the Ser552 phosphorylation of ß-catenin, thus enhancing its activity. Reducing ß-catenin levels could partially rescue heart defects of Pten deletion mice. We conclude that Akt signaling regulates the cell proliferation of SHF progenitors through coordination of BMP signaling and ß-catenin activity.

Circulating LncRNAs as Novel, Non-Invasive Biomarkers for Prenatal Detection of Fetal Congenital Heart Defects.

OBJECTIVES: To explore the clinical value of circulating long non-coding RNAs (lncRNAs) as biomarkers to predict fetal congenital heart defects (CHD) in pregnant women. METHODS: Differential expression of lncRNAs isolated from the plasma of pregnant women with typical fetal CHD or healthy controls was analyzed by microarray. Gene ontology (GO), pathway and network analysis were performed to study the function of the lncRNAs. Differentially expressed lncRNAs were validated in plasma samples from 62 pregnant women with typical CHD and 62 matched controls by RT-PCR. The sensitivity and specificity of each lncRNA in the diagnosis of fetal CHD was determined by ROC curve analysis. RESULTS: Microarray analysis identified 3694 up-regulated and 3919 down-regulated (fold change ≥2.0) lncRNAs. The top ten significantly differentially expressed, CHD-associated lncRNAs were validated by RT-PCR. Five significantly up-regulated or down-regulated lncRNAs were identified: ENST00000436681, ENST00000422826, AA584040, AA709223 and BX478947 with the AUC of ROC curves calculated as 0.892, 0.817, 0.755, 0.882 and 0.886, respectively. CONCLUSIONS: Specific lncRNAs aberrantly expressed in the plasma of pregnant women with typical fetal CHD may play a key role in the development of CHD and may be used as novel biomarkers for prenatal diagnosis of fetal CHD.

Cardiac-specific PID1 overexpression enhances pressure overload-induced cardiac hypertrophy in mice.

BACKGROUND/AIMS: PID1 was originally described as an insulin sensitivity relevance protein, which is also highly expressed in heart tissue. However, its function in the heart is still to be elucidated. Thus this study aimed to investigate the role of PID1 in the heart in response to hypertrophic stimuli. METHODS: Samples of human failing hearts from the left ventricles of dilated cardiomyopathy (DCM) patients undergoing heart transplants were collected. Transgenic mice with cardiomyocyte-specific overexpression of PID1 were generated, and cardiac hypertrophy was induced by transverse aortic constriction (TAC). The extent of cardiac hypertrophy was evaluated by echocardiography as well as pathological and molecular analyses of heart samples. RESULTS: A significant increase in PID1 expression was observed in failing human hearts and TAC-treated wild-type mouse hearts. When compared with TAC-treated wild-type mouse hearts, PID1-TG mouse showed a significant exacerbation of cardiac hypertrophy, fibrosis, and dysfunction. Further analysis of the signaling pathway in vivo suggested that these adverse effects of PID1 were associated with the inhibition of AKT, and activation of MAPK pathway. CONCLUSION: Under pathological conditions, over-expression of PID1 promotes cardiac hypertrophy by regulating the Akt and MAPK pathway.

Overexpression of miR-19b impairs cardiac development in zebrafish by targeting ctnnb1.

BACKGROUND: MicroRNAs are broadly accepted as crucial regulators of cardiovascular development, and dysregulation of their expression has been linked to cardiac disease. MicroRNA cluster miR-17-92 has been implicated in cardiac development and function, yet its defined mechanisms of action in this context are uncertain. Here, we focused on miR-19b, a key component of the miR-17-92 cluster proven to induce cardiomyocyte proliferation in vitro. We aimed to identify the biological significance of miR-19b in cardiac development and its underlying molecular mechanism of action in vivo. METHODS: We micro-injected zebrafish embryos with different concentrations (0, 2, 4 and 8 µm) of miR-19b mimics or a negative control, and assessed the embryo malformation rate, mortality rate, hatching rate and heart abnormalities at 72 hours post-fertilization (72 hpf). RESULTS: We found that overexpression of miR-19b impacted left-right symmetry and cardiac development of zebrafish embryos, characterized by pericardial edema, slower heart rate and cardiac looping defects in a dose-dependent manner. Moreover, several important signaling molecules in the Wnt signaling pathway were abnormally expressed, suggesting that overexpression of miR-19b induces the inhibition of the Wnt signaling pathway by directly targeting ctnnb1. Interestingly, the deformed cardiac phenotype was partially rescued by treatment with the GSK3ß inhibitor lithium chloride. CONCLUSION: Our findings suggest that miR-19b regulates laterality development and heart looping in zebrafish embryos by targeting ctnnb1.

Genetic and pharmacological inhibition of Rheb1-mTORC1 signaling exerts cardioprotection against adverse cardiac remodeling in mice.

