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1.  Metabolic flux responses to genetic modification for shikimic acid production by Bacillus subtilis strains 
Background
Shikimic acid (SA) is a key chiral starting molecule for the synthesis of the neuramidase inhibitor GS4104 against viral influenza. Microbial production of SA has been extensively investigated in Escherichia coli, and to a less extent in Bacillus subtilis. However, metabolic flux of the high SA-producing strains has not been explored. In this study, we constructed with genetic manipulation and further determined metabolic flux with 13C-labeling test of high SA-producing B. subtilis strains.
Results
B. subtilis 1A474 had a mutation in SA kinase gene (aroI) and accumulated 1.5 g/L of SA. Overexpression of plasmid-encoded aroA, aroB, aroC or aroD in B. subtilis revealed that aroD had the most significantly positive effects on SA production. Simultaneous overexpression of genes for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroA) and SA dehydrogenase (aroD) in B. subtilis BSSA/pSAAroA/pDGSAAroD resulted in SA production of 3.2 g/L. 13C-Metabolic flux assay (MFA) on the two strains BSSA/pHCMC04/pDG148-stu and BSSA/pSAAroA/pDGSAAroD indicated the carbon flux from glucose to SA increased to 4.6% in BSSA/pSAAroA/pDGSAAroD from 1.9% in strain BSSA/pHCMC04/pDG148-stu. The carbon flux through tricarboxylic acid cycle significantly reduced, while responses of the pentose phosphate pathway and the glycolysis to high SA production were rather weak, in the strain BSSA/pSAAroA/pDGSAAroD. Based on the results from MFA, two potential targets for further optimization of SA production were identified. Experiments on genetic deletion of phosphoenoylpyruvate kinase gene confirmed its positive influence on SA production, while the overexpression of the transketolase gene did not lead to increase in SA production.
Conclusion
Of the genes involved in shikimate pathway in B. subtilis, aroD exerted most significant influence on SA accumulation. Overexpression of plasmid-encoded aroA and aroD doubled SA production than its parent strain. MFA revealed metabolic flux redistribution among phosphate pentose pathway, glycolysis, TCA cycle in the low and high SA-producing B. subtilis strains. The high SA producing strain BSSA/pSAAroA/pDGSAAroD had increased carbon flux into shikimate pathway and reduced flux into TCA cycle.
doi:10.1186/1475-2859-13-40
PMCID: PMC4003833  PMID: 24628944
Shikimic acid production; Shikimate pathway; Bacillus subtilis; Metabolic flux assay (MFA); aroA; aroD; tkt; pyk
2.  Production of salidroside in metabolically engineered Escherichia coli 
Scientific Reports  2014;4:6640.
Salidroside (1) is the most important bioactive component of Rhodiola (also called as “Tibetan Ginseng”), which is a valuable medicinal herb exhibiting several adaptogenic properties. Due to the inefficiency of plant extraction and chemical synthesis, the supply of salidroside (1) is currently limited. Herein, we achieved unprecedented biosynthesis of salidroside (1) from glucose in a microorganism. First, the pyruvate decarboxylase ARO10 and endogenous alcohol dehydrogenases were recruited to convert 4-hydroxyphenylpyruvate (2), an intermediate of L-tyrosine pathway, to tyrosol (3) in Escherichia coli. Subsequently, tyrosol production was improved by overexpressing the pathway genes, and by eliminating competing pathways and feedback inhibition. Finally, by introducing Rhodiola-derived glycosyltransferase UGT73B6 into the above-mentioned recombinant strain, salidroside (1) was produced with a titer of 56.9 mg/L. Interestingly, the Rhodiola-derived glycosyltransferase, UGT73B6, also catalyzed the attachment of glucose to the phenol position of tyrosol (3) to form icariside D2 (4), which was not reported in any previous literatures.
doi:10.1038/srep06640
PMCID: PMC4200411  PMID: 25323006
3.  Development of a Real-Time Resistance Measurement for Vibrio parahaemolyticus Detection by the Lecithin-Dependent Hemolysin Gene 
PLoS ONE  2013;8(8):e72342.
The marine bacterium Vibrio parahaemolyticus (V. parahaemolyticus) causes gastroenteritis in humans via the ingestion of raw or undercooked contaminated seafood, and early diagnosis and prompt treatment are important for the prevention of V. parahaemolyticus-related diseases. In this study, a real-time resistance measurement based on loop-mediated isothermal amplification (LAMP), electrochemical ion bonding (Crystal violet and Mg2+), real-time monitoring, and derivative analysis was developed. V. parahaemolyticus DNA was first amplified by LAMP, and the products (DNA and pyrophosphate) represented two types of negative ions that could combine with a positive dye (Crystal violet) and positive ions (Mg2+) to increase the resistance of the reaction liquid. This resistance was measured in real-time using a specially designed resistance electrode, thus permitting the quantitative detection of V. parahaemolyticus. The results were obtained in 1–2 hours, with a minimum bacterial density of 10 CFU.mL−1 and high levels of accuracy (97%), sensitivity (96.08%), and specificity (97.96%) when compared to cultivation methods. Therefore, this simple and rapid method has a potential application in the detection of V. parahaemolyticus on a gene chip or in point-of-care testing.
doi:10.1371/journal.pone.0072342
PMCID: PMC3753338  PMID: 23991096
4.  Deletion of C7L and K1L Genes Leads to Significantly Decreased Virulence of Recombinant Vaccinia Virus TianTan 
PLoS ONE  2013;8(7):e68115.
