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author:("Liu, taohong")
1.  A matched case–control study of preterm birth in one hospital in Beijing, China 
Reproductive Health  2015;12:1.
Preterm birth is an unresolved global health issue. The etiologies of preterm birth are complex and multifactorial. To examine risk factors related to preterm birth, a matched case–control study was conducted in a hospital in Beijing, China where little data on preterm birth have been published in the scientific literature.
A 1:1 matched case–control study was conducted in 172 pairs of women with preterm birth (case group) and term delivery (control group). Eligible subjects were interviewed in person by well-trained investigators using a questionnaire. Information on obstetric diagnosis and newborns were abstracted from inpatients’ medical records. Univariate and multivariate conditional logistic regression models were used to measure the associations between related factors and preterm birth.
Univariate analysis showed that 6 of 12 factors were associated with preterm birth. Multivariate results showed that gestational hypertension (OR = 7.76), low gestational weight gain (OR = 3.02), frequent prenatal care (OR = 0.16), balanced diet (OR = 0.36), and high gestational weight gain (OR = 0.41) were associated with preterm birth.
This study provides information on preterm birth in Beijing, China, and it also lends support to existing evidence about the role of maternal nutritional status, prenatal care and gestational hypertension as risk factors for preterm birth.
PMCID: PMC4290090  PMID: 25561377
Preterm birth; Case–control study; Risk factors
2.  Cecropin P1 inhibits porcine reproductive and respiratory syndrome virus by blocking attachment 
BMC Microbiology  2014;14(1):273.
Porcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pig industry, causing high economic losses worldwide. Current vaccines have specific limitations in terms of their safety and efficacy, so the development of novel antiviral drugs is urgently required. The aim of this study was to evaluate the inhibitory effects and underlying molecular mechanisms of the antimicrobial peptide cecropin P1 (CP1) against PRRSV infection in vitro.
CP1 not only displayed extracellular virucidal activity against PRRSV, but also exerted a potent inhibitory effect when added either before, simultaneously with, or after viral inoculation. The inhibitory effect of CP1 occurred during viral attachment, but not at viral entry into Marc-145 cells. CP1 also inhibited viral particle release and attenuated virus-induced apoptosis during the late phase of infection. CP1 exerted similar inhibitory effects against PRRSV infection in porcine alveolar macrophages, the cells targeted by the virus in vivo during its infection of pigs. The expression of interleukin 6 was elevated by CP1 in porcine alveolar macrophages, which might contribute to its inhibition of PRRSV infection.
Collectively, our findings provide a new direction for the development of potential therapeutic drugs against PRRSV infection.
PMCID: PMC4243277  PMID: 25403758
Cecropin P1; PRRSV; Antiviral activity; Antimicrobial peptide
3.  Gene Expression Profiling in Human Lung Development: An Abundant Resource for Lung Adenocarcinoma Prognosis 
PLoS ONE  2014;9(8):e105639.
A tumor can be viewed as a special “organ” that undergoes aberrant and poorly regulated organogenesis. Progress in cancer prognosis and therapy might be facilitated by re-examining distinctive processes that operate during normal development, to elucidate the intrinsic features of cancer that are significantly obscured by its heterogeneity. The global gene expression signatures of 44 human lung tissues at four development stages from Asian descent and 69 lung adenocarcinoma (ADC) tissue samples from ethnic Chinese patients were profiled using microarrays. All of the genes were classified into 27 distinct groups based on their expression patterns (named as PTN1 to PTN27) during the developmental process. In lung ADC, genes whose expression levels decreased steadily during lung development (genes in PTN1) generally had their expression reactivated, while those with uniformly increasing expression levels (genes in PTN27) had their expression suppressed. The genes in PTN1 contain many n-gene signatures that are of prognostic value for lung ADC. The prognostic relevance of a 12-gene demonstrator for patient survival was characterized in five cohorts of healthy and ADC patients [ADC_CICAMS (n = 69, p = 0.007), ADC_PNAS (n = 125, p = 0.0063), ADC_GSE13213 (n = 117, p = 0.0027), ADC_GSE8894 (n = 62, p = 0.01), and ADC_NCI (n = 282, p = 0.045)] and in four groups of stage I patients [ADC_CICAMS (n = 22, p = 0.017), ADC_PNAS (n = 76, p = 0.018), ADC_GSE13213 (n = 79, p = 0.02), and ADC_qPCR (n = 62, p = 0.006)]. In conclusion, by comparison of gene expression profiles during human lung developmental process and lung ADC progression, we revealed that the genes with a uniformly decreasing expression pattern during lung development are of enormous prognostic value for lung ADC.
PMCID: PMC4139381  PMID: 25141350
4.  Selection of competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing for patients undergoing preimplantation genetic screening: a prospective study with sibling oocytes 
BMC Medical Genomics  2014;7:38.
Recent advances in time-lapse monitoring in IVF treatment have provided new morphokinetic markers for embryonic competence. However, there is still very limited information about the relationship between morphokinetic parameters, chromosomal compositions and implantation potential. Accordingly, this study aimed at investigating the effects of selecting competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing on pregnancy and implantation outcomes for patients undergoing preimplantation genetic screening (PGS).
