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1.  High Resolution Melting Analysis: A Rapid Screening and Typing Tool for Common β-Thalassemia Mutation in Chinese Population 
PLoS ONE  2014;9(8):e102243.
β-thalassemia is a common inherited disorder worldwide including southern China, and at least 45 distinct β-thalassemia mutations have been identified in China. High-resolution melting (HRM) assay was recently introduced as a rapid, inexpensive and effective method for genotyping. However, there was no systemic study on the diagnostic capability of HRM to identify β-thalassemia. Here, we used an improved HRM method to screen and type 12 common β-thalassemia mutations in Chinese, and the rapidity and reliability of this method was investigated. The whole PCR and HRM procedure could be completed in 40 min. The heterozygous mutations and 4 kinds of homozygous mutations could be readily differentiated from the melting curve except c.-78A>G heterozygote and c.-79A>G heterozygote. The diagnostic reliability of this HRM assay was evaluated on 756 pre-typed genomic DNA samples and 50 cases of blood spots on filter paper, which were collected from seven high prevalent provinces in southern China. If c.-78A>G heterozygote and c.-79A>G heterozygote were classified into the same group (c.-78&79 A>G heterozygote), the HRM method was in complete concordance with the reference method (reverse dot blot/DNA-sequencing). In a conclusion, the HRM method appears to be an accurate and sensitive method for the rapid screening and identification of β-thalassemia mutations. In the future, we suggest this technology to be used in neonatal blood spot screening program. It could enlarge the coverage of β-thalassemia screening program in China. At the same time, its value should be confirmed in prospectively clinical and epidemiological studies.
doi:10.1371/journal.pone.0102243
PMCID: PMC4121066  PMID: 25089872
2.  Molecular Epidemiological Characterization and Health Burden of Thalassemia in Jiangxi Province, P. R. China 
PLoS ONE  2014;9(7):e101505.
Background
Thalassemia is the most common inherited disease in southern China. However, this disorder is usually ignored by Jiangxi provincial health system and government due to lack of epidemiological data.
Materials and Methods
A total of 9489 samples from Hakka Han and Gan-speaking Han in three geographical areas of Jiangxi Province were analyzed for both complete blood cell (CBC) count and reverse dot blot (RDB) gene chip for thalassemia.
Results
1182 cases of suspected thalassemia carriers with microcytosis (MCV<82 fL) were found by CBC count, and were tested by RDB gene chip to reveal a total of 594 mutant chromosomes, including 433 α-thalassemia mutant chromosomes and 172 β-thalassemia mutant chromosomes. Our results indicated a higher prevalence of thalassemia with the heterozygote frequency of 9.49% in southern Jiangxi province, whereas the low frequency was found in middle (3.90%) and northern Jiangxi (2.63%).
Conclusions
Based on the epidemiological data, the estimated numbers of pregnancies in Jiangxi province in which the fetus is at risk for β-thalassemia major or intermedia, Bart's hydrops fetalis and Hb H disease are 34 (95% CI, 16 to 58), 79 (95% CI, 50 to 114) and 39 (95% CI, 27 to 58) per year, respectively. We suggested that prevention network of thalassemia should be established, especially in high prevalent southern Jiangxi (Hakka Han), including establishment of thalassemia database collection, hematological analysis laboratories, genetic counselling clinics, prenatal diagnosis centers and neonatal screening centers.
doi:10.1371/journal.pone.0101505
PMCID: PMC4084819  PMID: 25000193
3.  Hemoglobinopathy: Molecular Epidemiological Characteristics and Health Effects on Hakka People in the Meizhou Region, Southern China 
PLoS ONE  2013;8(2):e55024.
Background
Hemoglobinopathies are the most common inherited diseases in southern China. However, there have been only a few epidemiological studies of hemoglobinopathies in Guangdong province.
Materials and Methods
Peripheral blood samples were collected from 15299 “healthy” unrelated subjects of dominantly ethnic Hakka in the Meizhou region, on which hemoglobin electrophoresis and routine blood tests were performed. Suspected cases with hemoglobin variants and hereditary persistence of fetal hemoglobin (HPFH) were further characterized by PCR, DNA sequencing, reverse dot blot (RDB) or multiplex ligation-dependent probe amplification (MLPA). In addition, 1743 samples were randomly selected from the 15299 subjects for thalassemia screening, and suspected thalassemia carriers were identified by PCR and RDB.
Results
The gene frequency of hemoglobin variants was 0.477% (73/15299). The five main subgroups of the ten hemoglobin variants were Hb E, Hb G-Chinese, Hb Q-Tahiland, Hb New York and Hb J-Bangkok. 277 cases (15.89%, 277/1743) of suspected thalassemia carriers with microcytosis (MCV<82 fl) were found by thalassemia screening, and were tested by a RDB gene chip to reveal a total of 196 mutant chromosomes: including 124 α-thalassemia mutant chromosomes and 72 β-thalassemia mutant chromosomes. These results give a heterozygote frequency of 11.24% for common α and β thalassemia in the Hakka population in the Meizhou region. 3 cases of HPFH/δβ-thalassemia were found, including 2 cases of Vietnamese HPFH (FPFH-7) and a rare Belgian Gγ(Aγδβ)0–thalassemia identified in Chinese.
