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1.  Uptake and Metabolism of N-Acetylglucosamine and Glucosamine by Streptococcus mutans 
Applied and Environmental Microbiology  2014;80(16):5053-5067.
Glucosamine and N-acetylglucosamine are among the most abundant sugars on the planet, and their introduction into the oral cavity via the diet and host secretions, and through bacterial biosynthesis, provides oral biofilm bacteria with a source of carbon, nitrogen, and energy. In this study, we demonstrated that the dental caries pathogen Streptococcus mutans possesses an inducible system for the metabolism of N-acetylglucosamine and glucosamine. These amino sugars are transported by the phosphoenolpyruvate:sugar phosphotransferase system (PTS), with the glucose/mannose enzyme II permease encoded by manLMN playing a dominant role. Additionally, a previously uncharacterized gene product encoded downstream of the manLMN operon, ManO, was shown to influence the efficiency of uptake and growth on N-acetylglucosamine and, to a lesser extent, glucosamine. A transcriptional regulator, designated NagR, was able to bind the promoter regions in vitro, and repress the expression in vivo, of the nagA and nagB genes, encoding N-acetylglucosamine-6-phosphate deacetylase and glucosamine-6-phosphate deaminase, respectively. The binding activity of NagR could be inhibited by glucosamine-6-phosphate in vitro. Importantly, in contrast to the case with certain other Firmicutes, the gene for de novo synthesis of glucosamine-6-phosphate in S. mutans, glmS, was also shown to be regulated by NagR, and NagR could bind the glmS promoter region in vitro. Finally, metabolism of these amino sugars by S. mutans resulted in the production of significant quantities of ammonia, which can neutralize cytoplasmic pH and increase acid tolerance, thus contributing to enhanced persistence and pathogenic potential.
PMCID: PMC4135778  PMID: 24928869
2.  Analysis of the cervical spine sagittal alignment in young idiopathic scoliosis: a morphological classification of 120 cases 
European Spine Journal  2013;22(11):2372-2381.
To analyze the relationship between the cervical spine and global spinal-pelvic alignment in young patients with idiopathic scoliosis based on a morphological classification, and to postulate the hypothesis that cervical kyphosis is a part of cervico-thoracic kyphosis in them.
120 young patients with idiopathic scoliosis were recruited retrospectively between 2006 and 2011. The following values were measured and calculated: cervical angles (CA), cervico-thoracic angles (CTA), pelvic incidence (PI), pelvic tilt (PT), sacral slope (SS), spinal sacral angle (SSA), hip to C7/hip to sacrum, thoracic kyphosis (TK), lumbar lordosis (LL), Roussouly sagittal classification, Lenke Type Curve and Lumbar Modifier. The cervical curves were classified as lordosis, straight, sigmoid and kyphosis. They were categorized into four groups as cervical non-kyphosis group (CNK Group), cervical kyphosis group (CK Group), cervical-middle-thoracic kyphosis group (CMTK Group), and cervical-lower-thoracic kyphosis group (CLTK Group) according to their morphological characters of sagittal alignments. All parameters were compared and analyzed among groups.
The incidence of cervical kyphosis was 40 % (48/120). The CA and the CTA were in significant correlation (r = 0.854, P = 0.00). The cervical spine alignments were revealed to be significantly different among groups (r = 85.04, P = 0.00). Significant differences among groups in CA, CTA and TK were also detected. A strong correlation between the group type and Lenke Lumbar Modifier was still seen (P < 0.05). Fisher’s exact test revealed that the individual vertebral body kyphosis and wedging were directly related to the overall cervical kyphosis (P = 0.00, respectively).
The cervical kyphosis is correlated with global sagittal alignment, and is a part of cervico-thoracic sagittal deformity in young patients with idiopathic scoliosis. Despite the deformity in cervical alignment, the global spine could still be well-balanced with spontaneous adjustment. The correlation between our grouping based on the morphological characteristics of the sagittal alignments and Lenke Lumbar Modifier suggests that the coupled motion principle be appropriate to explain the modifications both in coronal and sagittal planes.
