Hospitalization costs of pediatric community-acquired pneumonia in Shanghai / 中华儿科杂志

Objective: To investigate the epidemiology and hospitalization costs of pediatric community-acquired pneumonia (CAP) in Shanghai. Methods: A retrospective case summary was conducted on 63 614 hospitalized children with CAP in 59 public hospitals in Shanghai from January 2018 to December 2020. These children's medical records, including their basic information, diagnosis, procedures, and costs, were extracted. According to the medical institutions they were admitted, the patients were divided into the children's hospital group, the tertiary general hospital group and the secondary hospital group; according to the age, they were divided into <1 year old group, 1-<3 years old group, 3-<6 years old group, 6-<12 years old group and 12-18 years old group; according to the CAP severity, they were divided into severe pneumonia group and non-severe pneumonia group; according to whether an operation was conducted, the patients were divided into the operation group and the non-operation group. The epidemiological characteristics and hospitalization costs were compared among the groups. The χ2 test or Wilcoxon rank sum test was used for the comparisons between two groups as appropriate, and the Kruskal-Wallis H test was conducted for comparisons among multiple groups. Results: A total of 63 614 hospitalized children with CAP were enrolled, including 34 243 males and 29 371 females. Their visiting age was 4 (2, 6) years. The length of stay was 6 (5, 8) days. There were 17 974 cases(28.3%) in the secondary hospital group, 35 331 cases (55.5%) in the tertiary general hospital group and 10 309 cases (16.2%) in the children's hospital group. Compared with the hospitalizations cases in 2018 (27 943), the cases in 2019 (29 009) increased by 3.8% (1 066/27 943), while sharply declined by 76.2% (21 281/27 943) in 2020 (6 662). There were significant differences in the proportion of patients from other provinces and severe pneumonia cases, and the hospitalization costs among the children's hospital, secondary hospital and tertiary general hospital (7 146 cases(69.3%) vs. 2 202 cases (12.3%) vs. 9 598 cases (27.2%), 6 929 cases (67.2%) vs. 2 270 cases (12.6%) vs. 9 397 cases (26.6%), 8 304 (6 261, 11 219) vs. 1 882 (1 304, 2 796) vs. 3 195 (2 364, 4 352) CNY, χ2=10 462.50, 9 702.26, 28 037.23, all P<0.001). The annual total hospitalization costs of pediatric CAP from 2018 to 2020 were 110 million CNY, 130 million CNY and 40 million CNY, respectively. And the cost for each hospitalization increased year by year, which was 2 940 (1 939, 4 438), 3 215 (2 126, 5 011) and 3 673 (2 274, 6 975) CNY, respectively. There were also significant differences in the hospitalization expenses in the different age groups of <1 year old, 1-<3 years old, 3-<6 years old, 6-<12 years old and 12-18 years old (5 941 (2 787, 9 247) vs. 2 793 (1 803, 4 336) vs. 3 013 (2 070, 4 329) vs. 3 473 (2 400, 5 097) vs. 4 290 (2 837, 7 314) CNY, χ2=3 462.39, P<0.001). The hospitalization cost of severe pneumonia was significantly higher than that of non-severe cases (5 076 (3 250, 8 364) vs. 2 685 (1 780, 3 843) CNY, Z=109.77, P<0.001). The cost of patients who received operation was significantly higher than that of whom did not (10 040 (4 583, 14 308) vs. 3 083 (2 025, 4 747) CNY, Z=44.46, P<0.001). Conclusions: The number of children hospitalized with CAP in Shanghai decreased significantly in 2020 was significantly lower than that in 2018 and 2019.The proportion of patients from other provinces and with severe pneumonia are mainly admitted in children's hospitals. Hospitalization costs are higher in children's hospitals, and also for children younger than 1 year old, severe cases and patients undergoing operations.