The vaccinia virus TianTan (VTT) has been modified as an HIV vaccine vector in China and has shown excellent performance in immunogenicity and safety. However, its adverse effects in immunosuppressed individuals warrant the search for a safer vector in the following clinic trails. In this study, we deleted the C7L and K1L genes of VTT and constructed six recombinant vaccinia strains VTT△C7L, VTT△K1L, VTT△C7LK1L, VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag. The pathogenicity and immunogenicity of these recombinants were evaluated in mouse and rabbit models. Comparing to parental VTT, VTT△C7L and VTT△K1L showed significantly decreased replication capability in CEF, Vero, BHK-21 and HeLa cell lines. In particular, replication of VTT△C7LK1L decreased more than 10-fold in all four cell lines. The virulence of all these mutants were decreased in BALB/c mouse and rabbit models; VTT△C7LK1L once again showed the greatest attenuation, having resulted in no evident damage in mice and erythema of only 0.4 cm diameter in rabbits, compared to 1.48 cm for VTT. VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag elicited as strong cellular and humoral responses against HIV genes as did VTKgpe, while humoral immune response against the vaccinia itself was reduced by 4-8-fold. These data show that deletion of C7L and K1L genes leads to significantly decreased virulence without compromising animal host immunogenicity, and may thus be key to creating a more safe and effective HIV vaccine vector.
doi:10.1371/journal.pone.0068115
PMCID: PMC3698190  PMID: 23840887
5.  Highlights for α-fetoprotein in determining prognosis and treatment monitoring for hepatocellular carcinoma 
AIM: To explore the prognostic value in the monitoring of treatment efficacy of serial α-fetoprotein (AFP) in hepatocellular carcinoma (HCC) patients.
METHODS: We searched MEDLINE, EMBASE and COCHRANE LIBRARY through April 21, 2012, to find qualifying articles. Our overall search strategy included terms for HCC, AFP, treatment response, and prognosis. Literature was limited to English-language, human studies. Studies reporting cumulative survival rates were summarized qualitatively. For the prognostic meta-analysis, we undertook a series of meta-analyses that summarised the Cox proportional hazard ratios (HRs) by assuming a random effects model. With regards to the correlation of AFP change with radiologic response, the categorical dichotomous variables were assessed using Poisson relative risks (RRs), which were incorporated into the random effects model meta-analysis of accuracy prediction. Between-study heterogeneity was estimated by use of the I² statistic. Publication bias was evaluated using the Begg funnel plot and Egger plot. Sensitivity analyses were conducted first by separating systemic treatment estimates from locoregional therapy estimates, evaluating different AFP response cut-off point effects, and exploring the impact of different study sizes.
RESULTS: Of 142 titles identified in our original search, 11 articles (12 clinical studies) met our criteria. Six studies investigated outcome in a total of 464 cases who underwent systemic treatment, and six studies investigated outcome in a total of 510 patients who received locoregional therapy. A random-effects model meta-analysis showed that AFP response was associated with an mortality HR of 0.55 (95%CI, 0.47-0.65) across HCC in overall survival (OS) and 0.50 (95%CI, 0.38-0.65) in progression-free survival. Restricting analysis to the six eligible analyses of systemic treatment, the pooled HRs were 0.64 (95%CI, 0.53-0.77) for OS. Limiting analysis to the six analyses of locoregional therapy, the pooled HRs for OS was 0.39 (95%CI, 0.29-0.53). We showed a larger pooled HR in the 50% definition studies (HR, 0.67, 95%CI, 0.55-0.83) compared with that from the 20% definition studies (HR, 0.41, 95%CI, 0.32-0.53). Restricting analysis to the four studies including over 100 patients individually, the pooled HR was 0.65 (95%CI, 0.54-0.79), with a pooled HR for OS of 0.35 (95%CI, 0.23-0.46) in the studies of less than 100 patients. As to radiological imaging, 43.1% (155/360) of the patients in the AFP response group presented with a radiological overall response, while the response rate decreased to 11.5% (36/313) in the patients from the AFP nonresponse group. The RR of having no overall response was significantly lower in the AFP response group than the AFP nonresponse group (RR, 0.67; 95%CI, 0.61-0.75). In terms of disease control rate, 86.9% (287/330) in the AFP response group and 51.0% (153/300) in the AFP nonresponse group showed successful disease control, respectively. The RR of disease control failure, similarly, was significantly lower in the AFP response group (RR, 0.37; 95%CI, 0.23-0.58). But these findings could be overestimates because of publication and reporting bias.
CONCLUSION: HCC patients presenting with an AFP response are at decreased risk of mortality. In addition, patients with an AFP response also present with a higher overall response rate and disease control rate.
doi:10.3748/wjg.v18.i48.7242
PMCID: PMC3544026  PMID: 23326129
Liver cancer; α-fetoprotein; Response; Prognosis; Monitoring
6.  Genome sequence of the model medicinal mushroom Ganoderma lucidum 
Nature Communications  2012;3:913-.