Conclusions
Our results provide a detailed prevalence and molecular characterization of hemoglobinopathies in Hakka people of the Meizhou region. The estimated numbers of pregnancies each year in the Meizhou region, in which the fetus would be at risk for β thalassemia major or intermedia, Bart’s hydrops fetalis, and Hb H disease, are 25 (95% CI, 15 to 38), 40 (95% CI, 26 to 57), and 15 (95% CI, 8 to 23), respectively.
doi:10.1371/journal.pone.0055024
PMCID: PMC3562339  PMID: 23383304
4.  Application of air insufflation to prevent clinical pancreatic fistula after pancreaticoduodenectomy 
AIM: To introduce an air insufflation procedure and to investigate the effectiveness of air insufflation in preventing pancreatic fistula (PF).
METHODS: From March 2010 to August 2013, a total of 185 patients underwent pancreaticoduodenectomy (PD) at our institution, and 74 patients were not involved in this study for various reasons. The clinical outcomes of 111 patients were retrospectively analyzed. The air insufflation test was performed in 46 patients to investigate the efficacy of the pancreaticojejunal anastomosis during surgery, and 65 patients who did not receive the air insufflation test served as controls. Preoperative assessments and intraoperative outcomes were compared between the 2 groups. Univariate and multivariate analyses were performed to identify the risk factors for PF.
RESULTS: The two patient groups had similar baseline demographics, preoperative assessments, operative factors, pancreatic factors and pathological results. The overall mortality, morbidity, and PF rates were 1.8%, 48.6%, and 26.1%, respectively. No significant differences were observed in either morbidity or mortality between the two groups. The rate of clinical PF (grade B and grade C PF) was significantly lower in the air insufflation test group, compared with the non-air insufflation test group (6.5% vs 23.1%, P = 0.02). Univariate analysis identified the following parameters as risk factors related to clinical PF: estimated blood loss; pancreatic duct diameter ≤ 3 mm; invagination anastomosis technique; and not undergoing air insufflation test. By further analyzing these variables with multivariate logistic regression, estimated blood loss, pancreatic duct diameter ≤ 3 mm and not undergoing air insufflation test were demonstrated to be independent risk factors.
CONCLUSION: Performing an air insufflation test could significantly reduce the occurrence of clinical PF after PD. Not performing an air insufflation test was an independent risk factor for clinical PF.
doi:10.3748/wjg.v21.i6.1872
PMCID: PMC4323465
Pancreatic fistula; Pancreaticoduodenectomy; Air insufflation test; Surgery; Morbidity
5.  Mechanisms Involved in the Association between Periodontitis and Complications in Pregnancy 
The association between periodontitis and some of the problems with pregnancy such as premature delivery, low weight at birth, and preeclampsia (PE) has been suggested. Nevertheless, epidemiological data have shown contradictory data, mainly due to differences in clinical parameters of periodontitis assessment. Furthermore, differences in microbial composition and immune response between aggressive and chronic periodontitis are not addressed by these epidemiological studies. We aimed to review the current data on the association between some of these problems with pregnancy and periodontitis, and the mechanisms underlying this association. Shifts in the microbial composition of the subgingival biofilm may occur during pregnancy, leading to a potentially more hazardous microbial community. Pregnancy is characterized by physiological immune tolerance. However, the infection leads to a shift in maternal immune response to a pathogenic pro-inflammatory response, with production of inflammatory cytokines and toxic products. In women with periodontitis, the infected periodontal tissues may act as reservoirs of bacteria and their products that can disseminate to the fetus-placenta unit. In severe periodontitis patients, the infection agents and their products are able to activate inflammatory signaling pathways locally and in extra-oral sites, including the placenta-fetal unit, which may not only induce preterm labor but also lead to PE and restrict intrauterine growth. Despite these evidences, the effectiveness of periodontal treatment in preventing gestational complications was still not established since it may be influenced by several factors such as severity of disease, composition of microbial community, treatment strategy, and period of treatment throughout pregnancy. This lack of scientific evidence does not exclude the need to control infection and inflammation in periodontitis patients during pregnancy, and treatment protocols should be validated.
doi:10.3389/fpubh.2014.00290
PMCID: PMC4310218
periodontal diseases; pregnancy; preterm birth; inflammation
6.  A Pathway-Centric Survey of Somatic Mutations in Chinese Patients with Colorectal Carcinomas 
PLoS ONE  2015;10(1):e0116753.
Previous genetic studies on colorectal carcinomas (CRC) have identified multiple somatic mutations in four candidate pathways (TGF-β, Wnt, P53 and RTK-RAS pathways) on populations of European ancestry. However, it is under-studied whether other populations harbor different sets of hot-spot somatic mutations in these pathways and other oncogenes. In this study, to evaluate the mutational spectrum of novel somatic mutations, we assessed 41 pairs of tumor-stroma tissues from Chinese patients with CRC, including 29 colon carcinomas and 12 rectal carcinomas. We designed Illumina Custom Amplicon panel to target 43 genes, including genes in the four candidate pathways, as well as several known oncogenes for other cancers. Candidate mutations were validated by Sanger sequencing, and we further used SIFT and PolyPhen-2 to assess potentially functional mutations. We discovered 3 new somatic mutations in gene APC, TCF7L2, and PIK3CA that had never been reported in the COSMIC or NCI-60 databases. Additionally, we confirmed 6 known somatic mutations in gene SMAD4, APC, FBXW7, BRAF and PTEN in Chinese CRC patients. While most were previously reported in CRC, one mutation in PTEN was reported only in malignant endometrium cancer. Our study confirmed the existence of known somatic mutations in the four candidate pathways for CRC in Chinese patients. We also discovered a number of novel somatic mutations in these pathways, which may have implications for the pathogenesis of CRC.
doi:10.1371/journal.pone.0116753
PMCID: PMC4305320  PMID: 25617745
7.  Novel recombinant chimeric virus-like particle is immunogenic and protective against both enterovirus 71 and coxsackievirus A16 in mice 
Scientific Reports  2015;5:7878.