PMCID: PMC3886525  PMID: 23580056
Young idiopathic scoliosis; Cervical kyphosis; Cervico-thoracic sagittal alignment groups; Global sagittal alignment
3.  Efficacy and safety of ginger-salt-indirect moxibustion for urge urinary incontinence after stroke: protocol for a pilot multicentre randomised controlled trial 
BMJ Open  2014;4(10):e006326.
Ginger-salt-indirect moxibustion is widely applied to treat urge urinary incontinence after stroke, which is a common complication in stroke survivors. Moxa cone moxibustion and moxa box moxibustion are the main techniques of ginger-salt-indirect moxibustion. Our previous study had shown that ginger-salt-indirect moxibustion using moxa cones was feasible and effective for urination disorders post-stroke. This pilot study aims to assess the feasibility of conducting research to evaluate the efficacy and safety of ginger-salt-indirect moxibustion for patients with post-stroke urge urinary incontinence.
Methods and analysis
This is a multicentre, prospective, single-blinded, pilot randomised controlled trial. 120 eligible patients will be randomly allocated to three groups. Treatment group A (n=40) will receive moxa cone moxibustion and routine care; treatment group B (n=40) will receive moxa box moxibustion and routine care; control group (n=40) will only receive routine care for stroke recovery. The entire moxibustion treatment will consist of a total of 28 sessions during the course of 4 weeks. The primary outcome measure will be the increase in mean volume per void assessed at week 4 from the first moxibustion session (baseline). Secondary outcome measures will include mean frequency of urination per day and quality of life assessments measured by completion of the Incontinence Quality of Life Questionnaire and Barthel Index. All outcome measures will be assessed at baseline and at 4 and 16 weeks from baseline. Adverse events in the three groups will be recorded to assess the safety of moxibustion.
Ethics and dissemination
Research ethics was approved by the Research Ethical Committee of Beijing Hospital of Traditional Chinese Medicine Affiliated to the Capital Medical University (ref: 2013BL-094). Written informed consent will be obtained from all participants. Study results will be published in peer reviewed journals.
Trial registration number
ISRCTN 44706974.
PMCID: PMC4208054  PMID: 25335962
4.  Effects of ASKP1240 Combined With Tacrolimus or Mycophenolate Mofetil on Renal Allograft Survival in Cynomolgus Monkeys 
Transplantation  2014;98(3):267-276.
Supplemental digital content is available in the text.
Blocking the CD40-CD154 signal pathway has previously shown promise as a strategy to prevent allograft rejection. In this study, the efficacy of a novel fully human anti-CD40 monoclonal antibody—ASKP1240, administered as a monotherapy or combination therapy (subtherapeutic dose of tacrolimus or mycophenolate mofetil), on the prevention of renal allograft rejection was evaluated in Cynomolgus monkeys.
Heterotopic kidney transplants were performed in ABO-compatible, stimulation index 2.5 or higher in the two-way mixed lymphocyte reaction monkey pairs. Animals were divided into 12 groups and observed for a maximum of 180 days. Histopathologic, hematology, and biochemistry analyses were conducted in all groups. Cytokine release (interleukin [IL]-2, IL-4, IL-5, IL-6, tumor necrosis factor, and interferon-γ) was investigated in several groups.
ASKP1240 prolonged renal allograft survival in a dose-dependent manner in monotherapy. Low-dose (2 mg/kg) or high-dose (5 mg/kg) ASKP1240, in combination with mycophenolate mofetil (15 mg/kg) or tacrolimus (1 mg/kg), showed a significantly longer allograft survival time compared with monotherapy groups. No obvious side effects including drug-related thromboembolic complications were found. Cytokine release was not induced by ASKP1240 administration.
The present study indicates that ASKP1240, alone or in combination with other immunosuppressive drugs, could be a promising antirejection agent in organ transplantation.
