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author:("Hua, yiming")
1.  The Study of Fetal Rat Model of Intra-Amniotic Isoproterenol Injection Induced Heart Dysfunction and Phenotypic Switch of Contractile Proteins 
BioMed Research International  2014;2014:360687.
To establish a reliable isoproterenol induced heart dysfunction fetal rat model and understand the switches of contractile proteins, 45 pregnant rats were divided into 15 mg/kg-once, 15 mg/kg-twice, sham-operated once, sham-operated twice, and control groups. And 18 adult rats were divided into isoproterenol-treated and control groups. H&E staining, Masson staining, and transmission electron microscope were performed. Apoptotic rate assessed by TUNEL analysis and expressions of ANP, BNP, MMP-2, and CTGF of hearts were measured. Intra-amniotic injections of isoproterenol were supplied on E14.5 and E15.5 for fetuses and 7-day continuous intraperitoneal injections were performed for adults. Then echocardiography was performed with M-mode view assessment on E18.5 and 6 weeks later, respectively. Isoproterenol twice treated fetuses exhibited significant changes in histological evaluation, and mitochondrial damages were significantly severe with increased apoptotic rate. ANP and BNP increased and that of MMP-2 increased in isoproterenol twice treated group compared to control group, without CTGF. The isoforms transition of troponin I and myosin heavy chain of fetal heart dysfunction were opposite to adult procedure. The administration of intra-amniotic isoproterenol to fetal rats could induce heart dysfunction and the regulation of contractile proteins of fetuses was different from adult procedure.
PMCID: PMC4127273  PMID: 25136580
2.  Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide 
Electrophoresis  2013;34(5):753-760.
Sieving of proteins in silica colloidal crystals of mm dimensions is characterized for particle diameters of nominally 350 and 500 nm, where the colloidal crystals are chemically modified with a brush layer of polyacrylamide. A model is developed that relates the reduced electrophoretic mobility to the experimentally measurable porosity. The model fits the data with no adjustable parameters for the case of silica colloidal crystals packed in capillaries, for which independent measurements of the pore radii were made from flow data. The model also fits the data for electrophoresis in a highly ordered colloidal crystal formed in a channel, where the unknown pore radius was used as a fitting parameter. Plate heights as small as 0.4 μm point to the potential for miniaturized separations. Band broadening increases as the pore radius approaches the protein radius, indicating that the main contribution to broadening is the spatial heterogeneity of the pore radius. The results quantitatively support the notion that sieving occurs for proteins in silica colloidal crystals, and facilitate design of new separations that would benefit from miniaturization.
PMCID: PMC3753789  PMID: 23229163
3.  Pathological impact of SMN2 mis-splicing in adult SMA mice 
EMBO Molecular Medicine  2013;5(10):1586-1601.
Loss-of-function mutations in SMN1 cause spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. The related SMN2 gene expresses suboptimal levels of functional SMN protein, due to a splicing defect. Many SMA patients reach adulthood, and there is also adult-onset (type IV) SMA. There is currently no animal model for adult-onset SMA, and the tissue-specific pathogenesis of post-developmental SMN deficiency remains elusive. Here, we use an antisense oligonucleotide (ASO) to exacerbate SMN2 mis-splicing. Intracerebroventricular ASO injection in adult SMN2-transgenic mice phenocopies key aspects of adult-onset SMA, including delayed-onset motor dysfunction and relevant histopathological features. SMN2 mis-splicing increases during late-stage disease, likely accelerating disease progression. Systemic ASO injection in adult mice causes peripheral SMN2 mis-splicing and affects prognosis, eliciting marked liver and heart pathologies, with decreased IGF1 levels. ASO dose–response and time-course studies suggest that only moderate SMN levels are required in the adult central nervous system, and treatment with a splicing-correcting ASO shows a broad therapeutic time window. We describe distinctive pathological features of adult-onset and early-onset SMA.