Hand-foot-and-mouth disease (HFMD) has been recognized as an important global public health issue, which is predominantly caused by enterovirus 71 (EV-A71) and coxsackievirus A16 (CVA16). There is no available vaccine against HFMD. An ideal HFMD vaccine should be bivalent against both EV-A71 and CVA16. Here, a novel strategy to produce bivalent HFMD vaccine based on chimeric EV-A71 virus-like particles (ChiEV-A71 VLPs) was proposed and illustrated. The neutralizing epitope SP70 within the capsid protein VP1 of EV-A71 was replaced with that of CVA16 in ChiEV-A71 VLPs. Structural modeling revealed that the replaced CVA16-SP70 epitope is well exposed on the surface of ChiEV-A71 VLPs. These VLPs produced in Saccharomyces cerevisiae exhibited similarity in both protein composition and morphology as naive EV-A71 VLPs. Immunization with ChiEV-A71 VLPs in mice elicited robust Th1/Th2 dependent immune responses against EV-A71 and CVA16. Furthermore, passive immunization with anti-ChiEV-A71 VLPs sera conferred full protection against lethal challenge of both EV-A71 and CVA16 infection in neonatal mice. These results suggested that this chimeric vaccine, ChiEV-A71 might have the potential to be further developed as a bivalent HFMD vaccine in the near future. Such chimeric enterovirus VLPs provide an alternative platform for bivalent HFMD vaccine development.
doi:10.1038/srep07878
PMCID: PMC4297979  PMID: 25597595
8.  Clinical Outcomes with Alternative Dosing Strategies for Piperacillin/Tazobactam: A Systematic Review and Meta-Analysis 
PLoS ONE  2015;10(1):e0116769.
Objectives
A better dosing strategy can improve clinical outcomes for patients. We sought to compare the extended or continuous infusion with conventional intermittent infusion of piperacillin/tazobactam, investigating which approach is better and worthy of recommendation for clinical use.
Methods
Articles were gathered from PubMed, Web of Science, ProQuest, Science Direct, Cochrane, two Chinese literature databases (CNKI, Wan Fang Data) and related ICAAC and ACCP conferences. Randomized controlled and observational studies that compared extended or continuous infusion with conventional intermittent infusion of piperacillin/tazobactam were identified from the databases above and analyzed. Two reviewers independently extracted and investigated the data. A meta-analysis was performed using Revman 5.2 software. The quality of each study was assessed. Sensitivity analysis and publication bias were evaluated.
Results
Five randomized controlled trials and nine observational studies were included in this study. All included studies had high quality and no publication bias was found. Compared to the conventional intermittent infusion approach, the extended or continuous infusion group had a significantly higher clinical cure rate (OR 1.88, 95% CI 1.29-2.73, P = 0.0009) and a lower mortality rate (OR 0.67, 95% CI 0.50-0.89, P = 0.005). No statistical difference was observed for bacteriologic cure (OR 1.40, 95% CI 0.82-2.37, P = 0.22) between the two dosing regimens. The sensitivity analysis showed the results were stable.
Conclusions
Our systematic review and meta-analysis suggested that the extended or continuous infusion strategy of piperacillin/tazobactam should be recommended for clinical use considering its higher clinical cure rate and lower mortality rate in comparison with conventional intermittent strategy. Data from this study could be extrapolated for other β-lactam antimicrobials. Therefore, this dosing strategy could be considered in clinical practice.
doi:10.1371/journal.pone.0116769
PMCID: PMC4289069  PMID: 25575030
9.  Third Party Umbilical Cord Blood-Derived Regulatory T cells Prevent Xenogenic Graft-versus-Host Disease 
Cytotherapy  2013;16(1):90-100.
Naturally occurring regulatory T cells (Treg) are emerging as a promising approach for prevention of graft-versus-host Disease (GvHD), which remains an obstacle to the successful outcome of allogeneic hematopoietic stem cell transplantation. However, Treg only constitute 1-5% of total nucleated cells in cord blood (CB) (<3×106 cells) and therefore novel methods of Treg expansion to generate clinically-relevant numbers are needed. Several methodologies are currently being utilized for ex vivo Treg expansion. Here, we report a new approach to expand Treg from CB and demonstrate their efficacy in vitro by blunting allogeneic mixed lymphocyte reactions and in vivo by preventing GvHD using a xenogenic GvHD mouse model. Using magnetic cell sorting, naturally-occurring Treg were isolated from CB by the positive selection of CD25+ cells. These were expanded to clinically-relevant numbers using CD3/28 co-expressing Dynabeads and interleukin (IL)-2. Ex vivo-expanded Treg were CD4+25+FOXP3+127lo and expressed a polyclonal T-cell receptor Vβ repertoire. When compared to conventional T-lymphocytes (CD4+25- cells), Treg consistently showed demethylation of the FOXP3 TSDR promoter region and suppression of allogeneic proliferation responses in vitro. In our NOD-SCID IL-2Rγnull (NSG) xenogeneic model of GvHD, prophylactic injection of 3rd party CB-derived, ex vivo-expanded Treg led to the prevention of GvHD that translated into improved GvHD score, decreased circulating inflammatory cytokines and significantly superior overall survival. This model of xenogenic GvHD can be used to study the mechanism of action of CB Treg as well as other therapeutic interventions.
doi:10.1016/j.jcyt.2013.07.009
PMCID: PMC4124936  PMID: 24480547
10.  Outcomes of Secondary Laminoplasty for Patients with Unsatisfactory Results after Anterior Multilevel Cervical Surgery 
Objective
To investigate the causes for failed anterior cervical surgery and the outcomes of secondary laminoplasty.
