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1.  Transcriptional analysis of porcine circovirus-like virus P1 
BMC Veterinary Research  2014;10:287.
Recently identified porcine circovirus-like virus P1 has the smallest DNA viral genome. In this study, we identified the viral genes and their corresponding mRNA transcripts.
The RNAs of P1, synthesized in porcine kidney cells, were examined with northern blotting and PCR analyses.
Eight virus-specific RNAs were detected. Four mRNAs (open reading frames (ORFs) 1, 2, 4, and 5) are encoded by the viral (−) strand and four (ORFs 3, 6, 7, and 8) are encoded by the viral (+) strand. All proteins encoded by the ORFs of the P1 virus are less than 50 amino acids in length, except that encoded by ORF1 (113 amino acids).
We show a very complex viral transcription pattern in P1-infected cells.
PMCID: PMC4258304  PMID: 25440084
Porcine circovirus like virus P1; Transcriptional analysis; Northern blotting; RACE
2.  Monitoring the Quality of HIV-1 Viral Load Testing through a Proficiency Testing Program Using Dried Tube Specimens in Resource-Limited Settings 
Journal of Clinical Microbiology  2015;53(4):1129-1136.
HIV-1 viral load (VL) levels are used for monitoring disease progression and antiretroviral therapy outcomes in HIV-infected patients. To assess the performance of laboratories conducting HIV-1 VL testing in resource-limited settings, the U.S. Centers for Disease Control and Prevention implemented a voluntary, free-of-charge, external quality assurance program using dried tube specimens (DTSs). Between 2010 and 2012, DTS proficiency testing (PT) panels consisting of 5 specimens were distributed at ambient temperature to participants. The results from the participants (n ≥ 6) using the same assay were grouped, analyzed, and graded as acceptable within a group mean ± 3 standard deviations. Mean proficiency scores were calculated by dividing the combined PT scores by the number of testing cycles using a linear regression model. Between 2010 and 2012, the number of participants enrolled increased from 32 in 16 countries to 114 in 44 countries. A total of 78.2% of the participants reported results using 10 different VL assays. The rates of reporting of acceptable results by the participants were 96.6% for the Abbott assay, 96.3% for the Roche Cobas assay, 94.5% for the Roche Amplicor assay, 93.0% for the Biocentric assay, and 89.3% for the NucliSens assay. The overall mean proficiency scores improved over time (P = 0.024). DTSs are a good alternative specimen type to plasma specimens for VL PT programs, as they do not require cold chain transportation and can be used on PCR-based assays. Our data suggest that the CDC HIV-1 VL PT program using DTSs positively impacts the testing performance of the participants, which might translate into better and more accurate VL testing services for patients.
PMCID: PMC4365191  PMID: 25609733
3.  Development of real-time PCR assay for detection of porcine circovirus-like virus P1 in domestic pigs in China 
BMC Veterinary Research  2015;11:240.
The porcine circovirus-like agent P1 is a newly discovered DNA virus with a single-stranded circular genome that is highly homologous to that of porcine circovirus type 2. P1 infection can cause symptoms resembling postweaning multisystemic wasting syndrome. This study aims to develop a rapid, sensitive and specific method to detect P1.
A pair of primers was designed and used to amplify a 119 bp DNA fragment to generate a recombinant plasmid which was served as the standard. A SYBR I qPCR protocol was established using the P1 recombinant plasmid standard and the sensitivity, specificity and stability of this method was analyzed. The results demonstrate a strong correlation with P1 recombinant plasmid titers when virus DNA copy numbers fall in between 100 ~ 109 copies/μL. This method doesn’t detect pseudo rabies, porcine parvovirus or porcine reproductive and respiratory syndrome virus; moreover it can distinguish porcine circovirus type 2 from P1 by melting temperature analysis. Coefficient of variation for each batch of reaction is less than 5 %. The serum virus titers of P1 positive in this study were measured by this protocol to be 103 to 107 copies/mL.
The established qPCR is sensitive, specific, and reliable, which could be a useful tool when applied to quantification of P1 in a variety of samples from infected pigs.
PMCID: PMC4583164  PMID: 26404908
Porcine circovirus-like virus P1; PCV2; Fluorescence quantitative PCR; Melting curve
4.  The CD133+CD44+ Precancerous Subpopulation of Oval Cells Is a Therapeutic Target for Hepatocellular Carcinoma 
Stem Cells and Development  2014;23(18):2237-2249.