Ganoderma lucidum is a widely used medicinal macrofungus in traditional Chinese medicine that creates a diverse set of bioactive compounds. Here we report its 43.3-Mb genome, encoding 16,113 predicted genes, obtained using next-generation sequencing and optical mapping approaches. The sequence analysis reveals an impressive array of genes encoding cytochrome P450s (CYPs), transporters and regulatory proteins that cooperate in secondary metabolism. The genome also encodes one of the richest sets of wood degradation enzymes among all of the sequenced basidiomycetes. In all, 24 physical CYP gene clusters are identified. Moreover, 78 CYP genes are coexpressed with lanosterol synthase, and 16 of these show high similarity to fungal CYPs that specifically hydroxylate testosterone, suggesting their possible roles in triterpenoid biosynthesis. The elucidation of the G. lucidum genome makes this organism a potential model system for the study of secondary metabolic pathways and their regulation in medicinal fungi.
Ganoderma lucidum is a macrofungus in traditional Chinese medicine known to produce different bioactive compounds. In this study, the genome of G. lucidum is sequenced, making this organism a potential model system for future studies of secondary metabolic pathways and their regulation in medicinal fungi.
doi:10.1038/ncomms1923
PMCID: PMC3621433  PMID: 22735441
7.  Central obesity and nonalcoholic fatty liver disease risk after adjusting for body mass index 
AIM: To investigate whether central obesity is associated with nonalcoholic fatty liver disease (NAFLD) formation after adjusting for general obesity.
METHODS: The online databases PubMed, EMBASE, and ISI Web of Science were searched for studies estimating the influence of central obesity on NAFLD occurrence published through April 2014. Studies that did not adjust for body mass index (BMI) were excluded. In addition, the independent effect of BMI was also assessed with the included studies. The pooled effect sizes and 95% confidence intervals (CIs) were calculated using random- or fixed-effects models based on the degree of heterogeneity. Furthermore, subgroup analyses, meta-regression, sensitivity analyses, and publication bias were performed.
RESULTS: Twenty eligible studies were identified. The summary odds ratio (OR) values per-unit increase in waist circumference (WC) and BMI for NAFLD formation were 1.07 (95%CI: 1.03-1.10, I2 = 73.9%, n = 11 studies) and 1.25 (95%CI: 1.13-1.38, I2 = 88.7%, n = 11 studies), respectively. When the indices were expressed as binary variables (with the non-obesity group as reference), the pooled OR in WC, waist-to-hip ratio, and BMI were 2.34 (95%CI: 1.83-3.00, I2 = 41.8%, n = 7 studies), 4.06 (95%CI: 1.53-10.79, I2 = 65.7%, n = 3 studies), and 2.85 (95%CI: 1.60-5.08, I2 = 57.8%, n = 5 studies), respectively. Using the same studies as the latter (n = 5), pooled OR in WC was 3.14 (95%CI: 2.07-4.77), which is greater than that in BMI.
CONCLUSION: Central obesity may pose a greater threat to national health than general obesity, although both are independently associated with increased risk of NAFLD.
doi:10.3748/wjg.v21.i5.1650
PMCID: PMC4316109  PMID: 25663786
Central obesity; General obesity; Nonalcoholic fatty liver disease; Body mass index; Waist circumference
8.  Serotonin Deficiency Exacerbates Acetaminophen-Induced Liver Toxicity In Mice 
Scientific Reports  2015;5:8098.
Acetaminophen (APAP) overdose is a major cause of acute liver failure. Peripheral 5-hydroxytryptamine (serotonin, 5-HT) is a cytoprotective neurotransmitter which is also involved in the hepatic physiological and pathological process. This study seeks to investigate the mechanisms involved in APAP-induced hepatotoxicity, as well as the role of 5-HT in the liver's response to APAP toxicity. We induced APAP hepatotoxicity in mice either sufficient of serotonin (wild-type mice and TPH1-/- plus 5- Hydroxytryptophan (5-HTP)) or lacking peripheral serotonin (Tph1-/- and wild-type mice plus p-chlorophenylalanine (PCPA)).Mice with sufficient 5-HT exposed to acetaminophen have a significantly lower mortality rate and a better outcome compared with mice deficient of 5-HT. This difference is at least partially attributable to a decreased level of inflammation, oxidative stress and endoplasmic reticulum (ER) stress, Glutathione (GSH) depletion, peroxynitrite formation, hepatocyte apoptosis, elevated hepatocyte proliferation, activation of 5-HT2B receptor, less activated c-Jun NH2-terminal kinase (JNK) and hypoxia-inducible factor (HIF)-1α in the mice sufficient of 5-HT versus mice deficient of 5-HT. We thus propose a physiological function of serotonin that serotonin could ameliorate APAP-induced liver injury mainly through inhibiting hepatocyte apoptosis ER stress and promoting liver regeneration.
doi:10.1038/srep08098
PMCID: PMC4309973  PMID: 25631548
9.  FoxM1 overexpression promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma 
AIM: To investigate the expression of forkhead box protein M1 (FoxM1) in the process of epithelial mesenchymal transition in hepatocellular carcinoma (HCC) and its role in metastasis.
METHODS: FoxM1 and E-cadherin expression in HCC tissue microarray specimens was evaluated by immunohistochemical staining, and statistical methods were applied to analyze the correlation between FoxM1 and epithelial-mesenchymal transition (EMT). Kaplan-Meier analysis of the correlation between the FoxM1 expression level and recurrence or overall survival of HCC patients was performed. The expression of FoxM1, E-cadherin and snail homologue 1 (SNAI1) in HCC cell lines was evaluated by real-time reverse transcription-polymerase chain reaction and Western blot. Hepatocyte growth factor (HGF) was used to induce EMT and stimulate cell migration in HCC cells. The expression of FoxM1 and SNAI1 was regulated by transfection with plasmids pcDNA3.1 and siRNAs in vitro. The occurrence of EMT was evaluated by Transwell assay, morphologic analysis and detection of the expression of EMT markers (E-cadherin and vimentin). Luciferase and chromatin immunoprecipitation assays were used to evaluate whether SNAI1 is a direct transcriptional target of FoxM1.