Methods
Seventeen patients failed anterior multilevel cervical surgery and the following conservative treatments between Feb 2003 and May 2011 underwent secondary laminoplasty. Outcomes were evaluated by the Japanese Orthopaedic Association (JOA) Scale and visual analogue scale (VAS) before the secondary surgery, at 1 week, 2 months, 6 months, and the final visit. Cervical alignment, causes for revision and complications were also assessed.
Results
With a mean follow-up of 29.7±12.1 months, JOA score, recovery rate and excellent to good rate improved significantly at 2 months (p<0.05) and maintained thereafter (p>0.05). Mean VAS score decreased postoperatively (p<0.05). Lordotic angle maintained during the entire follow up (p>0.05). The causes for secondary surgery were inappropriate approach in 3 patients, insufficient decompression in 4 patients, adjacent degeneration in 2 patients, and disease progression in 8 patients. Complications included one case of C5 palsy, axial pain and cerebrospinal fluid leakage, respectively.
Conclusion
Laminoplasty has satisfactory results in failed multilevel anterior surgery, with a low incidence of complications.
doi:10.3340/jkns.2015.57.1.36
PMCID: PMC4323503
Cervical spondylosis; Anterior discectomy and fusion; Anterior corpectomy and fusion; Laminoplasty
11.  Expression of PD-L1, PD-L2, PD-1 and CTLA4 in myelodysplastic syndromes is enhanced by treatment with hypomethylating agents 
Leukemia  2013;28(6):1280-1288.
Blockade of immune checkpoints is emerging as new form of anticancer therapy. We studied the expression of PD-L1, PD-L2, PD-1 and CTLA4 mRNA expression in CD34+ cells from MDS, CMML and AML patients (N=124). Aberrant up-regulation (≥2 fold) was observed in 34%, 14%, 15% and 8% of the patients respectively. Increased expression of these 4 genes was also observed in PBMNC (N=61). The relative expression of PD-L1 from PBMNC was significantly higher in MDS (p=0.018) and CMML (p=0.0128) compared to AML. By immunohistochemical (IHC) analysis, PD-L1 protein expression was observed in MDS CD34+ cells, whereas stroma/non-blast cellular compartment was positive for PD-1. In a cohort of patients treated with epigenetic therapy, PD-L1, PD-L2, PD-1 and CTLA4 expression was upregulated. Patients resistant to therapy had relative higher increments in gene expression compared to patients that achieved response. Treatment of leukemia cells with decitabine resulted in a dose dependent up-regulation of above genes. Exposure to decitabine resulted in partial demethylation of PD-1 in leukemia cell lines and human samples. This study suggests PD-1 signaling may be involved in MDS pathogenesis and resistance mechanisms to HMAs. Blockade of this pathway can be a potential therapy in MDS and AML.
doi:10.1038/leu.2013.355
PMCID: PMC4032802  PMID: 24270737
programmed death-1; myelodysplastic syndromes; DNA methylation
12.  Rapid identification of apolipoprotein E genotypes by high-resolution melting analysis in Chinese Han and African Fang populations 
Apolipoprotein E (APOE) gene polymorphism can affect APOE gene transcription, serum lipid levels and repair of tissue damage, which could place individuals at serious risk of cardiovascular disease or certain infectious diseases. Recently, high-resolution melting (HRM) analysis was reported to be a simple, inexpensive, accurate and sensitive method for the genotyping or/and scanning of rare mutations. For this reason, an HRM analysis was used in the present study for APOE genotyping in the Southern Chinese Han and African Fang populations. A total of 100 healthy Southern Chinese Han and 175 healthy African Fang individuals attended the study. Polymerase chain reaction-DNA sequencing was used as a reference method for the genotyping of these samples. The six APOE genotypes could all be rapidly and efficiently identified by HRM analysis, and 100% concordance was found between the HRM analysis and the reference method. The allele frequencies of APOE in the Southern Chinese Han population were 7.0, 87.5 and 5.5% for ɛ2, ɛ3 and ɛ4, respectively. In the African Fang population, the allele frequencies of APOE were 24.3, 65.7 and 10.0% for ɛ2, ɛ3 and ɛ4, respectively. A statistically significant difference was found between the allele frequencies between the populations (P<0.05). In conclusion, the present study revealed the molecular characterization of APOE gene polymorphism in the Han population from the Chaozhou region of Southern China and the Fang population from Equatorial Guinea. The findings of the study indicated that HRM analysis could be used as an accurate and sensitive method for the rapid screening and identification of APOE genotypes in prospective clinical and population genetic analyses.
doi:10.3892/etm.2014.2097
PMCID: PMC4280925  PMID: 25574218
apolipoprotein E; genotype; high-resolution melting; Chinese Han; African Fang
13.  All levels versus alternate levels plate fixation in expansive open door cervical laminoplasty 
Indian Journal of Orthopaedics  2014;48(6):582-586.