Hepatocellular carcinoma (HCC) is a malignant tumor associated with a generally poor prognosis and a high rate of recurrence. HCC usually develops in the context of chronic liver diseases, and long-lasting premalignant conditions precede cancer development. A promising therapeutic approach is to eliminate precancerous cells, which are considered as the precursors of cancer stem cells, to prevent further malignant transformation. In this study, we identified a subpopulation of precancerous cells in a rat liver carcinogenesis model, which were enriched in CD133+CD44+CD45−HIS49− cells that formed part of the hepatic oval cells fraction. Prospective isolation of the precancerous cells using flow cytometry identified stem cell properties such as the ability to expand clonally and differentiate into bi-lineage cell types. Furthermore, an acyclic retinoid, which was recently shown to improve overall survival after HCC resection, directly inhibited the extensive expansion of the isolated precancerous cells in vitro and decreased the emergence of the precancerous cells and their progeny in vivo. Long-term follow-up after the acyclic retinoid treatment confirmed reduction in precancerous changes, ultimately resulting in suppression of HCC development. These findings, together with data from recent clinical trials showing marked reduction in intrahepatic recurrence, suggest that acyclic retinoid directly prevents de novo HCC by inhibiting the development of precancerous cells. Given recent advances in diagnostic techniques and the establishment of surveillance programs, the targeting of precancerous cells may have a huge impact on preventative cancer therapies.
PMCID: PMC4155481  PMID: 24804872
5.  Human Cytomegalovirus Exploits Interferon-Induced Transmembrane Proteins To Facilitate Morphogenesis of the Virion Assembly Compartment 
Journal of Virology  2014;89(6):3049-3061.
Recently, interferon-induced transmembrane proteins (IFITMs) have been identified to be key effector molecules in the host type I interferon defense system. The invasion of host cells by a large range of RNA viruses is inhibited by IFITMs during the entry step. However, the roles of IFITMs in DNA virus infections have not been studied in detail. In this study, we report that human cytomegalovirus (HCMV), a large human DNA virus, exploits IFITMs to facilitate the formation of the virion assembly compartment (vAC) during infection of human fibroblasts. We found that IFITMs were expressed constitutively in human embryonic lung fibroblasts (MRC5 cells). HCMV infection inhibited IFITM protein accumulation in the later stages of infection. Overexpression of an IFITM protein in MRC5 cells slightly enhanced HCMV production and knockdown of IFITMs by RNA interference reduced the virus titer by about 100-fold on day 8 postinfection, according to the findings of a virus yield assay at a low multiplicity of infection. Virus gene expression and DNA synthesis were not affected, but the typical round structure of the vAC was not formed after the suppression of IFITMs, thereby resulting in defective virion assembly and the production of less infectious virion particles. Interestingly, the replication of herpes simplex virus, a human herpesvirus that is closely related to HCMV, was not affected by the suppression of IFITMs in MRC5 cells. These results indicate that IFITMs are involved in a specific pathway required for HCMV replication.
IMPORTANCE HCMV is known to repurpose the interferon-stimulated genes (ISGs) viperin and tetherin to facilitate its replication. Our results expand the range of ISGs that can be exploited by HCMV for its replication. This is also the first report of a proviral function of IFITMs in DNA virus replication. In addition, whereas previous studies showed that IFITMs modulate virus entry, which is a very early stage in the virus life cycle, we identified a new function of IFITMs during the very late stage of virus replication, i.e., virion assembly. Virus entry and assembly both involve vesicle transport and membrane fusion; thus, a common biochemical activity of IFITMs is likely to be involved. Therefore, our findings may provide a new platform for dissecting the molecular mechanism of action of IFITMs during the blocking or enhancement of virus infection, which are under intense investigation in this field.
PMCID: PMC4337551  PMID: 25552713
6.  External Application of Traditional Chinese Medicine for Venous Ulcers: A Systematic Review and Meta-Analysis 
Objective. To evaluate the effectiveness of external application of traditional Chinese medicine (EA-TCM) on venous ulcers. Methods. Seven databases were searched until April 2015 for randomized controlled trials (RCTs) of EA-TCM for venous ulcers. Risk of bias was assessed using Cochrane Handbook guidelines. Study outcomes were presented as risk ratios (RRs) for dichotomous data or mean differences (MDs) for continuous data. Results. Sixteen of 193 potentially relevant trials met the inclusion criteria; however, their methodological qualities were low. Comparison of the same intervention strategies revealed significant differences in total effectiveness rates between EA-TCM and conventional therapy groups (RR = 1.22, 95% confidence interval [CI] = 1.16–1.29, and P < 0.00001). Compared to conventional therapy, EA-TCM combined with conventional therapy had a superior total effectiveness rate (RR = 1.11, 95% CI = 1.04–1.19, and P = 0.003). There were no significant differences in recurrence rates during followup and final pain measurements between the experimental and those in the control groups (RR = 0.86, 95% CI = 0.31–2.39, and P = 0.85; MD −0.75, 95% CI = −2.15–0.65, and P = 0.29). Conclusion. The evidence that EA-TCM is an effective treatment for venous ulcers is encouraging, but not conclusive due to the low methodological quality of the RCTs. Therefore, more high-quality RCTs with larger sample sizes are required.
PMCID: PMC4576005  PMID: 26435725
8.  Pyrvinium attenuates Hedgehog signaling downstream of Smoothened 
Cancer research  2014;74(17):4811-4821.