RESULTS: FoxM1 expression was increased significantly in HCC compared with para-carcinoma (10.7 ± 0.9 vs 8.2 ± 0.7, P < 0.05) and normal hepatic (10.7 ± 0.9 vs 2.7 ± 0.4, P < 0.05) tissues. Overexpression of FoxM1 was correlated with HCC tumor size, tumor number, macrovascular invasion and higher TNM stage, but was negatively correlated with E-cadherin expression in microarray specimens and in cell lines. FoxM1 overexpression was correlated significantly with HCC metastasis and EMT. In vitro, we found that FoxM1 plays a key role in HGF-induced EMT, and overexpression of FoxM1 could suppress E-cadherin expression and induce EMT changes, which were associated with increased HCC cell invasiveness. Next, we confirmed that FOXM1 directly binds to and activates the SNAI1 promoter, and we identified SNAI1 as a direct transcriptional target of FOXM1. Moreover, inhibiting the expression of SNAI1 significantly inhibited FoxM1-mediated EMT.
CONCLUSION: FoxM1 overexpression promotes EMT and metastasis of HCC, and SNAI1 plays a critical role in FoxM1-mediated EMT.
doi:10.3748/wjg.v21.i1.196
PMCID: PMC4284335  PMID: 25574092
Forkhead box protein M1; Epithelial-mesenchymal transition; Hepatocellular carcinoma; Snail homolog 1; E-cadherin
10.  Silencing of HIV-1 gag gene from epidemic strains among men who have sex with men (MSM) in Tianjin, China by a broad-spectrum short hairpin RNA 
Virusdisease  2014;25(3):294-301.
RNA interference (RNAi) has been successfully used as a promising method to inhibit the replication of different viruses, including human immunodeficiency virus (HIV). Gene mutation is a hurdle for the anti-HIV by RNAi. Although prone to mutation, some genes are conserved and limited in functionally important regions. The gag gene is conserved in different subtypes and plays an important role in the assembly of HIV viral particle. Here, we identified subtypes and conserved sequences within forty-four gag genes from the epidemic strains among men who have sex with men. Three subtypes of gag gene, including CRF01_AE (47.7 %), CRF07_BC (40.9 %) and B (11.4 %) were analyzed by online blast. We designed five small hairpin RNAs (shRNAs) based on the conserved sequences. The gag–EGFP fusion transcript reporter system was used to select the most efficient shRNA. Among the five candidate shRNAs, gag-shRNA-3 represented a broad-spectrum inhibition against all chosen targets. This broad-spectrum shRNA diminished the titer of subtypes B and C of HIV-1 for a hundred orders of magnitude. The gag-shRNA-3 described here is a potential therapeutic agent in the HIV-1 gene therapy.
doi:10.1007/s13337-014-0209-9
PMCID: PMC4188215
HIV-1; MSM; gag; shRNA
11.  Peripheral Blood Mononuclear Cell Traffic Plays a Crucial Role in Mother-to-Infant Transmission of Hepatitis B Virus 
The role of peripheral blood mononuclear cells (PBMCs) in HBV intrauterine infection is not fully defined. Particularly the origin of PBMCs in HBV-infected neonates remains to be addressed. We carried out a population-based nested case-control study by enrolling 312 HBsAg-positive mothers and their babies. PBMC HBV DNA as well as serum HBsAg and HBV DNA was tested in cohort entry samples. Totally, 45.5% (142/312) of the newborns were found to be infected with HBV in perinatal transmission. 119 mother-infant pairs were identified to be different in the genetic profile of maternal and fetal PBMCs by AS-PCR and hemi-nested PCR. Among them, 57.1% (68/119) of the maternal PBMCs in index cases were positive for HBV DNA while 83.8% (57/68) of the HBV DNA positive maternal PBMCs passed the placental barrier and entered the fetus. Furthermore, maternal PBMC HBV infection was significantly associated with newborn infants HBV infection. PBMC traffic from mother to fetus resulted in a 9.5-fold increased risk of HBV infection in PBMC HBV DNA positive newborn infants. These data indicate that maternal PBMCs infected with HBV contribute to HBV intrauterine infection of newborn infants via PBMC traffic from mother to fetus.
doi:10.7150/ijbs.10813
PMCID: PMC4323366
Hepatitis B virus; mother-to-infant transmission; peripheral blood mononuclear cell; fetomaternal cellular traffic.
12.  Melatonin lowers edema after spinal cord injury 
Neural Regeneration Research  2014;9(24):2205-2210.