A total of 1163 metaphase II (MII) oocytes were retrieved from 138 PGS patients at a mean age of 36.6 ± 2.4 years. These sibling MII oocytes were then randomized into two groups after ICSI: 1) Group A, oocytes (n = 582) were cultured in the time-lapse system and 2) Group B, oocytes (n = 581) were cultured in the conventional incubator. For both groups, whole genomic amplification and array CGH testing were performed after trophectoderm biopsy on day 5. One to two euploid blastocysts within the most predictive morphokinetic parameters (Group A) or with the best morphological grade available (Group B) were selected for transfer to individual patients on day 6. Ongoing pregnancy and implantation rates were compared between the two groups.
There were significant differences in clinical pregnancy rates between Group A and Group B (71.1% vs. 45.9%, respectively, p = 0.037). The observed implantation rate per embryo transfer significantly increased in Group A compared to Group B (66.2% vs. 42.4%, respectively, p = 0.011). Moreover, a significant increase in ongoing pregnancy rates was also observed in Group A compared to Group B (68.9% vs. 40.5%. respectively, p = 0.019). However, there was no significant difference in miscarriage rate between the time-lapse system and the conventional incubator (3.1% vs. 11.8%, respectively, p = 0.273).
This is the first prospective investigation using sibling oocytes to evaluate the efficiency of selecting competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing for PGS patients. Our data clearly demonstrate that the combination of these two advanced technologies to select competent blastocysts for transfer results in improved implantation and ongoing pregnancy rates for PGS patients.
PMCID: PMC4077552  PMID: 24954518
Time-lapse monitoring; Array CGH; PGS; Ploidy; Implantation; Miscarriage
5.  Simultaneous Detection and Differentiation of Highly Virulent and Classical Chinese-Type Isolation of PRRSV by Real-Time RT-PCR 
Journal of Immunology Research  2014;2014:809656.
Porcine reproductive and respiratory syndrome (PRRS) is a leading disease in pig industry worldwide and can result in serious economic losses each year. The PRRS epidemic situation in China has been very complicated since the unprecedented large-scale highly pathogenic PRRS (HP-PRRS) outbreaks in 2006. And now the HP-PRRS virus (HP-PRRSV) and classical North American type PRRSV strains have coexisted in China. Rapid differential detection of the two strains of PRRSV is very important for effective PRRS control. The real-time RT-PCR for simultaneous detection and differentiation of HP-PRRSV and PRRSV by using both SYBR Green and TaqMan probes was developed and validated. Both assays can be used for rapid detection and strain-specific identification of HP-PRRSV and PRRSV. However, the TaqMan probe method had the highest detection rate whereas the conventional RT-PCR was the lowest. The real-time RT-PCR developed based on SYBR Green and TaqMan probe could be used for simultaneous detection and differentiation of HP-PRRSV and PRRSV in China, which will benefit much the PRRS control and research.
PMCID: PMC4119655  PMID: 25114934
6.  Expression of Wnt-5a and β-catenin in primary hepatocellular carcinoma 
It has been reported that changes in Wnt5a expression are closely related to hepatocellular carcinoma (HCC) development, while decreased or abnormal β-catenin expression may promote the invasion and metastasis of tumor cells. In this study, the roles and clinical significance of Wnt-5a and β-catenin expression were analyzed in primary HCC. Real-time PCR (RT-PCR) analysis of Wnt-5a mRNA expression was performed in 26 fresh HCC samples and the corresponding para-carcinoma tissues. Wnt-5a and β-catenin protein expression was detected by immunohistochemical staining of paraffin-embedded tissues of 85 cases of HCC and corresponding para-carcinoma tissues and 15 cases of hepatic cirrhosis. Results showed that Wnt-5a mRNA levels were significantly higher in HCC tissue than in the para-carcinoma tissue (0.102 ± 0.159 and 0.020 ± 0.022, respectively; P < 0.05), while Wnt-5a protein was absent or low in HCC. Wnt-5a expression was detected in significantly fewer HCC tissue samples than in the para-carcinoma and hepatic cirrhosis tissue samples (21.2% (18/85), 81.26% (69/85) and 86.7% (13/15), respectively; P < 0.01). Abnormal localization of β-catenin protein shown by intracytoplasmic or intranuclear staining was observed in 72.94% (62/85) of HCC samples. These observations indicate that the role of Wnt-5a in HCC is mediated at the protein level rather than the transcriptional level. Furthermore, the abnormal localization of β-catenin observed in HCC tissues may be associated with gene mutation leading to the generation of truncated β-catenin proteins, which in turn, may represent an initiating or contributing factor in the development of HCC.