The Hedgehog (HH) signaling pathway represents an important class of emerging developmental signaling pathways that play critical roles in the genesis of a large number of human cancers. The pharmaceutical industry is currently focused on developing small molecules targeting Smoothened (Smo), a key signaling effector of the HH pathway that regulates the levels and activity of the Gli family of transcription factors. Although one of these compounds vismodegib is now FDA-approved for advanced basal cell carcinoma patients, acquired mutations in Smo can result in rapid relapse. Furthermore, many cancers also exhibit a Smo-independent activation of Gli proteins, an observation that may underlie the limited efficacy of Smo inhibitors in clinical trials against other types of cancer. Thus, there remains a critical need for HH inhibitors with different mechanisms of action, in particularly those that act downstream of Smo. Recently, we identified the FDA-approved anti-pinworm compound pyrvinium as a novel, potent (IC50 ~ 10nM) Casein Kinase-1α (CK1α) agonist. We show here that pyrvinium is a potent inhibitor of HH signaling, which acts by reducing the stability of the Gli family of transcription factors. Consistent with CK1α agonists acting on these most distal components of the HH signaling pathway, pyrvinium is able to inhibit the activity of a clinically relevant, vismodegib resistant Smo mutant, as well as the Gli activity resulting from loss of the negative regulator Suppressor of fused. We go on to demonstrate the utility of this small-molecule in vivo, against the HH dependent cancer medulloblastoma, attenuating its growth and reducing the expression of HH biomarkers.
PMCID: PMC4321822  PMID: 24994715
Hedgehog; cancer; pyrvinium; casein kinase 1α
9.  New benzimidazole acridine derivative induces human colon cancer cell apoptosis in vitro via the ROS-JNK signaling pathway 
Acta Pharmacologica Sinica  2015;36(9):1074-1084.
To investigate the mechanisms underlying anticancer action of the benzimidazole acridine derivative N-{(1H-benzo[d]imidazol-2-yl)methyl}-2-butylacridin-9-amine(8m) against human colon cancer cells in vitro.
Human colon cancer cell lines SW480 and HCT116 were incubated in the presence of 8m, and then the cell proliferation and apoptosis were measured. The expression of apoptotic/signaling genes and proteins was detected using RT-PCR and Western blotting. ROS generation and mitochondrial membrane depolarization were visualized with fluorescence microscopy.
8m dose-dependently suppressed the proliferation of SW480 and HCT116 cells with IC50 values of 6.77 and 3.33 μmol/L, respectively. 8m induced apoptosis of HCT116 cells, accompanied by down-regulation of Bcl-2, up-regulation of death receptor-5 (DR5), truncation of Bid, cleavage of PARP, and activation of caspases (including caspase-8 and caspase-9 as well as the downstream caspases-3 and caspase-7). Moreover, 8m selectively activated JNK and p38 without affecting ERK in HCT116 cells. Knockout of JNK1, but not p38, attenuated 8m-induced apoptosis. In addition, 8m induced ROS production and mitochondrial membrane depolarization in HCT116 cells. Pretreatment with the antioxidants N-acetyl cysteine or glutathione attenuated 8m-induced apoptosis and JNK activation in HCT116 cells.
The new benzimidazole acridine derivative, 8m exerts anticancer activity against human colon cancer cells in vitro by inducing both intrinsic and extrinsic apoptosis pathways via the ROS-JNK1 pathway.
PMCID: PMC4561968  PMID: 26235743
benzimidazole acridine; anticancer drug; colon cancer; apoptosis; death receptor-5; JNK1; ROS
10.  FOXP3+ regulatory T cells and their functional regulation 
Cellular and Molecular Immunology  2015;12(5):558-565.
FOXP3+ regulatory T (Treg) cells are critical in maintaining immune tolerance and homeostasis of the immune system. The molecular mechanisms underlying the stability, plasticity and functional activity of Treg cells have been much studied in recent years. Here, we summarize these intriguing findings, and provide insight into their potential use or manipulation during Treg cell therapy for the treatment of autoimmune diseases, graft-versus-host disease (GVHD) and cancer.
PMCID: PMC4579651  PMID: 25683611
autoimmune diseases; FOXP3; immunosuppressive activity; Treg cells
11.  Tissue resident regulatory T cells: novel therapeutic targets for human disease 
Cellular and Molecular Immunology  2015;12(5):543-552.
Over the past decade, the ability of regulatory T cells (Tregs) to suppress multiple types of immune cells has received tremendous attention. Mounting evidence has revealed that tissue resident Tregs control non-immunological processes of their target tissues and contribute to a plethora of human diseases. The identification of novel tissue-specific Tregs has highlighted their heterogeneity and complexity. This review summarizes the recent findings for visceral adipose tissue CD4+Foxp3+ regulatory T cells (VAT Tregs), muscle Tregs, bone Tregs and skin memory Tregs, with a focus on their unique functions in local tissues. This interpretation of the roles of tissue-specific Tregs and of their involvement in disease progression provides new insight into the discovery of potential therapeutic targets of human diseases.
PMCID: PMC4579654  PMID: 25891216
Regulatory T cells; Tissue Tregs; VAT Tregs; Muscle Repair; Areg; Tumor; Bone; Skin memory Tregs
12.  How regulatory T cells sense and adapt to inflammation 
Cellular and Molecular Immunology  2015;12(5):519-520.