Melatonin has been shown to diminish edema in rats. Melatonin can be used to treat spinal cord injury. This study presumed that melatonin could relieve spinal cord edema and examined how it might act. Our experiments found that melatonin (100 mg/kg, i.p.) could reduce the water content of the spinal cord, and suppress the expression of aquaporin-4 and glial fibrillary acidic protein after spinal cord injury. This suggests that the mechanism by which melatonin alleviates the damage to the spinal cord by edema might be related to the expression of aquaporin-4 and glial fibrillary acidic protein.
doi:10.4103/1673-5374.147954
PMCID: PMC4316455  PMID: 25657743
nerve regeneration; spinal cord injury; melatonin; edema; aquaporin; glial fibrillary acidic protein; immunohistochemistry; western blot assay; neural regeneration
13.  Acoustic radiation force impulse elastography in differentiating renal solid masses: a preliminary experience 
New diagnostic methods are required to diagnose renal mass. Thus, we assessed virtual tissue quantification (VTQ) of acoustic radiation force impulse (ARFI) elastography in differentiation of renal solid masses. Forty-two patients with renal masses were assessed by VTQ in terms of measurement of the shear wave velocity (SWV). The masses were divided into three groups. They were clear cell carcinoma (CCC) angiomyolipoma (AML), and pseudotumor. The differences among the three groups in SWV, as well as between masses and its surrounding parenchyma, were investigated. Receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic performance. We found that the SWV among the three groups were significant different (F = 6.976, P = 0.003) and the SWV of pseudotumor (3.14 ± 0.75 m/s) was significantly higher than CCC (2.46 ± 0.45 m/s) and AML (2.49 ± 0.63 m/s) (P = 0.007 and 0.001 respectively). There were no significant difference between CCC and AML in SWV (P = 0.719). For each group, there was no significant difference between the mass and its surrounding parenchyma (P = 0.693, 0.892, and 0.714, respectively). Between pseudotumor and CCC, the optimal cut-off value of SWV for differential diagnoses was 3.07 m/s; and the area under the ROC curve (AUC) was 0.78 (95% CI: 0.560 to 0.924) (P = 0.004), the sensitivity and specificity were 100% and 58.3%, respectively. Between pseudotumor and AML, the optimal cut-off value of SWV for differential diagnoses was 3.03 m/s, thus AUC curve was 0.786 (95% CI: 0.591 to 0.918) (P = 0.002), the sensitivity and specificity were 100% and 58.3%, respectively. No significant difference was found between AML and CCC (P = 0.587) and the AUC was 0.562. To conclude, our results support that ARFI has potential value in differentiation between CCC and pseudotumor, or between AML and pseudotumor, however, it fails to make a distinction between CCC and AML.
PMCID: PMC4270564  PMID: 25550782
Acoustic radiation force impulse elastography; renal solid mass; ultrasound
14.  Analysis of primary resistance mutations to HIV-1 entry inhibitors in therapy naive subtype C HIV-1 infected mother–infant pairs from Zambia 
Background
Small molecular CCR5 inhibitors represent a new class of drugs for treating HIV-1 infection. The evaluation of the primary resistance mutations associated with entry inhibitors during HIV-1 perinatal transmission is required because they may have a profound impact on the clinical management in MTCT.
Objectives
To evaluate the primary resistance mutations to maraviroc and vicriviroc during perinatal transmission and analyze the sensitivity of Env derived from mother–infant pairs to maraviroc.
Study design
Nine MIPs infected by subtype C HIV-1 were recruited to analyze the prevalence and transmission of primary resistance mutations to maraviroc and vicriviroc. Moreover, Env derived from six MIPs were employed to construct provirus clones and to analyze the sensitivity to maraviroc.
Results
Mutations A316T, conferring partial resistance to maraviroc, T307I and R315Q, both conferring partial resistance to vicriviroc are prevalent in mother and infant cohorts, indicating the transmission of primary resistance mutations during HIV-1 perinatal transmission. However, the mutations of acutely infected mothers seem to directly transmit to their corresponding infants, while some mutations at low frequency of chronically infected mothers would be lost during transmission. Moreover, provirus clones derived from acutely infected MIPs are less susceptible to maraviroc than those from chronically infected MIPs.
Conclusions
Our study suggests that the transmission mode of primary resistance mutations and the sensitivity to maraviroc are dependent on infection status of MIPs either acutely or chronically infected. These results may indicate that higher dose of maraviroc could be needed for treatment of acutely infected MIPs compared to chronically infected MIPs.
doi:10.1016/j.jcv.2013.05.022
PMCID: PMC4017664  PMID: 23809473
MTCT; HIV-1; Subtype C; Primary resistance; Maraviroc; Vicriviroc
15.  Model based on γ-glutamyltransferase and alkaline phosphatase for hepatocellular carcinoma prognosis 
World Journal of Gastroenterology : WJG  2014;20(31):10944-10952.
AIM: To determine the prognostic value of alkaline phosphatase (ALP) and γ-glutamyltransferase (GGT) for hepatocellular carcinoma (HCC) .
METHODS: We analyzed the outcome of 172 HCC patients who underwent liver resection. Receiver operating characteristic (ROC) curve analysis was performed to determine the cut-off value of ALP and GGT. Then, preoperative risk factors for survival were evaluated by multivariate analysis. Based on the significant factors, a prognostic score model was established.
RESULTS: By ROC curve analysis, ALP > 120 U/L and GGT > 115 U/L were considered elevated. Overall survival (OS) and tumor-free survival (TFS) for patients with elevated ALP and GGT were significantly worse than for patients with ALP and GGT within the normal range. Multivariate analysis showed that the elevated levels of ALP, GGT and tumor size were independent prognostic factors. Giving each positive factor as a score of 1, we established a preoperative prognostic score model. The 5-year OS for patients with a score of 0, 1, 2 and 3 were 84.0%, 45.9%, 44.1% and 0%, respectively, while the TFS was 80.6%, 40.0%, 38.8% and 0%, respectively. When combining patients with scores of 1 and 2 into the middle risk group, and patients with scores of 0 and 3 into the low-risk and high-risk groups, respectively, different outcomes would be significantly distinguished by the risk groups.