PMCID: PMC4097247  PMID: 25031739
Hepatocellular carcinoma; Wnt-5a; β-catenin; RT-PCR; immunohistochemistry
7.  Histone preconditioning protects against obstructive jaundice-induced liver injury in rats 
A major consequence of obstructive jaundice (OJ) in clinical practice is the development of severe liver injury, and at present, no effective treatments have been developed to protect against it. Preconditioning with damage-associated molecular pattern (DAMP) molecules has been demonstrated to protect multiple organs from injury, and histones have been recently identified as DAMP molecules. The aim of the present study was to investigate the protective effect of histone preconditioning against OJ-induced liver injury in rats and the involvement of Toll-like receptors. Rats were administered histone proteins (200 μg/kg; 1 ml) or physiological saline (1 ml) intraperitoneally 24 h prior to being subjected to bile duct ligation (BDL). The serum levels of liver enzymes and bilirubin, as well as the histopathology were analyzed. The mRNA expression of interleukin-6 (IL-6) in the liver tissue was analyzed using quantitative polymerase chain reaction. BDL in the control group caused severe OJ-induced liver injury, as indicated by the significantly elevated levels of liver enzymes and mRNA levels of IL-6, and confirmed by histopathological alterations. However, histone preconditioning significantly ameliorated the OJ-induced liver injury caused by BDL, as shown by an improvement in the levels of liver enzymes, a suppression of IL-6 production, as well as histopathological alterations. Therefore, these results suggested that histone preconditioning is able to protect against OJ-induced liver injury in rats.
PMCID: PMC4061184  PMID: 24944590
histone; precondition; obstructive jaundice; liver injury
8.  Secular trends of low birthweight and macrosomia and related maternal factors in Beijing, China: a longitudinal trend analysis 
Information tracking changes of birthweight is scarce in China. To examine trends of low birthweight (birthweight < 2500 g) and macrosomia (birthweight ≥ 4000 g) and potential risk factors in Beijing, hospital records from two major obstetrics and gynecology hospitals in urban districts in Beijing were analyzed.
Hospital records from 1996 to 2010 were retrieved. Information of prenatal examination and birth outcomes was entered into a structured database. Live births were used for trend analysis. Information of live births in 2010 was used to identify potential risk factors.
A total of 63 661 live births were delivered during 1996–2010 in the study hospitals. The average birthweight increased from 3271 g in 1996 to 3 359 g in 2000 and slightly declined to 3 331 in 2010. The percentage of low birthweight fluctuated around 4.0%. No significant increase or decrease was observed. Preterm birth was the main cause of low birthweight, accounting for more than 73% of low birthweight. The average percentage of macrosomia was 7.6%. The percentages of macrosomia increased from 6.6% in 1996 to 9.5% in 2000 and declined to 7.0% in 2010. Excessive gestational weight gain and gestational diabetes were significantly associated with macrosomia.
Continuously monitoring abnormal birthweight is needed and intervention should focus on appropriate gestational weight gain and reduction of preterm birth and gestational diabetes.
PMCID: PMC4003827  PMID: 24641671
Low birthweight; Macrosomia; Trends; Gestational weight gain; Gestational diabetes
9.  Inhibition of HSP70 reduces porcine reproductive and respiratory syndrome virus replication in vitro 
BMC Microbiology  2014;14:64.
Successful viral infection requires the involvement of host cellular factors in their life cycle. Heat shock protein 70 (HSP70) can be recruited by numerous viruses to promote the folding, maturation, or assembly of viral proteins. We have previously shown that HSP70 is significantly elevated in porcine reproductive and respiratory syndrome virus (PRRSV)-infected lungs, suggesting HSP70 may play a potential role during PRRSV infection. In this study, we tried to investigate the role of HSP70 during PRRSV infection.
In this study, we observed that PRRSV infection induced the expression of HSP70. The down-regulation of HSP70 using quercetin, a HSPs synthesis inhibitor, or small interfering RNAs (siRNA) reduced the viral protein level and viral production. Notably, these inhibitory effects on PRRSV infection could be attenuated by heat shock treatment. In addition, HSP70 was found to colocalize with the viral double-stranded RNA (dsRNA) and knockdown of HSP70 decreased the dsRNA levels, suggesting HSP70 is involved in the formation of viral replication and transcription complex (RTC) and thus affects the viral replication.
Our study revealed that HSP70 is an essential host factor required for the replication of PRRSV. The inhibition of HSP70 significantly reduced PRRSV replication, which may be applied as an effective antiviral strategy.
PMCID: PMC3984673  PMID: 24625230
PRRSV; HSP70; DsRNA; Replication; Antiviral
10.  Wnt5a involved in regulation of the biological behavior of hepatocellular carcinoma 
Objective: Wnt5a has been shown to be involved in cancer progression in a variety of tumor types. Previous experimental studies have indicated that it has been shown to be down-regulated in hepatocellular carcinoma (HCC). The goal of this study was to explore the effect of Wnt5a overexpression in an HCC cell line. Methods: We transfected the human HCC cell line Huh7 with a pcDNA3.1-Wnt5a overexpression vector or an empty vector control. The integration of the plasmid DNA and the expression of Wnt5a in Huh7 cells were confirmed by real-time RT-PCR and Western blot. A plate colony formation test was used to calculate the clone formation rate and the cell cycle was analyzed by flow cytometry. The effect of Wnt5a overexpression on cell migration was studied in vitro using a scratch assay and in vivo by xenograft studies in nude mice. Results: Our results showed that in Huh7 cells with overexpression of Wnt5a, the fraction of cells in the G1 and S phases of the cell cycle was significantly increased compared with untransfected cells. In agreement with this finding, overexpression of Wnt5a was associated with a lower colony formation rate compared with control cells. In our xenograft studies, nude mice injected with Huh7 cells with overexpression of Wnt5a had decreased tumor volumes compared with controls. The vitro scratch assay revealed that Wnt5a overexpression cells had a diminished capacity for cell migration. Furthermore, we studied the expression of important proteins associated with Wnt5a signaling pathway, and it was found that Ror2 and E-cadherin were both increased in Huh7 cells with overexpression of Wnt5a, whereas p53 expression was unaffected. Conclusion: Overexpression of Wnt5a in Huh7 cells was associated with decrease of cell proliferation and migration. Wnt5a may act as a tumor-suppressor gene in HCC, which works through the non-canonical Wnt signaling pathway by binding to the Ror2 and E-cadherin receptor.