PMCID: PMC4579659  PMID: 26277895
13.  Efficacy of high-dose rosuvastatin preloading in patients undergoing percutaneous coronary intervention: a meta-analysis of fourteen randomized controlled trials 
Numerous studies have evidenced that statins can reduce the incidence of cardiovascular disease. However, the effects of high-dose rosuvastatin (RSV) preloading in patients undergoing percutaneous coronary intervention (PCI) are controversial.
We attempted to identify and quantify the potential cardioprotective benefits of high-dose RSV preloading on final thrombolysis in myocardial infarction (TIMI) flow grade, major adverse cardiac events (MACE), and peri-procedural myocardial injury (PMI) in patients undergoing PCI.
Pubmed, EMBASE, Cochrane Central Register of Controlled Trials and ISI Web of Science databases were systematically searched for randomized controlled trials (RCTs) up to June 2015. We assessed the incidence of MACE and PMI in all enrolled patients for subgroups stratified by clinical presentation and previous statin therapy during the follow-up period.
Fourteen trials with 3368 individuals were included in our meta-analysis. High-dose RSV preloading before PCI lead to a 58 % reduction in MACE (odds ratio [OR] = 0.42, 95 % confidence intervals [CI]: 0.29-0.61, P < 0.00001) and a 60 % reduction in PMI (OR = 0.40, 95 % CI: 0.25–0.63, P < 0.0001). This procedure also improved the final TIMI flow grade in patients undergoing PCI (OR = 1.61, 95 % CI: 1.09–2.38, P = 0.02). The benefits on MACE were significant for both stable angina patients (OR = 0.42, 95 % CI: 0.21-0.87, P = 0.02) and acute coronary syndrome (ACS) patients (OR = 0.42, 95 % CI: 0.27-0.65, P < 0.0001); and for both statin naïve patients (OR = 0.42, 95 % CI: 0.28-0.64, P < 0.0001) and previous statin therapy patients (OR = 0.28, 95 % CI: 0.10-0.73, P = 0.01).
High-dose RSV preloading can significantly improve myocardial perfusion and reduce both MACE and PMI in patients undergoing PCI. The cardioprotective benefits of RSV preloading were significant in not only stable angina and ACS patients but also statin naïve and previous statin therapy patients. The cardioprotective benefits of RSV preloading in the follow-up period mainly resulted from a reduction in spontaneous MI and TVR, especially for ACS and statin naïve patients.
PMCID: PMC4549857  PMID: 26306625
Rosuvastatin; Clinical events; Peri-procedural myocardial infarction; Meta-analysis
14.  The association between metabolic syndrome and the risk of urothelial carcinoma of the bladder: a case-control study in China 
Evidence of the association of metabolic syndrome (MetS) with cancer risk is accumulating. However, uncertainties still exist as to the link of MetS with bladder cancer. This study aimed to assess the relationship between MetS and the risk of urothelial carcinoma of the bladder (UC) in a Chinese population.
We retrospectively analyzed clinicopathological data of 972 newly diagnosed UC patients and 1098 cancer-free controls matched to the cases by age and gender. Odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated using unconditional logistic regression in both unadjusted and adjusted models.
MetS was not significantly associated with the overall UC risk (p = 0.08). However, a significant association of MetS with UC was observed in female patients (p = 0.006). Diabetes mellitus (crude OR 1.339, 95 % CI 1.079–1.662, p = 0.008; adjusted OR 1.767, 95 % CI 1.308–2.386, p < 0.001) and hypertriglyceridemia (crude OR 1.245, 95 % CI 1.018–1.522, p = 0.033; adjusted OR 1.254, 95 % CI 1.020–1.542, p = 0.032) were significantly associated with UC risk. As the number of MetS components increased, the UC risk was elevated. Having three or more (versus zero) components of MetS was significantly related to risk of overall UC (OR 1.315; 95 % CI 1.006–1.719; p = 0.045) and non-muscle invasive bladder cancer (OR 1.354; 95 % CI 1.019–1.798; p = 0.037).
The present study indicated a marginal association between MetS and UC risk, and a significant association with UC risk in female patients. The results need to be evaluated in large-scale prospective cohorts.
PMCID: PMC4527224  PMID: 26246367
Urothelial carcinoma of the bladder; Metabolic syndrome; Diabetes mellitus; Hypertriglyceridemia; Epidemiology
15.  Huangqin-Tang Ameliorates TNBS-Induced Colitis by Regulating Effector and Regulatory CD4+ T Cells 
BioMed Research International  2015;2015:102021.