Effect of dhfr gene overexpression on ethanol-induced abnormal cardiovascular development in zebrafish embryos / 中国当代儿科杂志

OBJECTIVE@#To study the effect of dhfr gene overexpression on ethanol-induced abnormal cardiac and vascular development in zebrafish embryos and underlying mechanisms.@*METHODS@#dhfr mRNA was transcribed in vitro and microinjected into zebrafish fertilized eggs to induce the overexpression of dhfr gene, and the efficiency of overexpression was verified. Wild-type zebrafish were divided into a control group, an ethanol group, and an ethanol+dhfr overexpression group (microinjection of 6 nL dhfr mRNA). The embryonic development was observed for each group. The transgenic zebrafish Tg (cmlc2:mcherry) with heart-specific red fluorescence was used to observe atrial and ventricular development. Fluorescence microscopy was performed to observe the development of cardiac outflow tract and blood vessels. Heart rate and ventricular shortening fraction were used to assess cardiac function. Gene probes were constructed, and embryo in situ hybridization and real-time PCR were used to measure the expression of nkx2.5, tbx1, and flk-1 in the embryo.@*RESULTS@#Compared with the ethanol group, the ethanol+dhfr overexpression group had a significant reduction in the percentage of abnormal embryonic development and a significant increase in the percentage of embryonic survival (P<0.05), with significant improvements in the abnormalities of the atrium, ventricle, outflow tract, and blood vessels and cardiac function. Compared with the control group, the ethanol group had significant reductions in the expression of nkx2.5, tbx1, and flk-1 (P<0.05), and compared with the ethanol group, the ethanol+dhfr overexpression group had significant increases in the expression of nkx2.5, tbx1, and flk-1 (P<0.05), which were still lower than their expression in the control group.@*CONCLUSIONS@#The overexpression of the dhfr gene can partially improve the abnormal development of embryonic heart and blood vessels induced by ethanol, possibly by upregulating the decreased expression of nkx2.5, tbx1, and flk-1 caused by ethanol.

Research advances in the pathogenesis of familial Kawasaki disease / 中国当代儿科杂志

Kawasaki disease has become the leading cause of acquired heart disease in children in North America and Japan. The incidence rate of Kawasaki disease varies significantly across regions and races. The first-degree relatives of patients with Kawasaki disease have a significantly higher risk of this disease than the general population. This article reviews the onset of familial Kawasaki disease and possible pathogenesis.

Generation of tnnt2a knock-out zebrafish via CRISPR/Cas9 and phenotypic analysis / 生理学报

Cardiac troponin T (cTnT) serves as a structural protein of myocardial fiber, and participates in heart excitation-contraction coupling process. Here, we generated tnnt2a (cTnT-coding gene) deletion mutant zebrafish via CRISPR/Cas9 technique, and performed phenotypic analysis of the identified tnnt2a mutants. We observed that there was no significant difference between heterozygous mutant and wild type zebrafish, and the homozygous mutants displayed significant malformations in heart, including cardiac arrest, atrium and ventricle enlargement, pericardium effusion, and the individuals usually died before 7 day post fertilization (dpf). We further analyzed the expression alternations of heart sarcomere genes (tnnt2a, actc1a, tpm4a, myl7, vmhc) at transcriptional level in tnnt2a(Δ2) zebrafish by performing real time RT-PCR, and found that the RNA expression level of tnnt2a in tnnt2azebrafish decreased constantly at each time point of developmental stages, and actc1a, tpm4a, myl7 and vmhc all showed higher expressions at early developmental stages and lower expressions at late developmental stages, in comparison with those of wild type zebrafish. Lastly, electron microscopy on cardiac tissues suggested that there were significant changes of the thick or thin filament structures in tnnt2a(Δ2) zebrafish, which was further confirmed by F-actin and Tpm4 immunofluorescence staining. The tnnt2azebrafish generated by CRISPR/Cas9 bears the most common symptoms of patients with dilated cardiomyopathy, and therefore can be used as a tool to study TNNT2-deficiency related cardiomyopathy.

Research advances in the mechanism of congenital heart disease induced by pregestational diabetes mellitus / 中国当代儿科杂志

Congenital heart disease (CHD) is the most common birth defect at present and has a complex etiology which involves the combined effect of genetic and environmental factors. Pregestational diabetes mellitus is significantly associated with the development of CHD, but the detailed mechanism remains unknown. This article reviews the research advances in the molecular mechanism of CHD caused by pregestational diabetes mellitus.