PMCID: PMC4175122  PMID: 24992357
ASKP1240; Costimulation blockade; Kidney transplantation; Nonhuman primate
5.  “Out of Pollen” Hypothesis for Origin of New Genes in Flowering Plants: Study from Arabidopsis thaliana 
Genome Biology and Evolution  2014;6(10):2822-2829.
New genes, which provide material for evolutionary innovation, have been extensively studied for many years in animals where it is observed that they commonly show an expression bias for the testis. Thus, the testis is a major source for the generation of new genes in animals. The source tissue for new genes in plants is unclear. Here, we find that new genes in plants show a bias in expression to mature pollen, and are also enriched in a gene coexpression module that correlates with mature pollen in Arabidopsis thaliana. Transposable elements are significantly enriched in the new genes, and the high activity of transposable elements in the vegetative nucleus, compared with the germ cells, suggests that new genes are most easily generated in the vegetative nucleus in the mature pollen. We propose an “out of pollen” hypothesis for the origin of new genes in flowering plants.
PMCID: PMC4224333  PMID: 25237051
“Out of pollen” hypothesis; young gene evolution; Arabidopsis thaliana
6.  Use of 1-ml hollow tube-assisted radial artery catheterization in clinical anesthesiology 
Objective: To introduce a new modified technique for radial artery catheterization. Materials and Methods: A prolongated needle was made by using routine Vasocan Braunule needle and 1 ml syringe. A table of random digits was used for randomization of 32 interns. 14 interns were involved in group T and 18 interns were in group M. Each intern accomplished 20 cases. Then 640 patients were divided into 2 groups: group T included 280 patients with traditional direct technique, group M included 360 patients with 1 ml hollow tube-assisted technique. Results: Satisfactory results were obtained for 107 patients in group T and 292 patients in group M (P < 0.05). The success rates for catheter insertion after one attempt were 38.2% in group T and 81.1% in group M (P < 0.001). The blood flow times for observation were 1.7 ± 0.2 s in group T and 19.6 ± 1.8 s in group M (P < 0.001). Conclusion: The authors suggested the use of 1 ml hollow tube-assisted radial artery cannulation technique rather than a direct technique. This modified technique provided easy, safe, quick and less cost cannulation.
PMCID: PMC4211784  PMID: 25356134
Radial artery catheterization; modified; 1-ml hollow tube
7.  Safety and efficacy of S-1 chemotherapy in recurrent and metastatic nasopharyngeal carcinoma patients after failure of platinum-based chemotherapy: multi-institutional retrospective analysis 
This retrospective study evaluates the efficacy and safety of S-1 chemotherapy for recurrent and metastatic nasopharyngeal carcinoma patients after failure of platinum-based chemotherapy.
Patients and methods
Thirty-nine patients with recurrent and metastatic nasopharyngeal carcinoma who failed previous platinum-based chemotherapy received oral S-1 chemotherapy (twice daily from day 1 to 14) every 3 weeks. The dose of S-1 was determined according to the body surface area (BSA): 40 mg twice a day for BSA <1.25 m2; 50 mg twice a day for 1.25 m2 ≤BSA<1.5 m2; and 60 mg twice a day for BSA ≥1.5 m2.
Treatment was well tolerated. Most adverse events were mild. Grade 3 hematological toxicity occurred in 7.7%. There was one complete response (2.6%) and 12 partial responses (30.7%), giving an overall response rate of 33.3% (95% CI [confidence interval], 21.7–50.8). Median time-to-progression was 5.6 months, and median survival was 13.9 months. One- and 2-year survival rates were 60% and 26%, respectively.
S-1 monotherapy is considered a safe and effective treatment option for recurrent and metastatic nasopharyngeal carcinoma patients after failure of platinum-based chemotherapy.