Huangqin-Tang decoction (HQT) is a classic traditional Chinese herbal formulation that is widely used to ameliorate the symptoms of gastrointestinal disorders, including inflammatory bowel disease (IBD). This study was designed to investigate the therapeutic potential and immunological regulatory activity of HQT in experimental colitis in rats. Using an animal model of colitis by intrarectally administering 2,4,6-trinitrobenzenesulfonic acid (TNBS), we found that administration of HQT significantly inhibited the severity of TNBS-induced colitis in a dose-dependent manner. In addition, treatment with HQT produced better results than that with mesalazine, as shown by improvedweight loss bleeding and diarrhoea scores, colon length, and intestinal inflammation. As for potential immunological regulation of HQT action, the percentages of Th1 and Th17 cells were reduced, but those Th2 and Treg cells were enhanced in LPMCs after HQT treatment. Additionally, HQT lowered the levels of Th1/Th17-associated cytokines but increased production of Th2/Treg-associated cytokines in the colon and MLNs. Furthermore, we observed a remarkable suppression of the Th1/Th17-associated transcription factors T-bet and ROR-γt. However, expression levels of the Th2/Treg-associated transcription factors GATA-3 and Foxp3 were enhanced during treatment with HQT. Our results suggest that HQT has the therapeutic potential to ameliorate TNBS-induced colitis symptoms. This protective effect is possibly mediated by its effects on CD4+ T cells subsets.
PMCID: PMC4539427  PMID: 26347453
16.  Experimental and Theoretical Investigations on the Supermolecular Structure of Isoliquiritigenin and 6-O-α-d-Maltosyl-β-cyclodextrin Inclusion Complex 
Isoliquiritigenin (ILTG) possesses many pharmacological properties. However, its poor solubility and stability in water hinders its wide applications. The solubility of bioactive compounds can often be enhanced through preparation and delivery of various cyclodextrin (CD) inclusion complexes. The 6-O-α-d-maltosyl-β-CD (G2-β-CD), as one of the newest developments of CDs, has high aqueous solubility and low toxicity, especially stable inclusion characteristics with bioactive compounds. In this work, we for the first time construct and characterize the supermolecular structure of ILTG/G2-β-CD by scanning electron microscopy (SEM), ultraviolet-visible spectroscopy (UV), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffractometry (XRD). The solubility of ILTG in water at 25 °C rises from 0.003 to 0.717 mg/mL by the encapsulation with G2-β-CD. Our experimental observations on the presence of the ILTG/G2-β-CD inclusion complex are further supported by the ONIOM(our Own N-layer Integrated Orbital molecular Mechanics)-based QM/MM (Quantum Mechanics/Molecular Mechanics) calculations, typically substantiating these supermolecular characteristics, such as detailed structural assignments, preferred binding orientations, selectivity, solvent effects, interaction energies and forces of the ILTG/G2-β-CD inclusion complex. Our results have elucidated how ILTG interacts with G2-β-CD, demonstrating the primary host-guest interactions between ILTG and G2-β-CD, characterized by hydrogen bonds, hydrophobic interactions, electrostatic forces, and conformational effects, are favored for the formation of the ILTG/G2-β-CD inclusion.
PMCID: PMC4581232  PMID: 26247946
isoliquiritigenin; 6-O-α-d-Maltosyl-β-CD; inclusion complex; ONIOM calculation
17.  Favorable outcome of haploidentical hematopoietic stem cell transplantation in Philadelphia chromosome-positive acute lymphoblastic leukemia: a multicenter study in Southwest China 
Since the introduction of tyrosine kinase inhibitors (TKIs) into combination chemotherapy regimens, the majority of newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) patients have achieved complete remission (CR). However, without allogeneic hematopoietic stem cell transplantation (HSCT), long-term outcomes in adults remain unsatisfactory. Indeed, haploidentical HSCT has become a common treatment for adult patients who lack an HLA-matched donor, though limited data are available on the efficacy of haploidentical HSCT in Ph+ ALL patients.
We analyzed the clinical outcomes of 82 Ph+ ALL patients who underwent haploidentical HSCT (n = 47) or HLA-matched HSCT (n = 35). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to assess BCR-ABL expression. All of the patients were treated with an imatinib-based regimen before undergoing HSCT. Imatinib treatment was resumed in the patients’ posttransplantation following detection of BCR-ABL transcripts.
All of the patients achieved neutrophil and platelet engraftment, with the exception of five patients who died prior to engraftment. Haploidentical HSCT was associated with higher incidences of acute graft-versus-host disease (GVHD) (51.1 vs. 25.7 %, p < 0.05) and chronic GVHD (48.9 vs. 25.7 %, p < 0.05) compared with HLA-matched HSCT, but there was no difference in the incidence of either grades III–IV acute GVHD or extensive chronic GVHD. The incidence of cytomegalovirus (CMV) infection was significantly higher in the patients treated with haploidentical HSCT than in those treated with HLA-matched HSCT (38.3 vs. 14.3 %, p < 0.05). Haploidentical HSCT was associated with a significantly lower relapse rate compared with HLA-matched HSCT (44.8 vs. 19.1 %, p < 0.05). There were no differences in non-relapse mortality (NRM), leukemia-free survival (LFS), or overall survival (OS) between the patients who received HLA-matched HSCT and those who underwent haploidentical HSCT.
Our data indicate that the incidence of NRM after HSCT is similar between the patients who receive HLA-matched donor cells and those who receive haploidentical donor cells and that haploidentical HSCT reduces the relapse rate. Haploidentical HSCT represents an encouraging treatment option for Ph+ ALL patients who lack a suitable HLA-matched donor.