A previous study indicated that Rheb1 is required for mammalian target of TOR complex 1 (mTORC1) signaling in the brain. However, the function of Rheb1 in the heart is still elusive. In the present study, we deleted Rheb1 specifically in cardiomyocytes and found that reduced Rheb1 levels conferred cardioprotection against pathologic remodeling in myocardial infarction (MI) and pressure overload (transverse aortic constriction) mouse models. Cardiomyocyte apoptosis was reduced and mTORC1 activity was suppressed in cardiomyocyte Rheb1-deletion mice, suggesting that Rheb1 regulates mTORC1 activation in myocardium. Furthermore, we demonstrated that astragaloside IV (As-IV) could inhibit mTORC1, and As-IV treatment displayed similar protection against MI and transverse aortic constriction as Rheb1 genetic inhibition. This study indicates that Rheb1 is essential for mTORC1 activation in cardiomyocytes and suggests that targeting Rheb1-mTORC1 signaling, such as by As-IV treatment, may be an effective therapeutic method for treating patients with adverse cardiac remodeling after MI and hypertrophy.

Toxic effects of polychlorinated biphenyls on cardiac development in zebrafish.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that may pose significant health-risks to various organisms including humans. Although the mixed PCB Aroclor 1254 is widespread in the environment, its potential toxic effect on heart development and the mechanism underlying its developmental toxicity have not been previously studied. Here, we used the zebrafish as a toxicogenomic model to examine the effects of Aroclor 1254 on heart development. We found that PCB exposure during zebrafish development induced heart abnormalities including pericardial edema and cardiac looping defects. Further malformations of the zebrafish embryo were observed and death of the larvae occurred in a time- and dose-dependent manner. Our mechanistic studies revealed that abnormalities in the arylhydrocarbon receptor, Wnt and retinoic acid signaling pathways may underlie the effects of PCBs on zebrafish heart development. Interestingly, co-administration of Aroclor 1254 and diethylaminobenzaldehyde, an inhibitor of retinaldehyde dehydrogenase, partially rescued the toxic effects of PCBs on zebrafish heart development. In conclusion, PCBs can induce developmental defects in the zebrafish heart, which may be mediated by abnormal RA signaling.

Differential expression profile of long non-coding RNAs during differentiation of cardiomyocytes.

Many long non-coding RNAs (lncRNAs) are species specific and seem to be less conserved than protein-coding genes. Some of them are involved in the development of the lateral mesoderm in the heart and in the differentiation of cardiomyocytes. The purpose of the study was to investigate the expression profiles of lncRNAs during the differentiation of P19 cells into cardiomyocytes, with a view to studying the biological function of lncRNAs and their involvement in the mechanism of heart development. First, we observed the morphology of P19 cells during differentiation using an inverted microscope. Then, cardiac troponin T (cTnT) expression was detected to validate that the cells had successfully differentiated into cardiac myocytes by real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) and western blotting. Lastly, the expression profile of lncRNA genes was obtained using an lncRNA microarray and real-time RT-PCR analyses. The microarray results showed that 40 lncRNAs were differentially expressed, of which 28 were upregulated and 12 were downregulated in differentiated cardiomyocytes. The differentially expressed lncRNAs were further validated. Our results illustrated a critical role of lncRNAs during the differentiation of P19 cells into cardiac myocytes, which will provide the foundation for further study of the biological functions of lncRNAs and the mechanism of heart development.

Incidence and Risk Factors for Retinopathy of Prematurity in a Tertiary Hospital in China.

Purpose: To investigate the incidence and risk factors for the retinopathy of prematurity (ROP) in the neonatal intensive care unit (NICU) of Obstetrics and Gynecology Hospital of Affiliated to Nanjing Medical University, China. Methods: This retrospective case-control study included 611 preterm infants with birth weight (BW)<1500 grams admitted to the Department of Neonatology, Obstetrics and Gynecology Hospital of Affiliated to Nanjing Medical University between January 2019 and December 2022. The incidence and risk factors for any stage and severe ROP were analyzed. Results: Within 611 infants, 245(40.1%) developed ROP; 160(26.2%) infants were stage 1, 54(8.8%) were stage 2, and 31(5.1%) were stage 3, no stage 4 and 5. Among them, 22(3.6%) infants needed treatment. Multivariate analysis showed a higher gestational age (GA) was protective, whereas twin birth and moderate-to-severe BPD increased the hazard of any stage ROP; higher BW and male gender were significant risk factors for severe ROP. Conclusion: Compared to other tertiary hospitals, the incidence of any stage ROP in our NICU was higher, but the rate of ROP needed treatment was lower. A higher GA was protective, whereas twin birth and moderate-to-severe BPD increased the hazard of any stage ROP; higher BW was protective, whereas male gender were risk factors for the development of severe ROP.