Background:
Expansive open door laminoplasty with the use of titanium miniplate is becoming popular. Usually, the plate is applied at each level to prevent re-closure of the opened lamina. However, it is also used at alternating levels (i.e., C3, C5 and C7) in clinical settings in order to reduce the cost. Whether they have any difference in clinical efficacy? There is a lack of comparative data between the two kinds of plate fixation in the literature.
Materials and Methods:
83 patients who underwent cervical laminoplasty with alternating levels plate fixation (51 patients in Group A) or all levels plate fixation (32 patients in Group B) between January 2008 and October 2012 were evaluated in our institute retrospectively. Clinical and radiologic outcomes were assessed.
Results:
No statistical difference was found in the mean operation time, blood loss, incidence of significant axial symptoms and C5 palsy, preoperative anteroposterior diameter (APD) and preoperative Japanese Orthopedic Association score between the two groups. However, Group B showed a higher rate of neurologic recovery after surgery. Postoperative increased APD and open angle in Group B were significantly larger than Group A. The mean cost for Group B (12801 ± 460.6 USD) was higher than Group A (8906 ± 566.7 USD).
Conclusions:
Despite the higher cost of all level fixation, it is more effective in maintaining the expansion of the spinal canal and can obtain better clinical improvement compared to alternating levels fixation.
doi:10.4103/0019-5413.144225
PMCID: PMC4232827  PMID: 25404770
Cervical myelopathy; laminoplasty; open door; Spinal cord compression; compressive; myelopathy; cervical vertebrae
14.  Methyllycaconitine Alleviates Amyloid-β Peptides-Induced Cytotoxicity in SH-SY5Y Cells 
PLoS ONE  2014;9(10):e111536.
Alzheimer's disease (AD) is a chronic progressive neurodegenerative disorder. As the most common form of dementia, it affects more than 35 million people worldwide and is increasing. Excessive extracellular deposition of amyloid-β peptide (Aβ) is a pathologic feature of AD. Accumulating evidence indicates that macroautophagy is involved in the pathogenesis of AD, but its exact role is still unclear. Although major findings on the molecular mechanisms have been reported, there are still no effective treatments to prevent, halt, or reverse Alzheimer's disease. In this study, we investigated whether Aβ25–35 could trigger an autophagy process and inhibit the growth of SH-SY5Y cells. Furthermore, we examined the effect of methyllycaconitine (MLA) on the cytotoxity of Aβ25–35. MLA had a protective effect against cytotoxity of Aβ, which may be related to its inhibition of Aβ-induced autophagy and the involvement of the mammalian target of rapamycin pathway. Moreover, MLA had a good safety profile. MLA treatment may be a promising therapeutic tool for AD.
doi:10.1371/journal.pone.0111536
PMCID: PMC4216102  PMID: 25360664
15.  Retroviral manipulation of the expression of bone morphogenetic protein receptor Ia by SVZa progenitor cells leads to changes in their p19INK4d expression but not in their neuronal commitment 
Bone morphogenetic proteins (BMPs), a group of cytokines in the TGF-β superfamily, have complex regulatory roles in the control of neural proliferation and cell fate decision. In this study, we analyzed the potential role(s) of BMP signaling on the regulation of the proliferation and differentiation of the unique progenitor cells of the neonatal anterior subventricular zone (SVZa). Unlike other progenitor cells of the brain, SVZa progenitor cells have the capacity to divide even though they express a neuronal phenotype. In order to augment or inhibit endogenous BMP signaling, we injected into the neonatal rat SVZa replication-deficient retroviruses encoding for either the wild-type BMP receptor subtype Ia (wt-BMPR-Ia) or a mutated dominant-negative version of BMPR-Ia (dn-BMPR-Ia) in conjunction with a reporter gene, human alkaline phosphatase (AP) and perfused the pups 1, 4 and 7 days post injection. We analyzed whether changing the expression of BMPR-Ia has an effect on the spatial-temporal expression pattern of the cyclin dependent kinase inhibitor, p19INK4d, or on the phenotype of SVZa derived cells. The results of our study confirmed and extended our previous findings that in control (non injected) animals, the rostral migratory stream (RMS), traversed by the SVZa-derived cells en route to the olfactory bulb, exhibits an anteriorhigh-posteriorlow gradient of p19INK4d expression; p19INK4d expression is essentially absent in the SVZa and highest in the subependymal zone in the middle of the olfactory bulb. However, SVZa progenitor cells encoding the wt-BMPR-Ia gene express p19INK4d within the SVZa, suggesting that the BMPs induce SVZa cells to ectopically undergo cell cycle exit within the SVZa. Furthermore, unlike striatal SVZ progenitor cells, which acquire an astrocytic phenotype when exposed to BMPs, SVZa progenitor cells retain their neuronal commitment under augmented BMP signaling.
PMCID: PMC4211639  PMID: 11255035
Bone morphogenetic proteins; Cell cycle; p19INK4d; Progenitor cells; Retrovirus; Rostral migratory stream; Subverticular zone
16.  Development of a Finite Element Head Model for the Study of Impact Head Injury 
BioMed Research International  2014;2014:408278.