CONCLUSION: Elevated ALP and GGT levels were risk predictors in HCC patients. Our prognostic model might vary the outcomes of patients from different risk groups.
doi:10.3748/wjg.v20.i31.10944
PMCID: PMC4138475  PMID: 25152598
Alkaline phosphatase; γ-Glutamyltransferase; Prognosis; Hepatocellular carcinoma
16.  Acoustic Radiation Force Impulse Imaging: A New Tool for the Diagnosis of Papillary Thyroid Microcarcinoma 
BioMed Research International  2014;2014:416969.
Purpose. To evaluate the diagnostic performance of ARFI imaging in differentiating between benign and malignant thyroid nodules <1 cm. Materials and Methods. 173 pathologically proven thyroid nodules (77 benign, 96 malignant) in 157 patients were included in this study. Receiver-operating characteristic curve (ROC) analyses were performed to assess the diagnostic performance of conventional ultrasound (US) and ARFI imaging in papillary thyroid microcarcinoma (PTMC). The independent risk factors for predicting PTMC were evaluated. Results. The mean SWV value of benign and malignant thyroid nodules were 2.57 ± 0.79 m/s (range: 0.90–4.92 m/s) and 3.88 ± 2.24 m/s (range: 1.49–9.00 m/s) (P = 0.000). Az for VTI elastography score was higher than that for hypoechoic, absence of halo sign, and type III vascularity (P < 0.05). The optimal cut-offs for VTI elastography score and SWV were score 4 and 3.10 m/s. Gender, hypoechoic, taller than wide, VTI elastography score ≥ 4, and SWV > 3.10 m/s had been found to be independent risk factors for predicting PTMC. Conclusion. ARFI elastography can provide elasticity information of PTMC quantitatively (VTQ) and directly reflects the overall elastic properties (VTI). Gender, hypoechogenicity, taller than wide, VTI elastography score ≥ 4, and SWV > 3.10 m/s are independent risk factors for predicting PTMC. ARFI elastography seems to be a new tool for the diagnosis of PTMC.
doi:10.1155/2014/416969
PMCID: PMC4090516  PMID: 25045673
17.  The influence of four dual-cure resin cements and surface treatment selection to bond strength of fiber post 
In this study, we evaluate the influence of post surface pre-treatments on the bond strength of four different cements to glass fiber posts. Eighty extracted human maxillary central incisors and canines were endodontically treated and standardized post spaces were prepared. Four post pre-treatments were tested: (i) no pre-treatment (NS, control), (ii) sandblasting (SA), (iii) silanization (SI) and (iv) sandblasting followed by silanization (SS). Per pre-treatment, four dual-cure resin cements were used for luting posts: DMG LUXACORE Smartmix Dual, Multilink Automix, RelyX Unicem and Panavia F2.0. All the specimens were subjected to micro push-out test. Two-way analysis of variance and Tukey post hoc tests were performed (α=0.05) to analyze the data. Bond strength was significantly affected by the type of resin cement, and bond strengths of RelyX Unicem and Panavia F2.0 to the fiber posts were significantly higher than the other cement groups. Sandblasting significantly increased the bond strength of DMG group to the fiber posts.
doi:10.1038/ijos.2013.83
PMCID: PMC3967305  PMID: 24177170
air abrasion; field emission scanning electron microscope; micro push-out bond strength; silane; surface pre-treatment
18.  Detecting and understanding genetic and structural features in HIV-1 B subtype V3 underlying HIV-1 co-receptor usage 
Bioinformatics  2013;29(4):451-460.
Motivation: To define V3 genetic elements and structural features underlying different HIV-1 co-receptor usage in vivo.
Results: By probabilistically modeling mutations in the viruses isolated from HIV-1 B subtype patients, we present a unique statistical procedure that would first identify V3 determinants associated with the usage of different co-receptors cooperatively or independently, and then delineate the complicated interactions among mutations functioning cooperatively. We built a model based on dual usage of CXCR4 and CCR5 co-receptors. The molecular basis of our statistical predictions is further confirmed by phenotypic and molecular modeling analyses. Our results provide new insights on molecular basis of different HIV-1 co-receptor usage. This is critical to optimize the use of genotypic tropism testing in clinical practice and to obtain molecular-implication for design of vaccine and new entry-inhibitors.
Contact: jing.zhang.jz349@yale.edu or cf.perno@uniroma2.it
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btt002
PMCID: PMC3570207  PMID: 23297034
19.  Simultaneous Determination and Pharmacokinetic Comparisons of Multi-Ingredients after Oral Administration of Radix Salviae Miltiorrhizae Extract, Hawthorn Extract, and a Combination of Both Extracts to Rats 
A simple and sensitive HPLC method was developed for simultaneous determination of danshensu (DSS), rosmarinic acid (RA), lithospermic acid (LA), salvianolic acid B (SAB), and hyperoside (HP) in rat plasma. This method validated was successfully applied to the pharmacokinetic study of the main active ingredients after oral administration of Radix Salviae Miltiorrhizae extract (SME), hawthorn extract (HTE), and a combination of both extracts (2.5 : 1) to rats. The results indicated that there have been great differences in pharmacokinetics between a single extract and a combination of both extracts. A combination of both extracts can enhance their bioavailabilities and delay the elimination of SAB and DSS in rats.
doi:10.1155/2014/617367
PMCID: PMC3934716  PMID: 24660090
20.  Impact of Cigarette Smoking on Outcome of Hepatocellular Carcinoma after Surgery in Patients with Hepatitis B 
PLoS ONE  2014;9(1):e85077.