PMCID: PMC4140443  PMID: 25152614
S-1; nasopharyngeal carcinoma; chemotherapy; platinum
8.  Modification of Gene Expression and Virulence Traits in Streptococcus mutans in Response to Carbohydrate Availability 
The genetic and phenotypic responses of Streptococcus mutans, an organism that is strongly associated with the development of dental caries, to changes in carbohydrate availability were investigated. S. mutans UA159 or a derivative of UA159 lacking ManL, which is the EIIAB component (EIIABMan) of a glucose/mannose permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and a dominant effector of catabolite repression, was grown in continuous culture to steady state under conditions of excess (100 mM) or limiting (10 mM) glucose. Microarrays using RNA from S. mutans UA159 revealed that 174 genes were differentially expressed in response to changes in carbohydrate availability (P < 0.001). Glucose-limited cells possessed higher PTS activity, could acidify the environment more rapidly and to a greater extent, and produced more ManL protein than cultures grown with excess glucose. Loss of ManL adversely affected carbohydrate transport and acid tolerance. Comparison of the histidine protein (HPr) in S. mutans UA159 and the manL deletion strain indicated that the differences in the behaviors of the strains were not due to major differences in HPr pools or HPr phosphorylation status. Therefore, carbohydrate availability alone can dramatically influence the expression of physiologic and biochemical pathways that contribute directly to the virulence of S. mutans, and ManL has a profound influence on this behavior.
PMCID: PMC3911228  PMID: 24271168
9.  A galactose-specific sugar:phosphotransferase permease is prevalent in the non-core genome of Streptococcus mutans 
Molecular oral microbiology  2013;28(4):292-301.
Three genes predicted to encode the A, B and C domains of a sugar:phosphotransferase system (PTS) permease specific for galactose (EIIGal) were identified in the genomes of 35 of 57 recently-sequenced isolates of Streptococcus mutans, the primary etiological agent of human dental caries. Mutants defective in the EIIGal complex were constructed in 6 of the isolates and showed markedly reduced growth rates on galactose-based medium relative to the parental strains. An EIIGal-deficient strain constructed using the invasive serotype f strain OMZ175 (OMZ/IIGal) expressed significantly lower PTS activity when galactose was present as the substrate. Galactose was shown to be an effective inducer of catabolite repression in OMZ175, but not in the EIIGal-deficient strain. In a mixed-species competition assay with galactose as the sole carbohydrate source, OMZ/IIGal was less effective than the parental strain at competing with the oral commensal bacterium Streptococcus gordonii, which has a high-affinity galactose transporter. Thus, a significant proportion of S. mutans strains encode a galactose PTS permease that could enhance the ability of these isolates to compete more effectively with commensal streptococci for galactose in salivary constituents and the diet.
PMCID: PMC3661675  PMID: 23421335
phosphotransferase system; galactose-PTS; tagatose pathway; biofilm; dental caries
10.  Hepatitis B surface antigen levels during natural history of chronic hepatitis B: A Chinese perspective study 
AIM: To determine the baseline hepatitis B surface antigen (HBsAg) levels during the different phases of chronic hepatitis B (CHB) patients in China.
METHODS: Six hundred and twenty-three hepatitis B virus or un-infected patients not receiving antiviral therapy were analyzed in a cross-sectional study. The CHB patients were classified into five phases: immune-tolerant (IT, n = 108), immune-clearance (IC, n = 161), hepatitis B e antigen negative hepatitis (ENH, n = 149), low-replicative (LR, n = 135), and liver cirrhosis (LC, n = 70). HBsAg was quantified (Abbott ARCHITECT assay) and correlated with hepatitis B virus (HBV) DNA, and serum alanine aminotransferase/aspartate aminotransferase (ALT/AST) in each phase of CHB was also determined.
RESULTS: Median HBsAg titers were different in each phase of CHB (P < 0.001): IT (4.85 log10 IU/mL), IC (4.36 log10 IU/mL), ENH (2.95 log10 IU/mL), LR (3.18 log10 IU/mL) and LC (2.69 log10 IU/mL). HBsAg titers were highest in the IT phase and lowest in the LC phase. Serum HBsAg titers showed a strong correlation with HBV viral load in the IC phase (r = 0.683, P < 0.001). No correlation between serum HBsAg level and ALT/AST was observed.