PMCID: PMC4515001  PMID: 26208715
18.  Multiple B-cell epitope vaccine induces a Staphylococcus enterotoxin B-specific IgG1 protective response against MRSA infection 
Scientific Reports  2015;5:12371.
No vaccine against methicillin-resistant Staphylococcus aureus (MRSA) has been currently approved for use in humans. Staphylococcus enterotoxin B (SEB) is one of the most potent MRSA exotoxins. In the present study, we evaluated the efficacy and immunologic mechanisms of an SEB multiple B-cell epitope vaccine against MRSA infection. Synthetic overlapping peptide ELISA identified three novel B-cell immunodominant SEB epitopes (in addition to those previously known): SEB31–48, SEB133–150, and SEB193–210. Six B-cell immunodominant epitopes (amino acid residues 31–48, 97–114, 133–150, 193–210, 205–222, and 247–261) were sufficient to induce robust IgG1/IgG2b-specific protective responses against MRSA infection. Therefore, we constructed a recombinant MRSA SEB-specific multiple B-cell epitope vaccine Polypeptides by combining the six SEB immunodominant epitopes and demonstrated its ability to induce a robust SEB-specific IgG1 response to MRSA, as well as a Th2-directing isotype response. Moreover, Polypeptides-induced antisera stimulated synergetic opsonophagocytosis killing of MRSA. Most importantly, Polypeptides was more effective at clearing the bacteria in MRSA-infected mice than the whole SEB antigen, and was able to successfully protect mice from infection by various clinical MRSA isolates. Altogether, these results support further evaluation of the SEB multiple B-cell epitope-vaccine to address MRSA infection in humans.
PMCID: PMC4511869  PMID: 26201558
19.  TMEM140 is associated with the prognosis of glioma by promoting cell viability and invasion 
Gliomas are the most common types of primary brain tumors in the adult central nervous system. TMEM140 is identified as an amplified gene in the human gastric cancer genome. However, the function of TMEM140 in gliomas has not been thoroughly elucidated. The aim of the current study was to determine the clinical significance of TMEM140 expression in patients with gliomas and its effect on tumor cell malignant phenotypes.
Immunohistochemical analysis and real-time reverse transcription PCR were performed to detect the expression levels of TMEM140 in 70 glioma brain tissue samples. Next, the correlation between the TMEM140 expression levels and the clinical characteristics and outcomes of glioma patients was statistically analyzed. TMEM140 expression was inhibited in two glioma cell lines (i.e., U87 and U373) using a knockdown method with small interfering RNA. Cell Counting Kit-8 and Transwell assays were used to investigate TMEM140 function during cell proliferation, invasion, and migration, respectively. Using flow cytometry and Western blot analysis, we subsequently determined the cell cycle and apoptosis profile of the TMEM140-silenced cells.
TMEM140 protein expression was significantly higher in gliomas than in normal brain tissues (p < 0.0001). TMEM140 overexpression was strongly correlated with tumor size, histologic grade, and overall survival time (P < 0.05). TMEM140 decreased cell viability in vitro and dramatically decreased tumor volume in vivo. This phenomenon might be caused by G1 phase cell cycle arrest and cell apoptosis. TMEM140 silencing could suppress the viability, migration, and invasion of glioma cells.
Our results suggest that TMEM140 expression is a prognostic factor that might play an important role in the viability, migration, and invasion of glioma cells. This study highlights the importance of TMEM140 as a novel prognostic marker and as an attractive therapeutic target for gliomas.
PMCID: PMC4511541  PMID: 26198430
TMEM140; Glioma; Cell viability; Invasion; Prognosis
20.  Effects of alveolar ridge preservation on delayed implant osseointegration 
To evaluate the effects of alveolar ridge preservation with Bio-Oss bone substitute (Geistlich Pharma) on delayed implant osseointegration. The 3rd and 4th left and right mandibular premolars were extracted from four adult healthy male and female dogs. For the experimental group, we randomly selected two extraction sockets in each dog to be filled with Bio-Oss bone substitute (Geistlich Pharma). The two remaining extraction sockets remained untreated and served as the control group. Three months after Bio-Oss placement, dental implants were inserted into the alveolar bone of the experimental group and the control group. The osteogenic activity of the bone around the implants was assessed by evaluating the histological morphology and by estimating histomorphometric parameters at 3 and 6 months after delayed implantation. At 3 months, Goldner’s trichrome staining analysis showed that the bone-implant contact rate and mineralised bone area around the implant were significantly higher in the experimental group (75.98% ± 8.97% and 69.52% ± 9.63%, respectively) than in the control group (56.13% ± 8.18% and 52.82% ± 7.25%, respectively; P < 0.05). However, at 6 months, the two groups showed no significant difference. Fluorescence microscopy analysis revealed that the average mineralisation apposition rate of the bone tissue around the dental implant in the experimental group at 3 and 6 months was 6.80 ± 0.43 μm and 8.38 ± 0.84 μm, respectively, which was significantly higher than the rate in the control group (P < 0.05). These data indicated that alveolar ridge preservation by using Bio-Oss placement can promote osseointegration of delayed implantation. This may be a promising option for clinical use.