PMCID: PMC3971301  PMID: 24696716
Hepatocellular carcinoma; Wnt5a; Huh7; proliferation; migration
11.  Inhibition of HSP90 attenuates porcine reproductive and respiratory syndrome virus production in vitro 
Virology Journal  2014;11:17.
Porcine reproductive and respiratory syndrome virus (PRRSV) infection leads to substantial economic losses to the swine industry worldwide. However, no effective countermeasures exist to combat this virus infection so far. The most common antiviral strategy relies on directly inhibiting viral proteins. However, this strategy invariably leads to the emergence of drug resistance due to the error-prone nature of viral ploymerase. Targeting cellular proteins required for viral infection for developing new generation of antivirals is gaining concern. Recently, heat shock protein 90 (HSP90) was found to be an important host factor for the replication of multiple viruses and the inhibition of HSP90 showed significant antiviral effects. It is thought that the inhibition of HSP90 could be a promising broad-range antiviral approach. However, the effects of HSP90 inhibition on PRRSV infection have not been evaluated. In the current research, we tried to inhibit HSP90 and test whether the inhibition affect PRRSV infection.
We inhibit the function of HSP90 with two inhibitors, geldanamycin (GA) and 17- allylamono-demethoxygeldanamycin (17-AAG), and down-regulated the expression of endogenous HSP90 with specific small-interfering RNAs (siRNAs). Cell viability was measured with alamarBlue. The protein level of viral N was determined by western blotting and indirect immunofluorescence (IFA). Besides, IFA was employed to examine the level of viral double-stranded RNA (dsRNA). The viral RNA copy number and the level of IFN-β mRNA were determined by quantitative real-time PCR (qRT-PCR).
Our results indicated that both HSP90 inhibitors showed strong anti-PRRSV activity. They could reduce viral production by preventing the viral RNA synthesis. These inhibitory effects were not due to the activation of innate interferon response. In addition, we observed that individual knockdown targeting HSP90α or HSP90β did not show dramatic inhibitory effect. Combined knockdown of these two isoforms was required to reduce viral infection.
Our results shed light on the possibility of developing potential therapeutics targeting HSP90 against PRRSV infection.
PMCID: PMC3942275  PMID: 24490822
Porcine reproductive and respiratory syndrome virus; PRRSV; HSP90; Geldanamycin; 17-AAG; Antiviral
12.  Aortic valve replacement with autologous pericardium: long-term follow-up of 15 patients and in vivo histopathological changes of autologous pericardium 
The study aimed to assess the long-term follow-up of patients with an autologous pericardial aortic valve (APAV) replacement and to analyse in vivo histopathological changes in implanted APAVs.
From 1996 to 1997, 15 patients (mean age, 34 years) underwent aortic valve replacement with the glutaraldehyde-treated autologous pericardium. All patients were followed up after discharge. The excised APAVs were processed for haematoxylin–eosin, Victoria blue-van Gieson and immunohistochemical staining.
The mean clinical follow-up was 11.43 ± 4.50 years. APAV-related in-hospital and late mortalities were both 0%. Five (33%) patients required reoperation because of a prolapse of the right coronary cusp (n = 1), infective endocarditis (n = 1) or fibrocalcific degeneration (n = 3). Freedom from endocarditis, fibrocalcific degeneration and reoperation at the end of follow-up was 93, 80 and 67%, respectively. The remaining 10 patients were alive and well with a mean New York Heart Association class of 1.10 ± 0.32 and normally functioning aortic valves (peak pressure gradient: 7.70 ± 3.41 mmHg; mean pressure gradient: 1.79 ± 0.64 mmHg). Histopathology revealed that (i) a thin factor VIII-positive layer (endothelialization) was found on all non-endocarditis APAVs; (ii) pericardial cells in all APAVs were positive for α-smooth muscle actin (myofibroblast phenotype) and some cells in the fibrocalcific APAVs were positive for alkaline phosphatase (osteoblast phenotype) and (iii) an elastic band was found in 3 cases (in vivo >9 years).
APAV replacement is a procedure with a low mortality. APAVs adapt to new environmental demands by producing an elastic band and by endothelialization, whereas myofibroblast/osteoblast transdifferentiation seems to be responsible for the fibrocalcification of APAVs.