CONCLUSION: The mean baseline HBsAg levels differ significantly during the five phases of CHB, providing evidence on the natural history of HBV infection. HBsAg quantification may predict the effects of immune-modulator or oral nucleos(t)ide analogue therapy.
PMCID: PMC4112899  PMID: 25083092
Hepatitis B surface antigen quantification; Chronic hepatitis B; Natural history; Perspective
11.  HBsAg levels in HBeAg-positive chronic hepatitis B patients with different immune conditions 
AIM: To investigate hepatitis B surface antigen (HBsAg) levels in patients with HBeAg-positive chronic hepatitis B (CHB) and different immune conditions.
METHODS: HBeAg-positive CHB patients with different immune conditions were enrolled in this cross-sectional study. These patients were grouped according to the following criteria: immune-tolerant patients, IT group; patients with a mild immune response in the immune clearance phase, IC-Mild group; and patients with a dramatic immune response in the immune clearance phase and exhibiting acute on chronic liver failure (ACLF), ACLF group. All these patients had not previously received antiviral therapy and were enrolled at a pre-settled ratio of 2:2:1. Serum HBsAg levels and the correlation between serum HBsAg level and serum hepatitis B virus (HBV) DNA level were evaluated in these groups.
RESULTS: In total, 180 HBeAg-positive CHB patients [IT group (n = 72), IC-Mild group (n = 72), and ACLF group (n = 36)] were enrolled in this study. The median serum HBsAg levels varied among the groups (P < 0.001): IT, 4.86 log10 IU/mL; IC-Mild, 3.97 log10 IU/mL; and ACLF, 3.57 log10 IU/mL. Serum HBsAg level showed a moderate positive correlation with serum HBV-DNA level in the IC-Mild group (r = 0.60, P < 0.001), but exhibited a weaker correlation in the IT (r = 0.52, P < 0.001) and ACLF groups (r = 0.51, P = 0.001). The ratio of HBsAg/HBV DNA did not differ significantly among the IT, IC-Mild, and ACLF groups (medians: 0.56, 0.55, and 0.56, respectively; P = 0.179).
CONCLUSION: Serum HBsAg levels varied significantly in HBeAg-positive patients with different immune conditions. These findings may have important implications for understanding the immune clearance of HBV in HBeAg-positive CHB patients.
PMCID: PMC3989978  PMID: 24764680
Chronic hepatitis B; Hepatitis B surface antigen; Hepatitis B e antigen; Immune tolerance; Immune clearance; Acute on chronic liver failure
12.  Evolutionary and Population Genomics of the Cavity Causing Bacteria Streptococcus mutans 
Molecular Biology and Evolution  2012;30(4):881-893.
Streptococcus mutans is widely recognized as one of the key etiological agents of human dental caries. Despite its role in this important disease, our present knowledge of gene content variability across the species and its relationship to adaptation is minimal. Estimates of its demographic history are not available. In this study, we generated genome sequences of 57 S. mutans isolates, as well as representative strains of the most closely related species to S. mutans (S. ratti, S. macaccae, and S. criceti), to identify the overall structure and potential adaptive features of the dispensable and core components of the genome. We also performed population genetic analyses on the core genome of the species aimed at understanding the demographic history, and impact of selection shaping its genetic variation. The maximum gene content divergence among strains was approximately 23%, with the majority of strains diverging by 5–15%. The core genome consisted of 1,490 genes and the pan-genome approximately 3,296. Maximum likelihood analysis of the synonymous site frequency spectrum (SFS) suggested that the S. mutans population started expanding exponentially approximately 10,000 years ago (95% confidence interval [CI]: 3,268–14,344 years ago), coincidental with the onset of human agriculture. Analysis of the replacement SFS indicated that a majority of these substitutions are under strong negative selection, and the remainder evolved neutrally. A set of 14 genes was identified as being under positive selection, most of which were involved in either sugar metabolism or acid tolerance. Analysis of the core genome suggested that among 73 genes present in all isolates of S. mutans but absent in other species of the mutans taxonomic group, the majority can be associated with metabolic processes that could have contributed to the successful adaptation of S. mutans to its new niche, the human mouth, and with the dietary changes that accompanied the origin of agriculture.