Role of the canonical Wnt signaling pathway in heart valve development / 中国当代儿科杂志

Formation of the heart valves is one of critical steps in vertebrate cardiac development. Valvular heart anomaly can induce severe cardiac impairment, which is one of most common symptoms for congenital heart defects (CHD). The canonical Wnt/β-catenin signaling pathway, which is essential for numerous developmental processes, has also been suggested to be involved in the regulation of proliferation, differentiation, and migration of myocardium, endocardium and valve primordia at different stages. The canonical Wnt signaling also regulates the endocardial-mesenchymal transformation (EMT) process during the endocardial cushion formation. This paper reviews current knowledge about the canonical Wnt signaling pathway in heart valve development, including the functional diversities of Wnt activity in heart valve development at different stages and its interaction with other valve-relevant signaling pathways and the potential role of canonical Wnt activity in heart valve mesenchymal stem cells at the late developmental stage.

Retinol dehydrogenase, RDH1l, is essential for the heart development and cardiac performance in zebrafish / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Retinoic acid (RA) is a potent signaling molecule that plays pleiotropic roles in patterning, morphogenesis, and organogenesis during embryonic development. The synthesis from retinol (vitamin A) to retinoic acid requires two sequential oxidative steps. The first step involves the oxidation of retinol to retinal through the action of retinol dehydrogenases. Retinol dehydrogenases1l (RDH1l) is a novel zebrafish retinol dehydrogenase. Herein we investigated the role of zebrafish RDH1l in heart development and cardiac performance in detail.</p><p><b>METHODS</b>RDH1l specific morpholino was used to reduce the function of RDH1l in zebrafish. The gene expressions were observed by using whole mount in situ hybridization. Heart rates were observed and recorded under the microscope from 24 to 72 hours post fertilization (hpf). The cardiac performance was analyzed by measuring ventricular shortening fraction (VSF).</p><p><b>RESULTS</b>The knock-down of RDH1l led to abnormal neural crest cells migration and reduced numbers of neural crest cells in RDH1l morphant embryos. The reduced numbers of cardiac neural crest cells also can be seen in RDH1l morphant embryos. Furthermore, the morpholino-mediated knock-down of RDH1l resulted in the abnormal heart loop. The left-right determining genes expression pattern was altered in RDH1l morphant embryos. The impaired cardiac performance was observed in RDH1l morphant embryos. Taken together, these data demonstrate that RDH1l is essential for the heart development and cardiac performance in zebrafish.</p><p><b>CONCLUSIONS</b>RDH1l plays a important role in the neural crest cells development, and then ultimately affects the heart loop and cardiac performance. These results show for the first time that an enzyme involved in the retinol to retinaldehyde conversion participate in the heart development and cardiac performance in zebrafish.</p>

Post-transcriptional protein modification of Gata4 / 中国当代儿科杂志

Gata4 is an important transcription factor in heart development. Gata4 post-transcriptional protein modification regulates transcriptional activity and DNA binding, which in turn affects expression of downstream genes and transcription factors, differentiation of embryonic stem cells and cardiogenesis. This article summarizes the effect of post-transcriptional protein modification on transcriptional activity of Gata4 and the relationship between this effect and congenital heart disease. It was shown that acetylation, phosphorylation and SUMOylation upregulate transcriptional activity, DNA binding, downstream gene expression and embryonic stem cell differentiation. On the other hand, methylation and deacetylation downregulate Gata4 transcriptional activity. Post-transcriptional protein modification of Gata4 is very important in clinical research on congenital and other heart diseases.

The impacts of maternal gestational diabetes mellitus (GDM) on fetal hearts / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To evaluate the fetal cardiac function in gestational diabetes mellitus (GDM) pregnancies under different maternal glycemic controls.</p><p><b>METHODS</b>Forty four GDM mothers received 78 fetal echocardiographic evaluations at three gestational periods (<28, 28-34 and >34 weeks) and were divided into poorly-(DM1) and well-(DM2) controlled groups according to their glycemic control at examination. Seventy uncomplicated mothers were selected as controls. Parameters of fetal cardiac anatomy and function were measured and analyzed.</p><p><b>RESULTS</b>GDM fetuses' cardiac ventricular walls were thicker than controls', and the differences between DM1 and DM2 were not significant except for end-diastolic left ventricular walls. In both GDM groups, the aortic flow velocities increased earlier than pulmonary artery and DM1 fetuses changed earlier than DM2 ones. GDM fetuses' left atrial shortening fraction was smaller than the controls' in the period of ⩾34 weeks and negatively correlated with thicknesses of left ventricular walls and interventricular septum in DM1 fetuses (r=-0.438 and -0.506). The right ventricular diastolic function in DM1 and DM2 fetuses decreased after the period of 28-34 weeks and in the period of >34 weeks respectively. Tei index of both left and right ventricles increased in DM1 group after the period of <28 weeks and in DM2 group only in the period of ⩾34 weeks, with no significant differences between DM1 and DM2 groups in this period.</p><p><b>CONCLUSION</b>Fetuses of GDM mothers showed cardiac function impairments. Good maternal glycemic control may delay the impairments, but cannot reduce the degree. Some cardiac changes in GDM fetuses were similar to those in pregestational diabetic pregnancies except for several parameters and their changing time.</p>