PMCID: PMC3548525  PMID: 23143205
Autologous pericardial aortic valve; Fibrosis; Calcification; Endothelialization; Transdifferentiation
13.  Evidence for NG2-glia Derived, Adult-Born Functional Neurons in the Hypothalamus 
PLoS ONE  2013;8(10):e78236.
Accumulating evidence suggests that the adult murine hypothalamus, a control site of several fundamental homeostatic processes, has neurogenic capacity. Correspondingly, the adult hypothalamus exhibits considerable cell proliferation that is ongoing even in the absence of external stimuli, and some of the newborn cells have been shown to mature into cells that express neuronal fate markers. However, the identity and characteristics of proliferating cells within the hypothalamic parenchyma have yet to be thoroughly investigated. Here we show that a subset of NG2-glia distributed throughout the mediobasal hypothalamus are proliferative and express the stem cell marker Sox2. We tracked the constitutive differentiation of hypothalamic NG2-glia by employing genetic fate mapping based on inducible Cre recombinase expression under the control of the NG2 promoter, demonstrating that adult hypothalamic NG2-glia give rise to substantial numbers of APC+ oligodendrocytes and a smaller population of HuC/D+ or NeuN+ neurons. Labelling with the cell proliferation marker BrdU confirmed that some NG2-derived neurons have proliferated shortly before differentiation. Furthermore, patch-clamp electrophysiology revealed that some NG2-derived cells display an immature neuronal phenotype and appear to receive synaptic input indicative of their electrical integration in local hypothalamic circuits. Together, our studies show that hypothalamic NG2-glia are able to take on neuronal fates and mature into functional neurons, indicating that NG2-glia contribute to the neurogenic capacity of the adult hypothalamus.
PMCID: PMC3812154  PMID: 24205170
14.  Integrative Analysis of Porcine microRNAome during Skeletal Muscle Development 
PLoS ONE  2013;8(9):e72418.
Pig is an important agricultural animal for meat production and provides a valuable model for many human diseases. Functional studies have demonstrated that microRNAs (miRNAs) play critical roles in almost all aspects of skeletal muscle development and disease pathogenesis. To investigate the miRNAs involved in regulating different periods of skeletal muscle development, we herein performed a comprehensive research for porcine microRNAome (miRNAome) during 10 skeletal muscle developmental stages including 35, 49, 63, 77, 91 dpc (days post coitum) and 2, 28, 90, 120, 180 dpn (days postnatal) using Solexa sequencing technology. Our results extend the repertoire of pig miRNAome to 247 known miRNAs processed from 210 pre-miRNAs and 297 candidate novel miRNAs through comparison with known miRNAs in the miRBase. Expression analysis of the 15 most abundant miRNAs in every library indicated that functional miRNAome may be smaller and tend to be highly expressed. A series of muscle-related miRNAs summarized in our study present different patterns between myofibers formation phase and muscle maturation phase, providing valuable reference for investigation of functional miRNAs during skeletal muscle development. Analysis of temporal profiles of miRNA expression identifies 18 novel candidate myogenic miRNAs in pig, which might provide new insight into regulation mechanism mediated by miRNAs underlying muscle development.
PMCID: PMC3770649  PMID: 24039761
15.  Selection of euploid blastocysts for cryopreservation with array comparative genomic hybridization (aCGH) results in increased implantation rates in subsequent frozen and thawed embryo transfer cycles 
In assisted reproductive treatments, embryos remaining after fresh embryo transfer are usually selected for cryopreservation based on traditional morphology assessment. Our previous report has demonstrated that array comparative genomic hybridization (aCGH) screening for IVF patients with good prognosis significantly improves clinical and ongoing pregnancy rates in fresh embryo transfer cycles. The current study further investigates the efficiency of applying aCGH in the selection of euploid embryos for cryopreservation as related to pregnancy and implantation outcomes in subsequent frozen embryo transfer (FET) cycles.
First-time IVF patients with good prognosis undergoing fresh single embryo transfer and having at least one remaining blastocyst for cryopreservation were prospectively randomized into two groups: 1) Group A patients had embryos assessed by morphology first and then by aCGH screening of trophectoderm cells and 2) Group B patients had embryos evaluated by morphology alone. All patients had at least one blastocyst available for cryopreservation after fresh embryo transfer. There were 15 patients in Group A and 23 patients in Group B who failed to conceive after fresh embryo transfer and completed the FET cycles. Blastocyst survival and implantation rates were compared between the two groups.
There were no significant differences in blastocyst survival rates between Group A and Group B (90.9% vs. 91.3%, respectively; p >0.05). However, a significantly higher implantation rate was observed in the morphology assessment plus aCGH screening group compared to the morphology assessment alone group (65.0% vs. 33.3%, respectively; p = 0.038). There was no miscarriage observed in Group A while a 16.7% miscarriage rate was recorded in Group B (0% vs. 16.7%, respectively; p >0.05).
While aCGH screening has been recently applied to select euploid blastocysts for fresh transfer in young, low-risk IVF patients, this is the first prospective study on the impact of aCGH specifically on blastocyst survival and implantation outcomes in the subsequent FET cycles of IVF patients with good prognosis. The present study demonstrates that aCGH screening of blastocysts prior to cryopreservation significantly improves implantation rates and may reduce the risk of miscarriage in subsequent FET cycles. Further randomized clinical studies with a larger sample size are needed to validate these preliminary findings.