This study is aimed at developing a high quality, validated finite element (FE) human head model for traumatic brain injuries (TBI) prediction and prevention during vehicle collisions. The geometry of the FE model was based on computed tomography (CT) and magnetic resonance imaging (MRI) scans of a volunteer close to the anthropometry of a 50th percentile male. The material and structural properties were selected based on a synthesis of current knowledge of the constitutive models for each tissue. The cerebrospinal fluid (CSF) was simulated explicitly as a hydrostatic fluid by using a surface-based fluid modeling method. The model was validated in the loading condition observed in frontal impact vehicle collision. These validations include the intracranial pressure (ICP), brain motion, impact force and intracranial acceleration response, maximum von Mises stress in the brain, and maximum principal stress in the skull. Overall results obtained in the validation indicated improved biofidelity relative to previous FE models, and the change in the maximum von Mises in the brain is mainly caused by the improvement of the CSF simulation. The model may be used for improving the current injury criteria of the brain and anthropometric test devices.
doi:10.1155/2014/408278
PMCID: PMC4227498  PMID: 25405201
17.  Allele-Specific Induction of IL-1β Expression by C/EBPβ and PU.1 Contributes to Increased Tuberculosis Susceptibility 
PLoS Pathogens  2014;10(10):e1004426.
Mycobacterium tuberculosis infection is associated with a spectrum of clinical outcomes, from long-term latent infection to different manifestations of progressive disease. Pro-inflammatory pathways, such as those controlled by IL-1β, have the contrasting potential both to prevent disease by restricting bacterial replication, and to promote disease by inflicting tissue damage. Thus, the ultimate contribution of individual inflammatory pathways to the outcome of M. tuberculosis infection remains ambiguous. In this study, we identified a naturally-occurring polymorphism in the human IL1B promoter region, which alters the association of the C/EBPβ and PU.1 transcription factors and controls Mtb-induced IL-1β production. The high-IL-1β expressing genotype was associated with the development of active tuberculosis, the severity of pulmonary disease and poor treatment outcome in TB patients. Higher IL-1β expression did not suppress the activity of IFN-γ-producing T cells, but instead correlated with neutrophil accumulation in the lung. These observations support a specific role for IL-1β and granulocytic inflammation as a driver of TB disease progression in humans, and suggest novel strategies for the prevention and treatment of tuberculosis.
Author Summary
IL-1β is important for the initial establishment of antimicrobial adaptive immunity, but prolonged IL-1β expression can also cause progressive immunopathology during M. tuberculosis infection. The paradoxical activities of IL-1β in promoting both antimycobacterial immunity and chronic tissue damage have left the ultimate contribution of this cytokine to TB progression in human populations unclear. In this work, we address the role of IL-1β-mediated inflammation using a combination of human genetics and molecular biology, and suggest that exuberant IL-1β responses are causatively associated with TB progression and poor treatment outcome in humans. This work furthers our understanding of the immunological factors that underlie TB disease and provide a strong rationale for the development of specific anti-inflammatory adjunctive therapies that could improve the long-term outcome of TB treatment. In addition, these insights inform the design of future TB control efforts that include the rational design of disease-preventing vaccines and genotype-targeted delivery of TB chemotherapy.
doi:10.1371/journal.ppat.1004426
PMCID: PMC4199770  PMID: 25329476
18.  Cloning and characterization of a new β-Glucosidase from a metagenomic library of Rumen of cattle feeding with Miscanthus sinensis 
BMC Biotechnology  2014;14(1):85.
Background
The study on the second generation bio-fuel is a hot area of current research of renewable energy. Among series of key points in this area, the role of β-glucosidase in the degradation of intermediate gluco-oligosaccharides limits the rate of the complete saccharification of lignocellulose.
Results
In this study, a new β-glucosidase gene, unglu135B12, which was isolated from a metagenomic library of rumen of cattle feeding with Miscanthus sinensis by the function-based screening, encodes a 779 amino acid polypeptide that contains a catalytic domain belonging to glycoside hydrolase family 3 (GH3). It was recombinantly expressed, purified and biochemically characterized. The recombinant β-glucosidase, unglu135B12, displayed optimum enzymatic activity at pH 5.0 at 38°C, and showed the highest specific activity of 2.5 × 103 U/mg under this optimal condition to p-nitrophenyl-β-D-glucopyranoside (pNPG), and its Km and Vmax values were 0.309 mmol/L and 7.292 μmol/min, respectively. In addition, the presence of Ca2+, K+, Na+ slightly improved β-glucosidase activity of unglu135B12 by about 5%, while about 10 ~ 85% loss of β-glucosidase activity was induced by addition of Mn2+, Fe3+, Zn2+, Cu2+. Interestingly, unglu135B12 was activated by glucose at the concentration lower than 40 mM.
Conclusions
Our findings indicate that unglu135B12 is a new β-glucosidase derived from rumen of cattle, and it might be a potent candidate for saccharification of lignocellulose in industrial application.
Electronic supplementary material
The online version of this article (doi:10.1186/1472-6750-14-85) contains supplementary material, which is available to authorized users.
doi:10.1186/1472-6750-14-85
PMCID: PMC4287584  PMID: 25274487
β-glucosidase; Rumen; Miscanthus sinensis; Metagenomic library
19.  Biomechanical characteristics of cement/gelatin mixture for prevention of cement leakage in vertebral augmentation 
European Spine Journal  2013;22(10):2249-2255.
Purpose
This study evaluated whether or not the addition of gelatin micro-particles into the polymethyl methacrylate (PMMA) could reduce cement infiltration in cancellous bone of vertebra.
Methods
Gelatin micro-particles were prepared in various sizes and mixed with PMMA in different densities. Dynamic viscosity of the mixture was measured by a rotational rheometer. Fresh bovine vertebral bodies were sectioned into cylindrical samples. Permeability of the mixture through the samples was tested on a mechanical test machine, and calculated using Darcy’s law. The PMMA/gelatin mixture also underwent compressive and bending tests, and their structures were examined by scanning electron microscopy.