Effect of Tbx1 knock-down on cardiac performance in zebrafish / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tbx1 is the major candidate gene for DiGeorge syndrome (DGS). Similar to defects observed in DGS patients, the structures disrupted in Tbx1(-/-) animal models are derived from the neural crest cells during development. Although the morphological phenotypes of some Tbx1 knock-down animal models have been well described, analysis of the cardiac performance is limited. Therefore, myocardial performance was explored in Tbx1 morpholino injected zebrafish embryos.</p><p><b>METHODS</b>To elucidate these issues, Tbx1 specific morpholino was used to reduce the function of Tbx1 in zebrafish. The differentiation of the myocardial cells was observed using whole mount in situ hybridization. Heart rates were observed and recorded under the microscope from 24 to 72 hours post fertilization (hpf). The cardiac performance was analyzed by measuring ventricular shortening fraction and atrial shortening fraction.</p><p><b>RESULTS</b>Tbx1 morpholino injected embryos were characterized by defects in the pharyngeal arches, otic vesicle, aortic arches and thymus. In addition, Tbx1 knock down reduced the amount of pharyngeal neural crest cells in zebrafish. Abnormal cardiac morphology was visible in nearly 20% of the Tbx1 morpholino injected embryos. The hearts in these embryos did not loop or loop incompletely. Importantly, cardiac performance and heart rate were reduced in Tbx1 morpholino injected embryos.</p><p><b>CONCLUSIONS</b>Tbx1 might play an essential role in the development of pharyngeal neural crest cells in zebrafish. Cardiac performance is impaired by Tbx1 knock down in zebrafish.</p>

Lipoprotein lipase gene mutations and the risk of cardiovascular diseases in children with obesity / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To inquire into the relationship between lipoprotein lipase (LPL) gene D9N, N291S and S447X polymorphisms and the development of cardiovascular diseases in children with obesity.</p><p><b>METHODS</b>The polymerase chain reaction (PCR) and restriction fragment length polymorphism (RLFP) techniques were used to detect three common mutations of LPL gene exon D9N, N291S and S447X in 157 obese children and 175 normal controls. Plasma lipid and lipoprotein levels between children with different genotypes were compared.</p><p><b>RESULTS</b>The D9N and N291S gene mutations were not detected in either the obese or the control groups. There were no significant differences in the frequency of S447X gene mutation between the two groups. There were no significant differences in the levels of plasma lipid and lipoprotein between children with S447 and X447 genotypes.</p><p><b>CONCLUSIONS</b>D9N and N291S gene mutations may not be risk factors associated with cardiovascular diseases in children with obesity. S447X gene mutation might not play an important role in the development of cardiovascular diseases in childhood.</p>