PMCID: PMC4565254  PMID: 26379871
Alveolar ridge; osseointegration; delayed implantation
21.  The Use of Unidirectional Barbed Suture for Urethrovesical Anastomosis during Robot-Assisted Radical Prostatectomy: A Systematic Review and Meta-Analysis of Efficacy and Safety 
PLoS ONE  2015;10(7):e0131167.
Unidirectional barbed suture (UBS) has been widely used for surgery in recent years, especially for urethrovesical anastomosis (UVA) during robot-assisted radical prostatectomy (RARP). However, the efficacy and safety comparing it with conventional non-barbed suture (CS) for UVA is still controversial.
The objective of this study is to assess the current evidence regarding the efficacy and safety of UBS compared with CS for UVA during RARP.
We comprehensively searched PubMed, Embase, The Cochrane Library, SinoMed (Chinese) and other databases on Oct. 9, 2014 to conduct a systematic review and meta-analysis of all randomized controlled trials (RCTs) and other comparative studies evaluating these two types of suture. The outcome measures included anastomosis time operative time, posterior reconstruction (PR) time, postoperative leakage (PL) rate and continence rates at different time points (4-6 weeks, 3 months, 6-12 months) after surgery. Secondary outcomes included estimated blood loss (EBL) and length of catheterization (LOC).
Three RCTs and six observational studies including 786 cases were identified. Meta-analysis of extractable data showed that use of UBS could significantly reduce anastomosis time (weighted mean difference [WMD]:-3.98min; 95% confidence interval [CI], -6.02 -1.95; p = 0.0001), operative time (WMD:-10.06min; 95% CI, -15.45–-4.67; p = 0.0003) and PR time (WMD:-0.93min; 95% CI, -1.52–-0.34; p = 0.002). No significant difference was found in PL rate, EBL, LOC, or continence rates at 4-6 weeks, 3 months and 6–12 months after surgery.
Our meta-analysis indicates that UBS appears to be safe and efficient as CS for UVA during RARP with not only shorter anastomosis time, operative time, PR time, but also equivalent PL rate, EBL, LOC, and continence rates at 4-6 weeks, 3 months and 6-12 months after surgery. For the inherent limitations of the eligible studies, future more persuasive RCTs are needed to confirm and update our findings.
PMCID: PMC4489906  PMID: 26135310
22.  Nrf2 enhances myocardial clearance of toxic ubiquitinated proteins 
Nuclear factor erythroid-2 related factor 2 (Nrf2) is a master transcription factor that controls the basal and inducible expression of a battery of antioxidant genes and other cytoprotective phase II detoxifying enzymes. While knockout of Nrf2 exaggerates cardiac pathological remodeling and dysfunction in diverse pathological settings, pharmacological activation of Nrf2 protects against cardiomyocyte injury and cardiac dysfunction. In contrast, there is also a concern that the chronic activation of Nrf2 secondary to oxidative stress is a contributing mechanism for the reductive stress-mediated heart failure. However, a direct link between cardiac specific activation of Nrf2 and cardiac protection or dysfunction in vivo remains to be established. Therefore, we investigated the effect of cardiomyocyte-specific transgenic activation of Nrf2 (Nrf2ctg) on cardiac pathological remodeling and dysfunction. We found that the cardiomyocyte-specific activation of Nrf2 suppressed myocardial oxidative stress as well as cardiac apoptosis, fibrosis, hypertrophy, and dysfunction in a setting of sustained pressure overload induced by transverse aortic arch constriction (TAC) in mice. Notably, the constitutive activation of Nrf2 increased the steady level of autophagosomes while decreasing the ubiquitinated protein aggregates in the heart after TAC. Nrf2 gene gain- and loss-of-function approaches revealed that Nrf2 enhances autophagosome formation and autophagic flux in cardiomyocytes. Unexpectedly, while Nrf2 minimally regulated apoptosis, it suppressed significantly the proteotoxic necrosis in cardiomyocytes. In addition, Nrf2 attenuated the proteocytotoxicity presumably via enhancing autophagy-mediated clearance of ubiquitinated protein aggregates in cardiomyocytes. Taken together, we demonstrated for the first time that cardiac specific activation of Nrf2 suppresses cardiac maladaptive remodeling and dysfunction most likely by enhancing autophagic clearance of toxic protein aggregates in the heart.