PMCID: PMC3799581  PMID: 24014320
adult-onset SMA; pathology; SMN2; spinal muscular atrophy; splicing
4.  Increased Risk for Congenital Heart Defects in Children Carrying the ABCB1 Gene C3435T Polymorphism and Maternal Periconceptional Toxicants Exposure 
PLoS ONE  2013;8(7):e68807.
The etiology of congenital heart defect (CHD) is commonly believed to involve the interaction of multiple environmental and genetic factors. This study aimed to explore the joint effects of the ABCB1 gene C3435T polymorphism and maternal periconceptional toxicants exposure on the CHD risk in a Han Chinese population.
An age and gender matched case-control study with standardized data collection involving 201 pairs was conducted. Periconceptional toxicants exposure was obtained through a structured questionnaire. A job exposure matrix (JEM) was used for toxicants exposure assessment. Genotyping of the ABCB1 C3435T polymorphism was performed by sequencing. Logistic regression analysis was performed to assess the joint effects of the ABCB1 gene C3435T polymorphism and toxicants exposure on the risk of CHD. Placenta tissues and umbilical cords were collected to investigate the impact of C3435T polymorphism on the transcription and translation activities of ABCB1 gene.
Maternal periconceptional exposures to phthalates (adjusted OR: 1.6; 95%CI: 1.0–2.6) and alkylphenolic compounds (adjusted OR:1.8; 95%CI:1.1–3.0) were associated with a higher incidence of CHDs in general. More cases were carriers of the ABCB1 CC/CT genotypes (OR: 2.0, 95%CI: 1.1–3.5, P-value: 0.021). Children carrying the CC/CT genotype and periconceptionally exposed to phthalates and alkylphenolic compounds suffered almost 3.5-fold increased risk of having CHD than non-exposed children with TT genotype (adjusted OR: 3.5, 95%CI: 1.5–7.9, P-value: 0.003), and the OR changed to 4.4 for septal defects (adjusted OR: 4.4,95%CI:1.8–10.9,P-value:0.001). The ABCB1 mRNA expression of the TT genotype was significantly higher than that of the CC genotype (P = 0.03). Compared with TT genotype, lower P-glycoprotein expression was observed for the CC/CT genotypes.
The C3435T polymorphism in the ABCB1 gene of fetus increases the risks of CHD in a Han Chinese population when the mothers are exposed to phthalates and alkylphenolic compounds during the periconceptional period, particularly for septal defects.
PMCID: PMC3714281  PMID: 23874772
5.  Performance of Different Scan Protocols of Fetal Echocardiography in the Diagnosis of Fetal Congenital Heart Disease: A Systematic Review and Meta-Analysis 
PLoS ONE  2013;8(6):e65484.
The rapid progress in fetal echocardiography has lead to early detection of congenital heart diseases. Increasing evidences have shown that prenatal diagnosis could be life saving in certain cases. However, there is no agreement on which protocol is most adaptive diagnostic one. Thus, we use meta-analysis to conduct a pooled performance test on 5 diagnostic protocols.
We searched PUBMED, EMBASE, the Cochrane Central Register of Controlled Trials and WHO clinical trails registry center to identify relevant studies up to August, 2012. We performed meta-analysis in a fixed/random-effect model using Meta-disc 1.4. We used STATA 11.0 to estimate the publication bias and SPSS 17.0 to evaluate variance.
We use results from 81 studies in 63 articles to analyze the pooled accuracy. The overall performance of pooled sensitivities of spatiotemporal image correlation (STIC), extend cardiac echography examination (ECEE) and 4 chambers view + outflow tract view + 3 vessels and trachea view (4 CV+OTV+3 VTV) were around 0.90, which was significant higher than that of 4 chambers view + outflow tract view or 3 vessels and trachea view (4 CV+OTV/3 VTV) and 4 chambers view (4 CV). Unfortunately the pooled specificity of STIC was 0.92, which was significant lower than that of other 4 protocols which reached at 1.00. The area under the summary receiver operating characteristic curves value of STIC, ECEE, 4 CV+OTV+3 VTV, 4 CV+OTV/3 VTV and 4 CV were 0.9700, 0.9971, 0.9983, 0.9929 and 0.9928 respectively.