Effects of folic acid on the development of heart of zebrafish / 中华儿科杂志

<p><b>OBJECTIVE</b>To construct the folic acid deficient model in zebrafish and observe the abnormal cardiac phenotypes, to find the optimal period for supplementing folic acid that can most effectively prevent the heart malformation induced by folic acid deficiency, and to investigate the possible mechanisms by which folic acid deficiency induces malformations of heart.</p><p><b>METHOD</b>The folic acid deficient zebrafish model was constructed by using both the folic acid antagonist methotrexate (MTX) and knocking-down dhfr (dihydrofolate reductase gene). Exogenous tetrahydrofolic acid rescue experiment was performed. Folic acid was given to folic acid deficient groups in different periods. The percent of cardiac malformation, the cardiac phenotypes, the heart rate and the ventricular shortening fraction (VSF) were recorded. The out flow tract (OFT) was observed by using fluorescein micro-angiography. Whole-mount in situ hybridization and real-time PCR were performed to detect vmhc, amhc, tbx5 and nppa expressions.</p><p><b>RESULT</b>About (78.00 ± 3.74)% embryos in MTX treated group and (68.00 ± 6.32)% embryos in dhfr knocking-down group had heart malformations, including the abnormal cardiac shapes, the hypogenesis of OFT and the reduced heart rate and VSF. Giving exogenous tetrahydrofolic acid rescued the above abnormalities. Given the folic acid on 8 - 12 hours post-fertilization (hpf), both the MTX treated group (20.20% ± 3.77%) and dhfr knocking-down group (43.40% ± 4.51%) showed the most significantly reduced percent of cardiac malformation and the most obviously improved cardiac development. In folic acid deficient group, the expressions of tbx5 and nppa were reduced while the expressions of vmhc and amhc appeared normal. After being given folic acid to MTX treated group and dhfr knocking-down group, the expressions of tbx5 and nppa were increased.</p><p><b>CONCLUSIONS</b>The synthesis of tetrahydrofolic acid was decreased in our folic acid deficient model. Giving folic acid in the middle period, which is the early developmental stage, can best prevent the abnormal developments of hearts induced by folic acid deficiency. Folic acid deficiency did not disrupt the differentiations of myosins in ventricle and atrium. The cardiac malformations caused by folic acid deficiency were related with the reduced expressions of tbx5 and nppa.</p>

Evaluation of cardiac function in fetuses from pregnant women with abnormal blood glucose levels by brain natriuretic peptide in umbilical cord blood / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the feasibility of umbilical cord brain natriuretic peptide (BNP) level measurement for the evaluation of perinatal cardiac function in fetuses from pregnant women with abnormal blood glucose levels and the influence of abnormal blood glucose on fetal cardiac function.</p><p><b>METHODS</b>Twenty-four mothers with gestational diabetes mellitus (n=18) or gestational impaired glucose tolerance (n=6) (diabetic group) were classified into two subgroups according to blood glucose level before delivery: good (n=17) and poor (n=7) glucose control. They underwent fetal echocardiography in their late pregnant periods and fetal cardiac sizes and function were measured. Twenty-five normal pregnant mothers served as the control group. Umbilical cord blood BNP concentrations were measured at delivery.</p><p><b>RESULTS</b>The umbilical cord blood BNP concentrations in the diabetic group were significantly higher than in the control group(114.0+/-39.0 pg/mL vs 80.6+/-13.7 pg/mL; p<0.01). The poor glucose control subgroup demonstrated higher umbilical cord blood BNP concentrations than the good glucose control subgroup (142.1+/-44.1 pg/mL vs 102.4+/-31.2 pg/mL; p<0.01). No difference was found between the gestational diabetes mellitus and the impaired glucose tolerance groups. The BNP concentration was positively correlated to the thicknesses of fetal left ventricular walls and the peak velocities of mitral A wave (r=0.715, 0.491 respectively, p<0.05), and negatively correlated to the mitral E/A ratio (r=-0.507, p<0.05).</p><p><b>CONCLUSIONS</b>The fetuses of pregnant women with abnormal blood glucose levels have an increased BNP level in umbilical cord blood. Umbilical cord BNP level is related to maternal blood glucose control and the changes in fetal cardiac function. It may reflex the latent impairments of fetal cardiac function. A good glucose control may decrease the impact of abnormal maternal blood glucose on fetal hearts.</p>

Association of nitric oxide and eNOS with the pathogenesis of vasovagal syncope / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the roles of nitric oxide (NO) and eNOS in the pathogenesis of vasovagal syncope (VVS).</p><p><b>METHODS</b>Fourteen children with VVS (group A), 10 children with syncope other than vasovagal (group B) and 20 healthy volunteers (group C) were enrolled. Plasma NO levels in groups A and B were determined before and at the termination of the head-up tilt table test (HUT). The G894T polymorphism within the eNOS gene was determined in the three groups.</p><p><b>RESULTS</b>Plasma NO levels in group A increased significantly when syncope attacked from 76.7+/-9.6 micromol/L (before HUT) to 90.0+/-11.4 micromol/L (P<0.05). After the syncope attack was improved, plasma NO level in group A was significantly reduced. There were no statistical differences in plasma NO levels before and after the HUT in group B. Determining the G894T polymorphism within the eNOS gene showed that group A was associated with a higher incidence of the GT gene type as compared to groups B and C (42.9% vs 10%; P<0.05).</p><p><b>CONCLUSIONS</b>Plasma NO may be involved in the pathogenesis of VVS. The increased plasma NO level may be associated with the G894T polymorphism of the eNOS gene.</p>