PMCID: PMC3603310  PMID: 23228887
Streptococcus mutans; demographic inference; cavities; bacterial evolution; pan and core genome; infectious disease
13.  Fueling the caries process: carbohydrate metabolism and gene regulation by Streptococcus mutans  
Journal of Oral Microbiology  2014;6:10.3402/jom.v6.24878.
The nature of the oral cavity and host behaviors has mandated that the oral microbiota evolve mechanisms for coping with environmental fluctuations, especially changes in the type and availability of carbohydrates. In the case of human dental caries, the presence of excess carbohydrates is often responsible for altering the local environment to be more favorable for species associated with the initiation and progression of disease, including Streptococcus mutans. Some of the earliest endeavors to understand how cariogenic species respond to environmental perturbations were carried out using chemostat cultivation, which provides fine control over culture conditions and bacterial behaviors. The development of genome-scale methodologies has allowed for the combination of sophisticated cultivation technologies with genome-level analysis to more thoroughly probe how bacterial pathogens respond to environmental stimuli. Recent investigations in S. mutans and other closely related streptococci have begun to reveal that carbohydrate metabolism can drastically impact pathogenic potential and highlight the important influence that nutrient acquisition has on the success of pathogens; inside and outside of the oral cavity. Collectively, research into pathogenic streptococci, which have evolved in close association with the human host, has begun to unveil the essential nature of careful orchestration of carbohydrate acquisition and catabolism to allow the organisms to persist and, when conditions allow, initiate or worsen disease.
PMCID: PMC4157138  PMID: 25317251
carbohydrate transport; sugar phosphotransferase system; dental caries; biofilms; catabolite repression
14.  Correction: Phenotypic Heterogeneity of Genomically-Diverse Isolates of Streptococcus mutans 
PLoS ONE  2013;8(11):10.1371/annotation/ffff8cd5-b8fa-4d3c-a993-e5169198f1e6.
PMCID: PMC3826765  PMID: 24250771
15.  Acupuncture as prophylaxis for menstrual-related migraine: study protocol for a multicenter randomized controlled trial 
Trials  2013;14:374.
Menstrual-related migraine is a common form of migraine affecting >50% of female migraineurs. Acupuncture may be a choice for menstrual-related migraine, when pharmacological prophylaxis is not suitable. However, the efficacy of acupuncture has not been confirmed. We design and perform a randomized controlled clinical trial to evaluate the efficacy of acupuncture compared with naproxen in menstrual-related migraine patients.
This is a multicenter, single blind, randomized controlled clinical trial. A total of 184 participants will be randomly assigned to two different groups. Participants will receive verum acupuncture and placebo medicine in the treatment group, while participants in the control group will be treated with sham acupuncture and medicine (Naproxen Sustained Release Tablets). All treatments will be given for 3 months (menstrual cycles).
The primary outcome measures are the change of migraine days inside the menstrual cycle and the proportion of responders (defined as the proportion of patients with at least a 50% reduction in the number of menstrual migraine days). The secondary outcome measures are the change of migraine days outside the menstrual cycle, duration of migraine attack, the Visual Analogue Scale (VAS), and intake of acute medication. The assessment will be made at baseline (before treatment), 3 months (menstrual cycles), and 4 months (menstrual cycles) after the first acupuncture session.
The results of this trial will be helpful to supply the efficacy of acupuncture for menstrual-related migraine prophylaxis.