PMCID: PMC3766007  PMID: 23937723
aCGH; Trophectoderm biopsy; Cryopreservation; Implantation
16.  Hematopoietic Lineage Cell-Specific Protein 1 Is Recruited to the Immunological Synapse by IL-2-Inducible T Cell Kinase and Regulates Phospholipase Cγ1 Microcluster Dynamics during T Cell Spreading1 
Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We showed previously that the actin-regulatory protein, hematopoietic lineage cell-specific protein 1 (HS1), is required for the stabilization of F-actin and Vav1 at the immunological synapse and for efficient calcium responses. The Tec family kinase IL-2-inducible T cell kinase (Itk) regulates similar aspects of T cell activation, suggesting that these proteins act in the same pathway. Using video microscopy, we show that T cells lacking Itk or HS1 exhibited similar defects in actin responses, extending unstable lamellipodial protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk’s Src homology 2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, or in T cells overexpressing an Itk Src homology 2 domain mutant, HS1 failed to localize to the immunological synapse, indicating that Itk serves to recruit HS1 to sites of TCR engagement. Because Itk is required for phospholipase C (PLC)γ1 phosphorylation and calcium store release, we examined the calcium signaling pathway in HS1−/− T cells in greater detail. In response to TCR engagement, T cells lacking HS1 exhibited diminished calcium store release, but TCR-dependent PLCγ1 phosphorylation was intact, indicating that HS1’s role in calcium signaling is distinct from that of Itk. HS1-deficient T cells exhibited defective cytoskeletal association of PLCγ1 and altered formation of PLCγ1 microclusters. We conclude that HS1 functions as an effector of Itk in the T cell actin-regulatory pathway, and directs the spatial organization of PLCγ1 signaling complexes.
PMCID: PMC3715746  PMID: 19917685
17.  Involvement of MoVMA11, a Putative Vacuolar ATPase c’ Subunit, in Vacuolar Acidification and Infection-Related Morphogenesis of Magnaporthe oryzae 
PLoS ONE  2013;8(6):e67804.
Many functions of vacuole depend on the activity of vacuolar ATPase which is essential to maintain an acidic lumen and create the driving forces for massive fluxes of ions and metabolites through vacuolar membrane. In filamentous fungus Magnaportheoryzae, subcellular colocalization and quinacrine staining suggested that the V1V0 domains of V-ATPase were fully assembled and the vacuoles were kept acidic during infection-related developments. Targeted gene disruption of MoVMA11 gene, encoding the putative c’ subunit of V-ATPase, impaired vacuolar acidification and mimicked the phenotypes of yeast V-ATPase mutants in the poor colony morphology, abolished asexual and sexual reproductions, selective carbon source utilization, and increased calcium and heavy metals sensitivities, however, not in the typical pH conditional lethality. Strikingly, aerial hyphae of the MoVMA11 null mutant intertwined with each other to form extremely thick filamentous structures. The results also implicated that MoVMA11 was involved in cell wall integrity and appressorium formation. Abundant non-melanized swollen structures and rare, small appressoria without penetration ability were produced at the hyphal tips of the ΔMovma11 mutant on onion epidermal cells. Finally, the MoVMA11 null mutant lost pathogenicity on both intact and wounded host leaves. Overall, our data indicated that MoVMA11, like other fungal VMA genes, is associated with numerous cellular functions and highlighted that V-ATPase is essential for infection-related morphogenesis and pathogenesis in M. oryzae.
PMCID: PMC3694887  PMID: 23826342
18.  Significant increase of oxidase activity through the genetic incorporation of a tyrosine-histidine cross-link in a myoglobin model of heme-copper oxidase 
Heme-copper oxidase (HCO) performs efficient four-electron reduction of oxygen to water without releasing toxic, reactive oxygen species (ROS). Essential for this function is a post-translationally modified histidine–tyrosine cross-link (Tyr-His) in its heme a3/CuB oxygen reduction center. Through the genetic incorporation of the Tyr-His ligand and CuB site into myoglobin, we recapitulated important features of HCO into this small soluble protein, which exhibits selective O2 reduction activity while generating less than 6% ROS, at more than 1000 turnovers. These results support that Tyr-His crosslink is indeed important for HCO function, and creates the exciting opportunity to rapidly evolve better HCO model proteins to achieve higher activity and selectivity, which may be suitable as alternatives to precious metal catalyst in fuel cells.
PMCID: PMC3511862  PMID: 22411709
post-translational modification; tyrosine-histidine crosslink; protein design; heme copper oxidase; oxygen reduction
19.  Large-Scale Functional Purification of Recombinant HIV-1 Capsid 
PLoS ONE  2013;8(3):e58035.
During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents.
PMCID: PMC3589475  PMID: 23472130
20.  Changes in compressed neurons from dogs with acute and severe cauda equina constrictions following intrathecal injection of brain-derived neurotrophic factor-conjugated polymer nanoparticles☆ 
Neural Regeneration Research  2013;8(3):233-243.