Expression of monocyte chemotactic protein-1 in peripheral blood mononuclear cells of children with Kawasaki disease and its relation to coronary artery impairment / 中华儿科杂志

<p><b>OBJECTIVE</b>Kawasaki disease (KD) is a febrile illness of childhood. The etiology of KD remains unknown. Multiple theories exist, including an infectious etiology and an immunological abnormality. Cardiac involvement ranges from myocarditis and pericarditis in the acute stage to the development of coronary artery aneurysms later in the course. The present study aimed to explore the effect of monocyte chemotactic protein-1 (MCP-1) in Kawasaki disease and its relationship with damage to the coronary arteries during the development of KD.</p><p><b>METHODS</b>Plasma MCP-1 concentrations were measured by enzyme-linked immunosorbent assay (ELISA), and MCP-1 mRNA expression in peripheral blood mononuclear cells (PBMC) was measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in comparison of three groups: 56 patients with KD, 60 age-matched patients with non-infectious diseases, and 66 age-matched febrile patients with various diseases.</p><p><b>RESULTS</b>Plasma MCP-1 concentration and MCP-1 mRNA expression in PBMC of patients with active KD [(409.55 +/- 97.42) pg/ml] and (1.97 +/- 0.77) were higher than those of control group. Plasma MCP-1 levels and MCP-1 mRNA expression of inactive KD group [(301.64 +/- 71.55) pg/ml] and (1.31 +/- 0.39) were significantly higher than those of non-infectious diseases patients. There was a marked increase in patients with inactive KD than those of non-infective patients, but there were no significant differences between inactive KD and febrile patients. Plasma MCP-1 levels and MCP-1 mRNA expression were markedly increased in KD patients with coronary artery lesions than those in patients without coronary artery lesions.</p><p><b>CONCLUSION</b>Plasma MCP-1 concentration and MCP-1 mRNA expression in PBMC were significantly increased in patients with KD, and they were higher in KD with coronary artery lesions. It indicates that MCP-1 may be a useful parameter for monitoring disease activity in patients with KD.</p>

Effect of external retinoic acid on Tbx1 gene during zebrafish embryogenesis / 中华儿科杂志

<p><b>OBJECTIVE</b>DiGeorge/del22q11 syndrome is one of the most common genetic causes of outflow tract and aortic arch defects in human. DiGeorge/del22q11 is thought to involve an embryonic defect restricted to the pharyngeal arches and the corresponding pharyngeal pouches. Previous studies have evidenced that retinoic acid (RA) signaling is definitely indispensable for the development of the pharyngeal arches. Tbx1, one of the T-box containing genes, is proved to be the most attractive candidate gene for DiGeorge/del22q11 syndrome. However, the interaction between RA and Tbx1 has not been fully investigated. Exploring the interaction will contribute to discover the molecular pathways disrupted in DiGeorge/del22q11 syndrome, and will also be essential for understanding genetic basis for congenital heart disease. It now seems possible that genes and molecular pathways disrupted in DiGeorge syndrome will also account for some isolated cases of congenital heart disease. Accordingly, the present study aimed to extensively study the effects of external RA on the cardiac development and Tbx1 expression during zebrafish embryogenesis.</p><p><b>METHODS</b>The chemical genetics approach was applied by treating zebrafish embryos with 5 x 10(-8) mol/L RA and 10(-7) mol/L RA at 12.5 hour post fertilization (hpf). The expression patterns of Tbx1 were monitored by whole-mount in situ hybridization and quantitative real-time RT-PCR, respectively.</p><p><b>RESULTS</b>The zebrafish embryos treated with 5 x 10(-8) mol/L RA and 10(-7) mol/L RA for 1.5 h at 12.5 hpf exhibited selective defects of abnormal heart tube. The results of whole-mount in situ hybridization with Tbx1 RNA probe showed that Tbx1 was expressed in cardiac region, pharyngeal arches and otic vesicle during zebrafish embryogenesis. RA treatment led to a distinct spatio-temporal expression pattern for Tbx1 from that in wild type embryo. The real-time PCR analysis showed that Tbx1 expression levels were markedly reduced by RA treatment. Tbx1 expression in the pharyngeal arches and heart were obviously down regulated compared to the wild type embryos. In contrast to 5 x 10(-8) mol/L RA-treated groups, 10(-7) mol/L RA caused a more severe effect on the Tbx1 expression level.</p><p><b>CONCLUSION</b>These results suggested that there was a genetic link between RA and Tbx1 during development of zebrafish embryo. RA could produce an altered Tbx1 expression pattern in zebrafish. RA may regulate the Tbx1 expression in a dose-dependant manner. RA could represent a major epigenetic factor to cause abnormal expression of Tbx1, secondarily, disrupt the pharyngeal arch and heart development.</p>