Trial registration
PMCID: PMC3830503  PMID: 24195839
16.  De novo combined lamivudine and adefovir dipivoxil therapy vs entecavir monotherapy for hepatitis B virus-related decompensated cirrhosis 
AIM: To compare efficacy of combined lamivudine (LAM) and adefovir dipivoxil (ADV) therapy with that of entecavir (ETV) monotherapy for hepatitis B virus (HBV)-related decompensated liver cirrhosis.
METHODS: A total of 120 naïve patients with HBV-related decompensated cirrhosis participated in this study. Sixty patients were treated with combined LAM and ADV therapy (LAM + ADV group), while the other 60 were treated with ETV monotherapy (ETV group) for two years. Tests for liver and kidney function, alpha-fetoprotein, HBV serum markers, HBV DNA load, prothrombin time (PT), and ultrasonography or computed tomography scan of the liver were performed every 1 to 3 mo. Repeated measure ANOVA and the χ2 test were performed to compare the efficacy, side effects, and the cumulative survival rates at 48 and 96 wk.
RESULTS: Forty-five patients in each group were observed for 96 wk. No significant differences in HBV DNA negative rates and alanine aminotransferase (ALT) normalization rates at weeks 48 (χ2 = 2.12 and 2.88) and 96 (χ2 = 3.21 and 3.24) between the two groups were observed. Hepatitis B e antigen seroconversion rate in the LAM + ADV group at week 96 was significantly higher in the ETV group (43.5% vs 36.4%, χ2 = 4.09, P < 0.05). Viral breakthrough occurred in 2 cases (4.4%) by week 48 and in 3 cases (6.7%) by week 96 in the LAM + ADV group, and no viral mutation was detected. In the ETV group, viral breakthrough occurred in 1 case (2.2%) at the end of week 96. An increase in albumin (F = 18.9 and 17.3), decrease in total bilirubin and in ALT (F = 16.5, 17.1 and 23.7, 24.8), reduced PT (F = 22.7 and 24.5), and improved Child-Turcotte-Pugh and the model for end-stage liver disease scores (F = 18.5, 17.8, and 24.2, 23.8) were observed in both groups. The cumulative rates of mortality and liver transplantation were 16.7% (10/60) and 18.3% (11/60) in the LAM + ADV and ETV groups, respectively.
CONCLUSION: Both LAM + ADV combination therapy and ETV monotherapy can effectively inhibit HBV replication, improve liver function, and decrease mortality.
PMCID: PMC3787360  PMID: 24115827
Chronic hepatitis B; Decompensated liver cirrhosis; Lamivudine; Adefovir dipivoxil; Combination therapy; Entecavir
17.  Comprehensive Mutational Analysis of Sucrose-Metabolizing Pathways in Streptococcus mutans Reveals Novel Roles for the Sucrose Phosphotransferase System Permease 
Journal of Bacteriology  2013;195(4):833-843.
Sucrose is perhaps the most efficient carbohydrate for the promotion of dental caries in humans, and the primary caries pathogen Streptococcus mutans encodes multiple enzymes involved in the metabolism of this disaccharide. Here, we engineered a series of mutants lacking individual or combinations of sucrolytic pathways to understand the control of sucrose catabolism and to determine whether as-yet-undisclosed pathways for sucrose utilization were present in S. mutans. Growth phenotypes indicated that gtfBCD (encoding glucan exopolysaccharide synthases), ftf (encoding the fructan exopolysaccharide synthase), and the scrAB pathway (sugar-phosphotransferase system [PTS] permease and sucrose-6-PO4 hydrolase) constitute the majority of the sucrose-catabolizing activity; however, mutations in any one of these genes alone did not affect planktonic growth on sucrose. The multiple-sugar metabolism pathway (msm) contributed minimally to growth on sucrose. Notably, a mutant lacking gtfBC, which cannot produce water-insoluble glucan, displayed improved planktonic growth on sucrose. Meanwhile, loss of scrA led to growth stimulation on fructooligosaccharides, due in large part to increased expression of the fruAB (fructanase) operon. Using the LevQRST four-component signal transduction system as a model for carbohydrate-dependent gene expression in strains lacking extracellular sucrases, a PlevD-cat (EIIALev) reporter was activated by pulsing with sucrose. Interestingly, ScrA was required for activation of levD expression by sucrose through components of the LevQRST complex, but not for activation by the cognate LevQRST sugars fructose or mannose. Sucrose-dependent catabolite repression was also evident in strains containing an intact sucrose PTS. Collectively, these results reveal a novel regulatory circuitry for the control of sucrose catabolism, with a central role for ScrA.