Results
The cement/gelatin mixture increased the viscosity. Significant reduction of cement permeability in cancellous bone was determined after the addition of the micro-particles. Micro-particles of 2 % in density and 125–250 μm in size decreased the permeability by 1/3 without any significant change of the cement viscosity. The biomechanical strength was unchanged in compression but decreased by up to 20 % in bending.
Conclusions
Gelatin micro-particles significantly increased the cement viscosity, reduced the permeability in cancellous bone of vertebra, decreased the flexural strength, but did not affect the compressive strength. Although it suggested a manageable approach in vertebral augmentation, the outcome should be further verified on a cadaveric model or an animal model before the mixture could be used safely and effectively in the clinical treatment.
doi:10.1007/s00586-013-2886-2
PMCID: PMC3804717  PMID: 23832385
PMMA; Gelatin; Vertebra; Augmentation; Biomechanics
20.  Human ABCB1 (P-glycoprotein) and ABCG2 Mediate Resistance to BI 2536, a Potent and Selective Inhibitor of Polo-like Kinase 1 
Biochemical pharmacology  2013;86(7):904-913.
The overexpression of the serine/threonine specific polo-like kinase 1 (Plk1) has been detected in various types of cancer, and thus has fast become an attractive therapeutic target for cancer therapy. BI 2536 is the first selective inhibitor of Plk1 that inhibits cancer cell proliferation by promoting G2/M cell cycle arrest at nanomolar concentrations. Unfortunately, alike most chemotherapeutic agents, the development of acquired resistance to BI 2536 is prone to present a significant therapeutic challenge. One of the most common mechanisms for acquired resistance in cancer chemotherapy is associated with the overexpression of ATP-binding cassette (ABC) transporters ABCB1, ABCC1 and ABCG2. Here, we discovered that overexpressing of either ABCB1 or ABCG2 is a novel mechanism of acquired resistance to BI 2536 in human cancer cells. Moreover, BI 2536 stimulates the ATPase activity of both ABCB1 and ABCG2 in a concentration-dependent manner, and inhibits the drug substrate transport mediated by these transporters. More significantly, the reduced chemosensitivity and BI 2536-mediated G2/M cell cycle arrest in cancer cells overexpressing either ABCB1 or ABCG2 can be significantly restored in the presence of selective inhibitor or other chemotherapeutic agents that also interact with ABCB1 and ABCG2, such as tyrosine kinase inhibitors nilotinib and lapatinib. Taken together, our findings indicate that in order to circumvent ABCB1 or ABCG2-mediated acquired resistance to BI 2536, a combined regimen of BI 2536 and inhibitors or clinically active drugs that potently inhibit the function of ABC drug transporters, should be considered as a potential treatment strategy in the clinic.
doi:10.1016/j.bcp.2013.08.004
PMCID: PMC3791609  PMID: 23962445
ABC transporter; multidrug resistance; Polo-like kinase 1; BI 2536
21.  PCI-24781 down-regulates EZH2 expression and then promotes glioma apoptosis by suppressing the PIK3K/Akt/mTOR pathway 
Genetics and Molecular Biology  2014;37(4):716-724.
PCI-24781 is a novel histone deacetylase inhibitor that inhibits tumor proliferation and promotes cell apoptosis. However, it is unclear whether PCI-24781 inhibits Enhancer of Zeste 2 (EZH2) expression in malignant gliomas. In this work, three glioma cell lines were incubated with various concentrations of PCI-24781 (0, 0.25, 0.5, 1, 2.5 and 5 μM) and analyzed for cell proliferation by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and colony formation, and cell cycle and apoptosis were assessed by flow cytometry. The expression of EZH2 and apoptosis-related proteins was assessed by western blotting. Malignant glioma cells were also transfected with EZH2 siRNA to examine how PCI-24781 suppresses tumor cells. EZH2 was highly expressed in the three glioma cell lines. Incubation with PCI-24781 reduced cell proliferation and increased cell apoptosis by down-regulating EZH2 in a concentration-dependent manner. These effects were simulated by EZH2 siRNA. In addition, PCI-24781 or EZH2 siRNA accelerated cell apoptosis by down-regulating the expression of AKT, mTOR, p70 ribosomal protein S6 kinase (p70s6k), glycogen synthase kinase 3A and B (GSK3a/b) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). These data suggest that PCI-24781 may be a promising therapeutic agent for treating gliomas by down-regulating EZH2 which promotes cell apoptosis by suppressing the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway.
doi:10.1590/S1415-47572014005000011
PMCID: PMC4261972  PMID: 25505847
EZH2; malignant gliomas; PCI-24781; PIK3K/Akt/mTOR signaling pathway
22.  Entecavir combined with furin inhibitor simultaneously reduces hepatitis B virus replication and e antigen secretion 
Virology Journal  2014;11(1):165.
Background
The antiviral therapy of chronic hepatitis B virus (HBV) infection pursues the dual goals, virological response (undetectable serum HBV DNA) and hepatitis B e antigen (HBeAg) serological response (serum HBeAg loss/seroconversion). It is relatively difficult, however, to realize the serological response, especially for nucleotide/nucleoside analogs. Furin, a proprotein convertase, is involved in HBeAg maturation. The suppression of furin using inhibitors accordingly reduces HBeAg secretion, but possibly enhances HBV replication. For these reasons, the strategy based on the combination of nucleoside analog entecavir (ETV) and furin inhibitors to inhibit HBV replication and HBeAg secretion simultaneously were studied here.