Folic acid antagonist methotrexate causes the development malformation of heart and down-regulates the BMP2b and HAS2 expressions in zebrafish / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effect of methotrexate (MTX), a folic acid antagonist which can lead to folic acid deficient, on the cardiac development and on the expressions of BMP2b and HAS2 in zebrafish.</p><p><b>METHODS</b>The zebrafish embryos at 6-48 hrs post fertilization (hpf) were treated with various concentrations of MTX (0.5 x 10(-3), 1.0 x 10(-3) and 2.0 x 10(-3) M). At 48 hpf, the percentage of cardiac malformation and heart rate were recorded. The zebrafish embryos at 6-10 hpf treated with 1.5 x 10(-3) M MTX were used as the MTX treatment group. At 24 and 48 hpf the cardiac morphology was observed under a microscope. The expressions of BMP2b and HAS2 in zebrafish were detected by in situ antisense RNA hybridization and real-time PCR.</p><p><b>RESULTS</b>6-12 hpf, the early embryonic developmental stage, was a sensitive period that MTX affected cardiac formation of zebrafish. The retardant cardiac development and the evidently abnormal cardiac morphology was found in the MTX treatment group. The results of in situ antisense RNA hybridization showed that the expressions of BMP2b and HAS2 in the zebrafish heart were reduced in the MTX treatment group at 36 and 48 hpf. The real-time PCR results demonstrated that the BMP2b expression decreased at 12, 24, 36 and 48 hpf, and that the HAS2 expression decreased at 24, 36 and 48 hpf in the treatment group compared with the control group without MTX treatment.</p><p><b>CONCLUSIONS</b>The inhibition of folic acid function may affect cardiac development of early embryos, resulting in a retardant development and a morphological abnormality of the heart in zebrafish, possibly by down-regulating the expressions of BMP2b and HAS2.</p>

Effect of dihydrofolate reductase gene knock-down on the expression of heart and neural crest derivatives expressed transcript 2 in zebrafish cardiac development / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Folic acid is very important for embryonic development and dihydrofolate reductase is one of the key enzymes in the process of folic acid performing its biological function. Therefore, the dysfunction of dihydrofolate reductase can inhibit the function of folic acid and finally cause the developmental malformations. In this study, we observed the abnormal cardiac phenotypes in dihydrofolate reductase (DHFR) gene knock-down zebrafish embryos, investigated the effect of DHFR on the expression of heart and neural crest derivatives expressed transcript 2 (HAND2) and explored the possible mechanism of DHFR knock-down inducing zebrafish cardiac malformations.</p><p><b>METHODS</b>Morpholino oligonucleotides were microinjected into fertilized eggs to knock down the functions of DHFR or HAND2. Full length of HAND2 mRNA which was transcribed in vitro was microinjected into fertilized eggs to overexpress HAND2. The cardiac morphologies, the heart rates and the ventricular shortening fraction were observed and recorded under the microscope at 48 hours post fertilization. Whole-mount in situ hybridization and real-time PCR were performed to detect HAND2 expression.</p><p><b>RESULTS</b>DHFR or HAND2 knock-down caused the cardiac malformation in zebrafish. The expression of HAND2 was obviously reduced in DHFR knock-down embryos (P < 0.05). Microinjecting HAND2 mRNA into fertilized eggs can induce HAND2 overexpression. HAND2 overexpression rescued the cardiac malformation phenotypes of DHFR knock-down embryos.</p><p><b>CONCLUSIONS</b>DHFR plays a crucial role in cardiac development. The down-regulation of HAND2 caused by DHFR knock-down is the possible mechanism of DHFR knock-down inducing the cardiac malformation.</p>