Background and Objectives
Cigarette smoking is a potential risk factor for hepatocellular carcinoma (HCC) initiation, partially through interaction with hepatitis B virus (HBV). We examined the hypothesis that cigarette smoking might be associated with HBV-related HCC recurrence and patient survival after curative surgery.
Patients and Methods
Data of 302 patients with HBV infection who had undergone curative resection for HCC were prospectively collected from 2008 to 2011. Smoking status and smoking quantity (pack-years, PY) were asked at admission. Factors affecting recurrence-free survival (RFS) were examined. RFS and liver-specific mortality (LSM) stratified by risk factors were compared with log-rank test.
Results
109 were current smokers. Current smokers were not different from non-smokers in tumor burden and surgical procedure. Univariate and multivariate analysis identified that heavy smoking (PY ≥20) was the most significant factor associated with HBV-related HCC recurrence after curative surgical resection (p = 0.001), followed by anti-HBV treatment (p<0.01), current smoking (p = 0.028), surgical margin <1 cm (p = 0.048) and blood transfusion >600 ml (p = 0.028). The median RFS in non-smokers, ex-smokers and current smokers was 34 months, 24 months and 26 months, respectively (p = 0.033). Current smokers had significantly worse RFS rate and increased 5-year cumulative LSM than non-smokers (p = 0.024, and p<0.001, respectively). Heavy smokers had significantly worse RFS than non- and light smokers (0
Conclusions
Smoking history and quantity appears to be risk factors for HBV-related HCC recurrence and LSM of patients after surgery. For smokers, continued smoking postoperatively might accelerate tumor recurrence and patient death. Therefore, smoking abstinence should be strongly recommended to patients pre- and postoperatively.
doi:10.1371/journal.pone.0085077
PMCID: PMC3893178  PMID: 24454795
AIM: To determine whether an elevated neutrophil-lymphocyte ratio (NLR) is negatively associated with tumor recurrence in patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) after liver transplantation (LT), and to determine the optimal predictive NLR cut-off value.
METHODS: The data of HCC patients who had undergone LT came from the China Liver Transplant Registry database. We collected data from 326 liver cancer patients who had undergone LT at our medical center. We divided the patients into groups based on their NLRs (3, 4 or 5). We then compared the clinicopathological data and long-time survival between these groups. Meanwhile, we used receiver operating characteristic analysis to determine the optimal NLR cut-off.
RESULTS: Of 280 HCC patients included in this study, 263 were HBV positive. Patients with an NLR < 3 and patients with an NLR ≥ 3 but < 4 showed no significant differences in overall survival (OS) (P = 0.212) or disease-free survival (DFS) (P = 0.601). Patients with an NLR ≥ 4 but < 5 and patients with an NLR ≥ 5 also showed no significant differences in OS (P = 0.208) or DFS (P = 0.618). The 1-, 3- and 5-year OS rates of patients with an NLR < 4 vs an NLR ≥ 4 were 87.8%, 63.8% and 61.5% vs 73.9%, 36.7% and 30.3%, respectively (P < 0.001). The 1-, 3- and 5-year DFS rates of patients with an NLR < 4 vs NLR ≥ 4 were 83.9%, 62.9% and 60.7% vs 64.9%, 30.1% and 30.1%, respectively (P < 0.001). Univariate and multivariate analyses demonstrated that three factors, including NLR ≥ 4 (P = 0.002), were significant predictors of tumor recurrence in HCC patients after LT.
CONCLUSION: A preoperative elevated NLR significantly increased the risk for tumor recurrence in HCC patients after LT.
doi:10.3748/wjg.v19.i45.8398
PMCID: PMC3857465  PMID: 24363533
Hepatocellular carcinoma; Liver transplantation; Inflammatory reaction; Neutrophil-lymphocyte ratio; Hepatitis B virus
BMC Psychiatry  2013;13:254.
Background
Malingering detection has emerged as an important issue in clinical and forensic settings. The Structured Interview of Reported Symptoms-2 (SIRS-2) was designed to assess the feigned symptoms in both clinical and non-clinical subjects. The aim of the study was to examine the reliability and validity of the Chinese version of this scale.
Methods
Two studies were conducted to evaluate the reliability and validity of the Chinese Version of SIRS-2. In Study one, with a simulation design, the subjects included a. 40 students asked to simulate symptoms of mental illness; b. 40 general psychiatric inpatients and c. 40 students asked to reply to questions honestly. Scales scores for feigning symptoms among three groups were carried out for discriminant validity of the Chinese Version of SIRS-2. Minnesota Multiphasic Personality Inventory-2(MMPI-2) was administered in 80 undergraduate students. In Study two, with a known-groups comparison design, scales scores for feigning symptoms were compared between 20 suspected malingerers and 80 psychiatric outpatients from two forensic centers using the Chinese Version of SIRS-2.