This study established a dog model of acute multiple cauda equina constriction by experimental constriction injury (48 hours) of the lumbosacral central processes in dorsal root ganglia neurons. The repair effect of intrathecal injection of brain-derived neurotrophic factor with 15 mg encapsulated biodegradable poly(lactide-co-glycolide) nanoparticles on this injury was then analyzed. Dorsal root ganglion cells (L7) of all experimental dogs were analyzed using hematoxylin-eosin staining and immunohistochemistry at 1, 2 and 4 weeks following model induction. Intrathecal injection of brain-derived neurotrophic factor can relieve degeneration and inflammation, and elevate the expression of brain-derived neurotrophic factor in sensory neurons of compressed dorsal root ganglion. Simultaneously, intrathecal injection of brain-derived neurotrophic factor obviously improved neurological function in the dog model of acute multiple cauda equina constriction. Results verified that sustained intraspinal delivery of brain-derived neurotrophic factor encapsulated in biodegradable nanoparticles promoted the repair of histomorphology and function of neurons within the dorsal root ganglia in dogs with acute and severe cauda equina syndrome.
PMCID: PMC4107517  PMID: 25206593
neural regeneration; peripheral nerve injury; cauda equina syndrome; dorsal root ganglion; brain-derived neurotrophic factor; multiple cauda equina constrictions; neurotrophic factors; neural protection; grants-supported paper; photographs-containing paper; neuroregeneration
21.  Differential Expression of Wnt Pathway Genes in Sporadic Hepatocellular Carcinomas Infected With Hepatitis B Virus Identified With OligoGE Arrays 
Hepatitis Monthly  2013;13(1):e6192.
Epidemiological evidence has clearly indicated that chronic infection with the hepatitis B virus (HBV) is the major risk factor for developing hepatocellular carcinoma (HCC). Nonetheless, the mechanisms by which HBV contributes to the pathogenesis of HCC have not been fully elucidated.
Our aim was to characterize differential gene expression profiles related to the Wnt signaling pathway between primary tumor and adjacent normal tissues in HCC patients with concomitant HBVinfection .
Materials and Methods
An oligoGEArray® (an oligonucleotide-based gene expression array platform) containing 126 Wnt signaling pathway-related genes was used to compare gene expressions between primary HCC and adjacent non-tumorous liver tissues from 10 patients with HCC. Selected differential genes were identified with real-time RT-PCR and immunohistochemistry (IHC). In particular, the protein of the differential gene DVL3 (disheveled, dsh homolog 3 [Drosophila]) was chosen to investigate whether it is up regulated in primary tumor correlated with the clinic pathological characteristics of HCC patients. For this purpose we examined 56 HCC tissue samples via IHC for the presence of DVL3 protein.
Sixteen genes were identified with significant differential expression between HCC and adjacent non-tumorous liver tissue. These genes have been previously associated with the Frizzled signaling pathway, cell cycle, transcription, or protein degradation. All (100%) of the tumor samples results from 56 HCC patients tested were positive for DVL3 via IHC. Based on the intensity of DVL3 immunoreactivity, 25 (44.6%) and 31 (55.4%) of the patients were classified aslow and high-DVL3, respectively, which correlated with tumor stage (P = 0.029).
This study clarified a number of Wnt pathway-related genes which are dysregulated in HBV-associated HCC. These genes may be contributedto the frequent activation of the Wnt signaling pathway. Our results promote the role of the Wnt signaling pathway in HBV-associated HCC.
PMCID: PMC3589879  PMID: 23483081
Carcinoma, Hepatocellular; Hepatitis B Virus; Oligonucleotide Array Sequence Analysis; Gene Expression Profiling; Disheveled Proteins
22.  MicroRNAome Comparison between Intramuscular and Subcutaneous Vascular Stem Cell Adipogenesis 
PLoS ONE  2012;7(9):e45410.
As an important factor affecting meat quality, intramuscular fat (IMF) content is a topic of worldwide concern. Emerging evidences indicate that microRNAs play important roles in adipocyte differentiation. However, miRNAome has neither been studied during porcine intramuscular preadipocyte differentiation, nor compared with subcutaneous preadipocytes. The objectives of this study were to identify porcine miRNAs involved in adipogenesis in primary preadipocytes, and to determine whether intramuscular and subcutaneous adipocytes differ in the expression and regulation of miRNAs.
miRNAomes in primary intramuscular and subcutaneous adipocytes during differentiation were first sequenced using the Solexa deep sequencing method. The sequences and relative expression levels of 224 known (98.2% in miRbase 18.0) and 280 potential porcine miRNAs were identified. Fifty-four of them changed in similar pattern between intramuscular vascular stem cells (IVSC) and subcutaneous vascular stem cells (SVSC) differentiation, such as miR-210, miR-10b and miR-99a. Expression levels of 10 miRNAs were reversely up-or down-regulated between IVSC and SVSC differentiation, 19 were up-or down-regulated only during IVSC differentiation and 55 only during SVSC differentiation. Additionally, 30 miRNAs showed fat-depot specific expression pattern (24 in cells of intramuscular origin and 6 in cells of subcutaneous origin). These adipogenesis-related miRNAs mainly functioned by targeting similar pathways in adipogenesis, obesity and syndrome.