Fetal echocardiography in diagnosing congenital heart disease prenatally: a multicenter clinical study / 中华儿科杂志

<p><b>OBJECTIVE</b>To evaluate the detection and accuracy of fetal echocardiography for congenital heart defects among high-risk populations.</p><p><b>METHODS</b>A prospective observational study of prenatal diagnosis of congenital heart disease was conducted in two tertiary obstetrics and gynecology hospitals between January 2003 and December 2004. Consecutive fetuses at risk of congenital heart disease underwent detailed fetal echocardiography during the study period. B-mode and colour/pulsed Doppler flow imaging were used in all cases. Follow-up was sought for all pregnancies. Indications for referral, maternal and gestational age at diagnosis, as well as prenatal and postnatal diagnosis were recorded prospectively. By comparing prenatal and postnatal diagnoses, sensitivity, specificity, and predictive values were estimated.</p><p><b>RESULTS</b>A series of 2063 high-risk fetuses underwent detailed fetal echocardiography during the study period. The mean gestational age at examination was 26.5 weeks, ranging from 16 to 42 weeks. The most common indications for fetal echocardiography were advanced maternal age (31.7%), fetal arrhythmias (13.5%) and maternal infections (10.4%). Forty-three cases of fetal congenital heart disease were detected. The mean gestational age at prenatal diagnosis was 27.3 weeks ranging from 16 to 40 weeks. There were 3 false-negatives and 1 false-positive. The sensitivity, specificity, positive and negative predictive values were 92.1%, 99.9%, 97.2%, and 99.8%, respectively. Diagnostic accuracy was 86.1%. A cardiac defect suspected on routine prenatal sonography accounted for the highest proportion of abnormal cases (67.4%). As for pregnancy outcome, there were 24 (52.1%) terminations; 2.2% died in utero, 13% postnatally, and 28.3% survived.</p><p><b>CONCLUSIONS</b>(1) Fetal congenital heart disease can be identified reliably by prenatal echocardiography. (2) Possible congenital heart disease or suspected heart defect noted on a screening obstetric sonogram is an important indication for fetal echocardiography. (3) A sequential segmental approach is critical for correct evaluation of the cardiac malformation. (4) The outcome of the patients with congenital heart disease is poor and a multidisciplinary approach is needed to the parental counseling and perinatal management planning.</p>

Expressions of plasma SDF-1 and its receptor CXCR4 in peripheral blood mononuclear cells of children with Kawasaki disease / 中国当代儿科杂志

<p><b>OBJECTIVE</b>This study examined the expressions of plasma stromal cell derived factor-1 (SDF-1) and CXCR4 mRNA in peripheral blood mononuclear cells (PBMC) of children with Kawasaki disease (KD) and aimed to explore the significance of SDF-1 and CXCR4 mRNA in KD.</p><p><b>METHODS</b>Fifty-six children with KD (12 cases complicated by coronary artery lesions) and 60 age and gender-matched healthy children (normal controls) were enrolled in this study. Plasma SDF-1 levels and CXCR4 mRNA expression in PBMC were measured using ELISA and real-time quantitative PCR at the acute and convalescence stages of KD.</p><p><b>RESULTS</b>Plasma SDF-1 levels (1833 +/- 395 ng/L vs 1126 +/- 408 ng/L; P < 0.05) and the CXCR4 mRNA expression in PBMC (6.57 +/- 2.81 vs 2.58 +/- 1.01; P < 0.01) in KD patients were significantly higher than those in normal controls at the acute stage. Both plasma SDF-1 levels and CXCR4 mRNA expression in KD patients decreased significantly at the convalescence stage, but nevertheless remained higher than those in the normal controls. The patients with concomitant coronary artery lesions showed higher CXCR4 mRNA levels than without at the acute stage (8.19 +/- 2.39 vs 6.13 +/- 2.77; P < 0.05).</p><p><b>CONCLUSIONS</b>Plasma SDF-1 concentration and CXCR4 mRNA expression in PBMC increased in KD patients. CXCR4 mRNA might be involved in the development of coronary artery lesions in KD.</p>