Methods
The suppression of furin was performed using inhibitors decanoyl-RVKR-chloromethylketone (CMK) and hexa-D-arginine (D6R) or the expression of furin inhibitory prosegment. The influence of furin suppression on HBV replication and the effect of CMK combined with nucleoside analog entecavir (ETV) on HBV replication and HBeAg secretion was investigated in HepG2.2.15 cells. HBeAg level in media was detected using enzyme-linked immunosorbent assay. Intracellular viral antigens and HBV DNA were detected using Western and Southern blotting analyses, respectively.
Results
CMK, D6R and the expression of inhibitory prosegment all significantly reduced HBeAg secretion, but only CMK enhance HBV replication. Concordantly, only CMK post-transcriptionally accumulated cytosolic HBV replication-essential hepatitis B core antigen (HBcAg). The HBcAg-accumulating effect of CMK was further found to be resulted from its redundant inhibitory effect on the trypsin-like activity of cellular proteasomes that are responsible for HBcAg degradation. Moreover, the viral replication-enhancing effect of CMK was abrogated by ETV and ETV combined with CMK reduced HBV replication and HBeAg secretion simultaneously.
Conclusion
The suppression of furin itself does not enhance HBV replication. Nucleotide/nucleoside analogs combined with furin inhibitors may be a potential easy way to realize the dual goals of the antiviral therapy for chronic hepatitis B in the future.
doi:10.1186/1743-422X-11-165
PMCID: PMC4177756  PMID: 25224377
Hepatitis B virus; Viral replication; Hepatitis B e antigen; Proprotein convertase; Furin; Antiviral therapy
23.  One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas mediated genome engineering 
Cell  2013;154(6):1370-1379.
SUMMARY
The type II bacterial CRISPR/Cas system is a novel genome engineering technology with the ease of multiplexed gene targeting. Here we created reporter and conditional mutant mice by co-injection of zygotes with Cas9 mRNA, different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2 and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.
doi:10.1016/j.cell.2013.08.022
PMCID: PMC3961003  PMID: 23992847
24.  A Ubiquitin Shuttle DC-UbP/UBTD2 Reconciles Protein Ubiquitination and Deubiquitination via Linking UbE1 and USP5 Enzymes 
PLoS ONE  2014;9(9):e107509.
The ubiquitination levels of protein substrates in eukaryotic cells are delicately orchestrated by various protein cofactors and enzymes. Dendritic cell-derived ubiquitin (Ub)-like protein (DC-UbP), also named as Ub domain-containing protein 2 (UBTD2), is a potential Ub shuttle protein comprised of a Ub-like (UbL) domain and a Ub-binding domain (UBD), but its biological function remains largely unknown. We identified two Ub-related enzymes, the deubiquitinating enzyme USP5 and the Ub-activating enzyme UbE1, as interacting partners of DC-UbP from HEK 293T cells. Biochemical studies revealed that the tandem UBA domains of USP5 and the C-terminal Ub-fold domain (UFD) of UbE1 directly interacted with the C-terminal UbL domain of DC-UbP but on the distinct surfaces. Overexpression of DC-UbP in HEK 293T cells enhanced the association of these two enzymes and thus prompted cellular ubiquitination, whereas knockdown of the protein reduced the cellular ubiquitination level. Together, DC-UbP may integrate the functions of USP5 and UbE1 through interacting with them, and thus reconcile the cellular ubiquitination and deubiquitination processes.
doi:10.1371/journal.pone.0107509
PMCID: PMC4160250  PMID: 25207809
25.  Upregulation of 5-Hydroxytryptamine Receptor Signaling in Coronary Arteries after Organ Culture 
PLoS ONE  2014;9(9):e107128.
Background
5-Hydroxytryptamine (5-HT) is a powerful constrictor of coronary arteries and is considered to be involved in the pathophysiological mechanisms of coronary-artery spasm. However, the mechanism of enhancement of coronary-artery constriction to 5-HT during the development of coronary artery disease remains to be elucidated. Organ culture of intact blood-vessel segments has been suggested as a model for the phenotypic changes of smooth muscle cells in cardiovascular disease.
Methodology/Principal Findings
We wished to characterize 5-HT receptor-induced vasoconstriction and quantify expression of 5-HT receptor signaling in cultured rat coronary arteries. Cumulative application of 5-HT produced a concentration-dependent vasoconstriction in fresh and 24 h-cultured rat coronary arteries without endothelia. 5-HT induced greater constriction in cultured coronary arteries than in fresh coronary arteries. U46619- and CaCl2-induced constriction in the two groups was comparable. 5-HT stimulates the 5-HT2A receptor and cascade of phospholipase C to induce coronary vasoconstriction. Calcium influx through L-type calcium channels and non-L-type calcium channels contributed to the coronary-artery constrictions induced by 5-HT. The contractions mediated by non-L-type calcium channels were significantly enhanced in cultured coronary arteries compared with fresh coronary arteries. The vasoconstriction induced by thapsigargin was also augmented in cultured coronary arteries. The decrease in Orai1 expression significantly inhibited 5-HT-evoked entry of Ca2+ in coronary artery cells. Expression of the 5-HT2A receptor, Orai1 and STIM1 were augmented in cultured coronary arteries compared with fresh coronary arteries.
Conclusions
An increased contraction in response to 5-HT was mediated by the upregulation of 5-HT2A receptors and downstream signaling in cultured coronary arteries.
doi:10.1371/journal.pone.0107128
PMCID: PMC4159325  PMID: 25202989

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