Results
The Chinese Version of SIRS-2 demonstrated satisfactory internal consistency in both study one and two. In study one, criterion validity of this scale was supported by its significantly positive correlation with the MMPI-2 (r = 0.282 ~ 0.481 for Infrequency), and by its significantly negative correlation with the MMPI-2 (r = -0.255 ~ -0.519 for Lie and -0.205 ~ 0.391 for Correction). Scores of 10 out of 13 subscales of the Chinese Version of SIRS-2 for simulators were significantly higher than scores of honest students and general psychiatric patients. In study two, the mean scores of the Chinese Version of 13 subscales for suspected malingerers were significantly higher than those of psychiatric outpatients. For discriminant validity, it yielded a large effect size (d = 1.80) for the comparison of the participant groups in study one and two. Moreover, the sensitivity (proportion of malingerers accurately identified by the measure) and specificity (proportion of people accurately classified as responding honestly) of the Chinese version of SIRS-2 in the detection of malingering in these two studies are acceptable.
Conclusions
The Chinese version of the SIRS-2 has good psychometric properties and is a valid and reliable tool for detection of malingering in Chinese populations.
doi:10.1186/1471-244X-13-254
PMCID: PMC3852949  PMID: 24106829
Malingering; The Chinese version of the structured interview of reported symptoms-2; Reliability; Validity
PLoS ONE  2013;8(3):e58574.
The epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells in the adult kidney is one of the key events in renal interstitial fibrosis. Glioma pathogenesis related-2 (GLIPR-2) has been shown to be up-regulated in proximal tubular cells (PTCs) in the fibrotic kidney. However, the biological function of GLIPR-2 remains unknown. In this study, we found that GLIPR-2 expression is elevated in the kidney tissue samples of patients with diabetic nephropathy (DN). Human proximal renal tubular epithelial cells (HK-2 cells) were transfected with pcDNA3.0-GLIPR-2 and selected with G418. To identify the biological function of GLIPR-2, an epithelial-to-mesenchymal transition (EMT) PCR array analysis was performed, and genes that had statistically significantly altered expression levels with more than a two-fold difference compared with the pcDNA3.0-transfected HK-2 cells were considered. Key elements of the EMT process, such as E-cadherin and vimentin, were transcriptionally activated in the pcDNA3.0-GLIPR-2-transfected sublines. In addition, α-SMA gene expression, which is a marker of myofibroblasts, increased in the pcDNA3.0-GLIPR-2-transfected HK-2 cells. The cell migration assay demonstrated that the transfection of HK-2 with GLIPR-2 promoted cell migration following an EMT. Additionally, consistent with the effects of increased EGFR expression levels, we found that the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was highly elevated in the pcDNA3.0-GLIPR-2-transfected group. Our study demonstrates that GLIPR-2 overexpression in HK-2 cells can potentiate EMT-like processes in this cell type through the ERK1/2 signaling pathway. GLIPR-2 may be responsible for the development of renal fibrosis by increasing the accumulation of interstitial fibroblasts.
doi:10.1371/journal.pone.0058574
PMCID: PMC3596275  PMID: 23516513
Journal of Medicinal Chemistry  2012;55(5):2242-2250.
Since the emergence of drug-resistant mutants has limited the efficacy of non-nucleoside reverse transcriptase inhibitors (NNRTIs), it is essential to develop new antivirals with better drug-resistance and pharmacokinetic profiles. Here we designed and synthesized a series of 1-[(2-benzyloxyl/alkoxyl)methyl]-5-halo-6-aryluracils, the HEPT analogues, and evaluated their biological activity using Nevirapine and 18 (TNK-651) as reference compounds. Most of these compounds, especially 6b, 7b, 9b, 11b and 7c, exhibited highly potent anti-HIV-1 activity against both wild-type and NNRTI-resistant HIV-1 strains. The compound 7b, that had the highest selectivity index (SI = 38,215), is more potent than Nevirapine and 18. These results suggest that introduction of halogen at the C-5 position may contribute to the effectiveness of these compounds against RTI-resistant variants. In addition, m-substituents on the C-6 aromatic moiety could significantly enhance activity against NNRTI-resistant HIV-1 strains. These compounds can be further developed as next-generation NNRTIs with improved antiviral efficacy and drug-resistance profile.
doi:10.1021/jm201506e
PMCID: PMC3312045  PMID: 22283377
HIV-1; Non-nucleoside reverse transcriptase inhibitors (NNRTIs); Drug-resistance
Scientific Reports  2012;2:971.
The growth of single-walled carbon nanotubes (SWCNTs) with predefined structure is of great importance for both fundamental research and their practical applications. Traditionally, SWCNTs are grown from a metal catalyst with a vapor-liquid-solid mechanism, where the catalyst is in liquid state with fluctuating structures, and it is intrinsically unfavorable for the structure control of SWCNTs. Here we report the heteroepitaxial growth of SWCNTs from a platelet boron nitride nanofiber (BNNF), which is composed of stacked (002) planes and is stable at high temperatures. SWCNTs are found to grow epitaxially from the open (002) edges of the BNNFs, and the diameters of the SWCNTs are multiples of the BN (002) interplanar distance. In situ transmission electron microscopy observations coupled with first principles calculations reveal that the growth of SWCNTs from the BNNFs follows a vapor-solid-solid mechanism. Our work opens opportunities for the control over the structure of SWCNTs by hetero-crystallographic epitaxy.
doi:10.1038/srep00971
PMCID: PMC3521219  PMID: 23240076

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