Differential expression profile of MicroRNAs during differentiation of cardiomyocytes exposed to polychlorinated biphenyls.

Exposure to persistent environmental pollutants, such as polychlorinated biphenyls (PCBs), is a risk factor for the development of congenital heart defects. MicroRNAs (miRNAs) have been shown to be involved in cardiac development. The objective of this study was to investigate changes in miRNA expression profiles during the differentiation of cardiomyocytes exposed to PCBs. For that purpose, PCBs (Aroclor 1254) at a concentration of 2.5 µmol/L were added on day 0 of differentiation of P19 mouse embryonal carcinoma cells into cardiac myocytes. The relative expression of miRNA genes was determined by miRNA microarray and real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) analyses. The microarray results revealed that 45 miRNAs, of which 14 were upregulated and 31 were downregulated, were differentially expressed in P19 cells treated with PCBs compared with control cells. The miRNA expression data was validated with real-time RT-PCR. The expression of certain potential target genes (Wnt1) was found to be reduced in P19 cells treated with PCBs, whereas the expression of other potential predicted target genes (GSK3ß) was increased. Our results demonstrate a critical role of miRNAs in mediating the effect of PCBs during the differentiation of P19 cells into cardiac myocytes.

The role of polypeptide PDTLN1 in suppression of PI3K/AKT signaling causes cardiogenetic disorders in vitro and in vivo.

AIMS: A new polypeptide, PDTLN1, derived from the human Talin-1 protein, which is highly expressed in both myocardial tissue and maternal peripheral blood of aborted fetuses with congenital heart disease (CHD). However, its role in cardiac developmental disorders has not been disclosed till now. In the present study, we aim to assess the functions of PDTLN1 in heart development of zebrafish and cellular viability, proliferation, and apoptosis of P19 cells. MAIN METHODS: Cellular viability was assessed by Cell Counting Kit-8, the EdU Kit was used to evaluate cellular proliferation, and apoptosic rate of P19 was examined using FITC Annexin-V staining followed by flow cytometry. The zebrafish embryos were divided into three groups: PEP group and NC group were microinjected with polypeptides, WT group without any intervention. The protein expression of PI3K/AKT were evaluated by western blotting. KEY FINDINGS: PDTLN1 could suppress the proliferation, and facilitate apoptosis. PDTLN1 caused abnormal heart development of zebrafish embryos and the PDTLN1 (50 µM)-injected group showed an aberrant expression pattern of vmhc, amhc and cmlc2. Compared to the CTL group and SC79 group of P19 cells, the PDTLN1 group had a lower phosphorylated PI3K/AKT proteins level, decreased cellular viability and lower proliferation activity. SIGNIFICANCE: PDTLN1 caused cardiac developmental defects in zebrafish, inhibited cellular viability, proliferation, and promoted apoptosis of P19 cells via suppressing the PI3K/AKT signaling pathway. Our findings provide a fresh perspective on the functional mechanism of human-derived peptides and may promote novel diagnostic biomarkers detection and therapeutic targets in CHD.

Effect of First Mother's Own Milk Feeding Time on the Risk of Moderate and Severe Bronchopulmonary Dysplasia in Infants With Very Low Birth Weight.

Objective: To explore the effect of mother's own milk (MOM) feeding time on the risk of moderate and severe bronchopulmonary dysplasia (BPD) in infants with very low birth weight (VLBW). Methods: Clinical data from 630 infants with VLBW were retrospectively analyzed. Participants were divided into the early mother's own milk (EMOM) feeding group (first mother's own milk feeding time ≤72 h after birth, n = 397) and the late mother's own milk (LMOM) feeding group (first mother's own milk feeding time >72 h after birth, n = 233). Differences in the incidence of moderate and severe BPD among the two groups were analyzed using the chi-square test. Effects of MOM feeding time on the incidence of moderate and severe BPD were evaluated using univariate and multivariate logistic regression analysis. Results: The incidences of moderate and severe BPD in the EMOM feeding group and the LMOM feeding group were 13.9% (55/397) and 21.0% (49/233), respectively (P = 0.019). Variate logistic regression analysis showed that the LMOM feeding group had an increased risk of moderate and severe BPD compared with the EMOM feeding group (OR = 1.656, 95% CI:1.083-2.532). The results of multivariate logistic regression analysis showed that the LMOM feeding group had an increased risk of moderate and severe BPD compared with the EMOM feeding group (OR = 1.894, 95% CI:1.127-3.185). Conclusion: The first time of MOM feeding within 72 h after birth and the persistence of mother's own milk feeding during hospitalization can reduce the incidence of moderate and severe BPD in infants with VLBW.