These results suggest a great diagnostic potential for fetal echocardiography detection as a reliable method of fetal congenital heart disease. But at least 3 sections view (4 CV, OTV and 3 VTV) should be included in scan protocol, while the STIC can be used to provide more information for local details of defects, and can not be used to make a definite diagnosis alone with its low specificity.
PMCID: PMC3672155  PMID: 23750263
6.  Antisense-based therapy for the treatment of spinal muscular atrophy 
The Journal of Cell Biology  2012;199(1):21-25.
One of the greatest thrills a biomedical researcher may experience is seeing the product of many years of dedicated effort finally make its way to the patient. As a team, we have worked for the past eight years to discover a drug that could treat a devastating childhood neuromuscular disease, spinal muscular atrophy (SMA). Here, we describe the journey that has led to a promising drug based on the biology underlying the disease.
PMCID: PMC3461520  PMID: 23027901
7.  Antisense-mediated exon inclusion 
Exon skipping induced by gene mutations is a common mechanism responsible for many genetic diseases. A practical approach to correct the aberrant splicing of defective genes is to use antisense oligonucleotides (ASOs). The recognition of splice sites and the regulation of splicing involve multiple positive or negative cis-acting elements and trans-acting factors. Base-pairing of ASOs to a negative element in a targeted pre-mRNA blocks the binding of splicing repressors to this cis-element and/or disrupts an unfavorable secondary structure; as a result, the ASO restores exon inclusion. For example, we have recently shown that appropriate 2’-O-(2-methoxyethyl) (MOE) phosphorothioate-modified ASOs can efficiently correct survival motor neuron 2 (SMN2) exon 7 splicing in a cell-free splicing assay, in cultured human cells—including patient fibroblasts—and in both peripheral tissues and the CNS of SMA mouse models. These ASOs are promising drug leads for SMA therapy.
PMCID: PMC3390937  PMID: 22454070
Exon skipping; antisense oligonucleotide; MOE; splicing; SMN2; SMA; ESS; ISS; cis-acting element; in vitro splicing assay; minigene; exon 7 inclusion; RT-PCR; ICV; ICV infusion; mouse tissue; spinal cord; CNS
8.  Notch Signaling Change in Pulmonary Vascular Remodeling in Rats with Pulmonary Hypertension and Its Implication for Therapeutic Intervention 
PLoS ONE  2012;7(12):e51514.
Pulmonary hypertension (PH) is a fatal disease that lacks an effective therapy. Notch signaling pathway plays a crucial role in the angiogenesis and vascular remodeling. However, its roles in vascular remodeling in PH have not been well studied. In the current study, using hypoxia-induced PH model in rat, we examined the expression of Notch and its downstream factors. Then, we used vessel strip culture system and γ-secretase inhibitor DAPT, a Notch signaling inhibitor to determine the effect of Notch signaling in vascular remodeling and its potential therapeutic value. Our results indicated that Notch 1–4 were detected in the lung tissue with variable levels in different cell types such as smooth muscle cells and endothelial cells of pulmonary artery, bronchia, and alveoli. In addition, following the PH induction, all of Notch1, Notch3, Notch4 receptor, and downstream factor, HERP1 in pulmonary arteries, mRNA expressions were increased with a peak at 1–2 weeks. Furthermore, the vessel wall thickness from rats with hypoxia treatment increased after cultured for 8 days, which could be decreased approximately 30% by DAPT, accompanied with significant increase of expression level of apoptotic factors (caspase-3 and Bax) and transformation of vascular smooth muscle cell (VSMC) phenotype from synthetic towards contractile. In conclusion, the current study suggested Notch pathway plays an important role in pulmonary vascular remodeling in PH and targeting Notch signaling pathway could be a valuable approach to design new therapy for PH.