Comparison of miRNAomes in IVSC and SVSC during differentiation revealed that many different miRNAs are involved in adipogenesis, and they regulate SVSC and IVSC differentiation through similar pathways. These miRNAs may serve as biomarkers or targets for enhancing IMF content, and uncovering their function in IMF development will be of great value in the near future.
PMCID: PMC3447870  PMID: 23028990
23.  The expression of KIT receptor dimers in gastrointestinal stromal tumors independent of c-kit mutation and SCF expression is associated with high-risk stratification 
Oncology Letters  2012;4(4):805-811.
Although the dimerization of KIT, a receptor tyrosine kinase, plays a major role in a number of tumors, correlations between the clinicopathological parameters and KIT receptor dimers have not been identified. In the current study, a method for the detection of KIT receptor dimer expression was described and correlations between the clinicopathological parameters and KIT receptor dimers were analyzed. A single center cohort study of 49 patients with gastrointestinal stromal tumors (GISTs) was conducted to analyze the expression of KIT receptor dimers by SDS-PAGE, Native-PAGE and modified Native-PAGE. Immunohistochemistry was used to examine the expression of ki-67, c-kit and stem cell factor (SCF). Mutations of the c-kit gene were examined in 48 GISTs according to the polymerase chain reaction (PCR) and direct sequencing methods. Based on the data, a signal for the KIT receptor monomer was obtained by SDS-PAGE. Faint bands were observed on the nitrocellulose membrane by Native-PAGE, while clear bands were identified for KIT receptor dimers and monomers using modified Native-PAGE (15 out of 49 cases). The tumor size was larger in KIT receptor dimer-positive cases compared with that in KIT receptor dimer-negative cases. Analysis of KIT receptor dimer expression levels and risk stratification demonstrated that KIT receptor dimer-positive cases belonged to the higher risk classification. In addition, there was no significant correlation between the existence of KIT receptor dimers and c-kit gene mutations, including SCF expression. In conclusion, this study established a method for the detection of the existence of KIT receptor dimers in tissues and confirmed that KIT receptor dimers were correlated with risk stratification. Data also indicated that ligand-dependent SCF/KIT dimerization is an independent crucial mechanism in GIST cell proliferation and increases the risk of GIST. Therefore, blocking KIT dimerization may prove to be an effective approach for the treatment of GISTs.
PMCID: PMC3506609  PMID: 23205104
gastrointestinal stromal tumors; dimerization; KIT-dimer
24.  The HCV Non-Nucleoside Inhibitor Tegobuvir Utilizes a Novel Mechanism of Action to Inhibit NS5B Polymerase Function 
PLoS ONE  2012;7(6):e39163.
Tegobuvir (TGV) is a novel non-nucleoside inhibitor (NNI) of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymatic activity in biochemical assays in vitro, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chemical activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further analysis reveals that the aberrantly migrating NS5B species contains the inhibitor molecule. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore analysis of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP- mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI) of the viral polymerase.
PMCID: PMC3374789  PMID: 22720059
25.  Array comparative genomic hybridization screening in IVF significantly reduces number of embryos available for cryopreservation 
During IVF, non-transferred embryos are usually selected for cryopreservation on the basis of morphological criteria. This investigation evaluated an application for array comparative genomic hybridization (aCGH) in assessment of surplus embryos prior to cryopreservation.
First-time IVF patients undergoing elective single embryo transfer and having at least one extra non-transferred embryo suitable for cryopreservation were offered enrollment in the study. Patients were randomized into two groups: Patients in group A (n=55) had embryos assessed first by morphology and then by aCGH, performed on cells obtained from trophectoderm biopsy on post-fertilization day 5. Only euploid embryos were designated for cryopreservation. Patients in group B (n=48) had embryos assessed by morphology alone, with only good morphology embryos considered suitable for cryopreservation.
Among biopsied embryos in group A (n=425), euploidy was confirmed in 226 (53.1%). After fresh single embryo transfer, 64 (28.3%) surplus euploid embryos were cryopreserved for 51 patients (92.7%). In group B, 389 good morphology blastocysts were identified and a single top quality blastocyst was selected for fresh transfer. All group B patients (48/48) had at least one blastocyst remaining for cryopreservation. A total of 157 (40.4%) blastocysts were frozen in this group, a significantly larger proportion than was cryopreserved in group A (p=0.017, by chi-squared analysis).
While aCGH and subsequent frozen embryo transfer are currently used to screen embryos, this is the first investigation to quantify the impact of aCGH specifically on embryo cryopreservation. Incorporation of aCGH screening significantly reduced the total number of cryopreserved blastocysts compared to when suitability for freezing was determined by morphology only. IVF patients should be counseled that the benefits of aCGH screening will likely come at the cost of sharply limiting the number of surplus embryos available for cryopreservation.
PMCID: PMC3398117  PMID: 22816070
Fertilization in vitro; Comparative genomic hybridization; Preimplantation genetic diagnosis; Cryopreservation

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