PMCID: PMC4418517  PMID: 24747945
Nrf2; Cardiac dysfunction; Autophagy; Proteinopathy; Necrosis; Oxidative stress
23.  Gene expression modulation in TGF-β3-mediated rabbit bone marrow stem cells using electrospun scaffolds of various stiffness 
Tissue engineering has recently evolved into a promising approach for annulus fibrosus (AF) regeneration. However, selection of an ideal cell source, which can be readily differentiated into AF cells of various regions, remains challenging because of the heterogeneity of AF tissue. In this study, we set out to explore the feasibility of using transforming growth factor-β3-mediated bone marrow stem cells (tBMSCs) for AF tissue engineering. Since the differentiation of stem cells significantly relies on the stiffness of substrate, we fabricated nanofibrous scaffolds from a series of biodegradable poly(ether carbonate urethane)-urea (PECUU) materials whose elastic modulus approximated that of native AF tissue. We cultured tBMSCs on PECUU scaffolds and compared their gene expression profile to AF-derived stem cells (AFSCs), the newly identified AF tissue-specific stem cells. As predicted, the expression of collagen-I in both tBMSCs and AFSCs increased with scaffold stiffness, whereas the expression of collagen-II and aggrecan genes showed an opposite trend. Interestingly, the expression of collagen-I, collagen-II and aggrecan genes in tBMSCs on PECUU scaffolds were consistently higher than those in AFSCs regardless of scaffold stiffness. In addition, the cell traction forces (CTFs) of both tBMSCs and AFSCs gradually decreased with scaffold stiffness, which is similar to the CTF change of cells from inner to outer regions of native AF tissue. Together, findings from this study indicate that tBMSCs had strong tendency to differentiate into various types of AF cells and presented gene expression profiles similar to AFSCs, thereby establishing a rationale for the use of tBMSCs in AF tissue engineering.
PMCID: PMC4511356  PMID: 25752910
bone marrow stem cells; annulus fibrosus-derived stem cells; TGF-β3 treatment; gene expression; electrospun scaffolds; stiffness
24.  Transcriptome Analysis and Discovery of Genes Involved in Immune Pathways from Coelomocytes of Sea Cucumber (Apostichopus japonicus) after Vibrio splendidus Challenge 
Vibrio splendidus is identified as one of the major pathogenic factors for the skin ulceration syndrome in sea cucumber (Apostichopus japonicus), which has vastly limited the development of the sea cucumber culture industry. In order to screen the immune genes involving Vibrio splendidus challenge in sea cucumber and explore the molecular mechanism of this process, the related transcriptome and gene expression profiling of resistant and susceptible biotypes of sea cucumber with Vibrio splendidus challenge were collected for analysis. A total of 319,455,942 trimmed reads were obtained, which were assembled into 186,658 contigs. After that, 89,891 representative contigs (without isoform) were clustered. The analysis of the gene expression profiling identified 358 differentially expression genes (DEGs) in the bacterial-resistant group, and 102 DEGs in the bacterial-susceptible group, compared with that in control group. According to the reported references and annotation information from BLAST, GO and KEGG, 30 putative bacterial-resistant genes and 19 putative bacterial-susceptible genes were identified from DEGs. The qRT-PCR results were consistent with the RNA-Seq results. Furthermore, many DGEs were involved in immune signaling related pathways, such as Endocytosis, Lysosome, MAPK, Chemokine and the ERBB signaling pathway.
PMCID: PMC4519954  PMID: 26193268
sea cucumber (Apostichopus japonicus); Vibrio splendidus; transcriptome sequencing; differentially expressed genes; bacteria-resistant gene; bacteria-susceptible gene
25.  Clinical evaluation of circulating microRNA-25 level change in sepsis and its potential relationship with oxidative stress 
Objective: to investigated the circulating microRNA expression profile in sepsis and its clinical evaluation. Methods: 70 patients with sepsis and 30 patients with SIRS were selected and their blood samples were collected. Using liquid bead array with 3 statistical analysis approaches analyzed the circulating microRNA expression profiles, for confirming the data of liquid bead array, qRT-PCR was performed. The prognostic value of the changed microRNA in sepsis was determined and compared with CRP and PCT by analyzing the receiver operating characteristic (ROC) curves. To reveal whether the selected microRNAs could predict the outcome of patients, 28 d survival rate were calculated using Kaplan-Meier curves. Furthermore, the level of malondialdehyde (MDA), activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in plasma were detected and the relationship with the changed microRNA was determined. Results: By integrating data from liquid bead array, we ultimately identified 6 microRNAs that were consistently changed in both of 3 statistical analysis approaches, however, only the change of microRNA-25 was significant according to the qPCR’s result. The area under ROC curve showed that the clinical accuracy of microRNA-25 for sepsis diagnosis was better than CRP and PCT (AUG=0.806, 0.676 and 0.726, P<0.05).The decrease in level of microRNA-25 was correlated with the severity of sepsis, SOFA score, CRP and PCT level, meanwhile, microRNA-25 level can be used for predicting the prognosis of patients, the patients with microRNA-25 level ≤0.492 had a lower 28 d survival rate. Moreover, Decreased microRNA-25 level was related to the level of oxidative stress indicators in sepsis patients. Conclusions: microRNA-25 can be used as a biomarker for the diagnosis and assessment of sepsis. Meanwhile, microRNA-25 level may be associated with oxidative stress in patients with sepsis, and it is expected to become a target for anti-oxidation therapy.
PMCID: PMC4555662  PMID: 26339334
Sepsis; circulating microRNA; liquid bead array; biomarker; diagnosis; prognosis; oxidative stress

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