PMCID: PMC3520790  PMID: 23251561
9.  Protective effects of indomethacin and dexamethasone in a goat model with intrauterine balloon aortic valvuloplasty 
Intrauterine balloon aortic valvuloplasty (IUBAV) has been used for critical aortic stenosis. However, it is necessary to determine the fetal impairments such as preterm birth after this approach and to find a way to prevent or reduce them.
In the present study, we evaluated the therapeutic value of indomethacin (IDM) and dexamethasone (DXS) on reducing the preterm birth rate in experimental goats after IUBAV.
Our results indicated that the administration of IDM/DXS significantly reduced the rate of premature birth. IDM/DXS treatment led to preservation of myocardial ultrastructure with less damage, and amelioration of the fetal and placental circulation. Furthermore, we found that norepinephrine (NE) level was positively associated with the degree of myocardial damage. IDM/DXS administration led to a significant decrease of operation-induced increase of NE levels, which may be associated with the protective effects of IDM/DXS. Lastly, we found that the administration of IDM/DXS did not induce the risk of ductus arteriosus closure or slow down fetal growth.
Our results indicate that IDM/DXS promotes a better gestational outcome at least partially by reducing stress response during and after the operation of IUBAV in the goat model. IDM/DXS may be a useful application in human patients during IUBAV intervention.
PMCID: PMC3438018  PMID: 22889399
Intrauterine balloon aortic valvuloplasty; Fetal goat; Gestational outcome; Ultrastructure; Hemodynamic; Indomethacin; Dexamethasone
10.  Field-free remobilization of proteins after isoelectric focusing in packed capillaries 
Analytical chemistry  2010;82(21):10.1021/ac101680z.
Pressure-driven remobilization without an applied electric field is shown to be possible with capillary isoelectric focusing by using packed capillaries. The capillary dimensions are 100 μm i.d. and 2 cm in length, and the packing is made of 0.9 μm nonporous silica particles that are chemically modified with a brush layer of polyacrylamide. Both reversible and irreversible adsorption are shown to be negligible. The packed capillaries eliminate the problem of unwanted hydrodynamic flow between reservoirs. Three proteins are focused: trypsin inhibitor, carbonic anhydrase II and myoglobin. The time required for focusing in the packed capillaries is increased by only a factor of two compared to the open capillary, giving complete focusing in less than 15 min at 200 V/cm. The packed capillaries allow the use of higher electric fields, with resolution continually increasing up to at least 1500 V/cm. The packing obstructs diffusional broadening after the field is turned off: for trypsin inhibitor, D=6.1(±0.3)×10-8 cm2/s for the packed capillary vs. D=28.8(±0.3)×10-8 cm2/s for the open capillary. The broadening contributed by the packing during remobilization is from eddy diffusion, and it is described by its plate height, H, which is the variance per unit length: H=σ2/L=0.64 μm. This limits the resolution to 0.1 pH unit for the 2-cm capillary having a pH range of 3-10, giving a theoretical peak capacity of 47.
PMCID: PMC3042039  PMID: 20931968
12.  Planar Cell Polarity Signaling Pathway in Congenital Heart Diseases 
Congenital heart disease (CHD) is a common cardiac disorder in humans. Despite many advances in the understanding of CHD and the identification of many associated genes, the fundamental etiology for the majority of cases remains unclear. The planar cell polarity (PCP) signaling pathway, responsible for tissue polarity in Drosophila and gastrulation movements and cardiogenesis in vertebrates, has been shown to play multiple roles during cardiac differentiation and development. The disrupted function of PCP signaling is connected to some CHDs. Here, we summarize our current understanding of how PCP factors affect the pathogenesis of CHD.
PMCID: PMC3205795  PMID: 22131815
13.  Antisense Oligonucleotides Delivered to the Mouse CNS Ameliorate Symptoms of Severe Spinal Muscular Atrophy 
Science translational medicine  2011;3(72):72ra18.
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the SMN1 gene that result in a deficiency of SMN protein. One approach to treat SMA is to use antisense oligonucleotides (ASOs) to redirect the splicing of a paralogous gene, SMN2, to boost production of functional SMN. Injection of a 2′-O-2-methoxyethyl–modified ASO (ASO-10-27) into the cerebral lateral ventricles of mice with a severe form of SMA resulted in splice-mediated increases in SMN protein and in the number of motor neurons in the spinal cord, which led to improvements in muscle physiology, motor function and survival. Intrathecal infusion of ASO-10-27 into cynomolgus monkeys delivered putative therapeutic levels of the oligonucleotide to all regions of the spinal cord. These data demonstrate that central nervous system–directed ASO therapy is efficacious and that intrathecal infusion may represent a practical route for delivering this therapeutic in the clinic.
PMCID: PMC3140425  PMID: 21368223
14.  Roles of planar cell polarity pathways in the development of neutral tube defects 
Neural tube defects (NTDs) are the second most common birth defect in humans. Despite many advances in the understanding of NTDs and the identification of many genes related to NTDs, the fundamental etiology for the majority of cases of NTDs remains unclear. Planar cell polarity (PCP) signaling pathway, which is important for polarized cell movement (such as cell migration) and organ morphogenesis through the activation of cytoskeletal pathways, has been shown to play multiple roles during neural tube closure. The disrupted function of PCP pathway is connected with some NTDs. Here, we summarize our current understanding of how PCP factors affect the pathogenesis of NTDs.
PMCID: PMC3175158  PMID: 21864354
Neural tube defects; planar cell polarity; organ morphogenesis; signaling pathway
15.  Enhancement of SMN2 Exon 7 Inclusion by Antisense Oligonucleotides Targeting the Exon 
PLoS Biology  2007;5(4):e73.
Several strategies have been pursued to increase the extent of exon 7 inclusion during splicing of SMN2 (survival of motor neuron 2) transcripts, for eventual therapeutic use in spinal muscular atrophy (SMA), a genetic neuromuscular disease. Antisense oligonucleotides (ASOs) that target an exon or its flanking splice sites usually promote exon skipping. Here we systematically tested a large number of ASOs with a 2′-O-methoxy-ethyl ribose (MOE) backbone that hybridize to different positions of SMN2 exon 7, and identified several that promote greater exon inclusion, others that promote exon skipping, and still others with complex effects on the accumulation of the two alternatively spliced products. This approach provides positional information about presumptive exonic elements or secondary structures with positive or negative effects on exon inclusion. The ASOs are effective not only in cell-free splicing assays, but also when transfected into cultured cells, where they affect splicing of endogenous SMN transcripts. The ASOs that promote exon 7 inclusion increase full-length SMN protein levels, demonstrating that they do not interfere with mRNA export or translation, despite hybridizing to an exon. Some of the ASOs we identified are sufficiently active to proceed with experiments in SMA mouse models.
Author Summary
Spinal muscular atrophy (SMA) is a severe genetic disease that causes motor-neuron degeneration. SMA patients lack a functional SMN1 (survival of motor neuron 1) gene, but they possess an intact SMN2 gene, which though nearly identical to SMN1, is only partially functional. The defect in SMN2 gene expression is at the level of pre-mRNA splicing (skipping of exon 7), and the presence of this gene in all SMA patients makes it an attractive target for potential therapy. Here we have surveyed a large number of antisense oligonucleotides (ASOs) that are complementary to different regions of exon 7 in the SMN2 mRNA. A few of these ASOs are able to correct the pre-mRNA splicing defect, presumably because they bind to regions of exon 7 that form RNA structures, or provide protein-binding sites, that normally weaken the recognition of this exon by the splicing machinery in the cell nucleus. We describe optimal ASOs that promote correct expression of SMN2 mRNA and, therefore, normal SMN protein, in cultured cells from SMA patients. These ASOs can now be tested in mouse models of SMA, and may be useful for SMA therapy.
Mutations inSMN1 cause spinal muscular atrophy; a nearly identical gene is not functional, but becomes functional in vitro and in vivo after addition of antisense oligos.
PMCID: PMC1820610  PMID: 17355180

Results 1-15 (15)