PMCID: PMC3562097  PMID: 23222725
18.  Genotypic variants at 2q33 and risk of esophageal squamous cell carcinoma in China: a meta-analysis of genome-wide association studies 
Abnet, Christian C. | Wang, Zhaoming | Song, Xin | Hu, Nan | Zhou, Fu-You | Freedman, Neal D. | Li, Xue-Min | Yu, Kai | Shu, Xiao-Ou | Yuan, Jian-Min | Zheng, Wei | Dawsey, Sanford M. | Liao, Linda M. | Lee, Maxwell P. | Ding, Ti | Qiao, You-Lin | Gao, Yu-Tang | Koh, Woon-Puay | Xiang, Yong-Bing | Tang, Ze-Zhong | Fan, Jin-Hu | Chung, Charles C. | Wang, Chaoyu | Wheeler, William | Yeager, Meredith | Yuenger, Jeff | Hutchinson, Amy | Jacobs, Kevin B. | Giffen, Carol A. | Burdett, Laurie | Fraumeni, Joseph F. | Tucker, Margaret A. | Chow, Wong-Ho | Zhao, Xue-Ke | Li, Jiang-Man | Li, Ai-Li | Sun, Liang-Dan | Wei, Wu | Li, Ji-Lin | Zhang, Peng | Li, Hong-Lei | Cui, Wen-Yan | Wang, Wei-Peng | Liu, Zhi-Cai | Yang, Xia | Fu, Wen-Jing | Cui, Ji-Li | Lin, Hong-Li | Zhu, Wen-Liang | Liu, Min | Chen, Xi | Chen, Jie | Guo, Li | Han, Jing-Jing | Zhou, Sheng-Li | Huang, Jia | Wu, Yue | Yuan, Chao | Huang, Jing | Ji, Ai-Fang | Kul, Jian-Wei | Fan, Zhong-Min | Wang, Jian-Po | Zhang, Dong-Yun | Zhang, Lian-Qun | Zhang, Wei | Chen, Yuan-Fang | Ren, Jing-Li | Li, Xiu-Min | Dong, Jin-Cheng | Xing, Guo-Lan | Guo, Zhi-Gang | Yang, Jian-Xue | Mao, Yi-Ming | Yuan, Yuan | Guo, Er-Tao | Zhang, Wei | Hou, Zhi-Chao | Liu, Jing | Li, Yan | Tang, Sa | Chang, Jia | Peng, Xiu-Qin | Han, Min | Yin, Wan-Li | Liu, Ya-Li | Hu, Yan-Long | Liu, Yu | Yang, Liu-Qin | Zhu, Fu-Guo | Yang, Xiu-Feng | Feng, Xiao-Shan | Wang, Zhou | Li, Yin | Gao, She-Gan | Liu, Hai-Lin | Yuan, Ling | Jin, Yan | Zhang, Yan-Rui | Sheyhidin, Ilyar | Li, Feng | Chen, Bao-Ping | Ren, Shu-Wei | Liu, Bin | Li, Dan | Zhang, Gao-Fu | Yue, Wen-Bin | Feng, Chang-Wei | Qige, Qirenwang | Zhao, Jian-Ting | Yang, Wen-Jun | Lei, Guang-Yan | Chen, Long-Qi | Li, En-Min | Xu, Li-Yan | Wu, Zhi-Yong | Bao, Zhi-Qin | Chen, Ji-Li | Li, Xian-Chang | Zhuang, Xiang | Zhou, Ying-Fa | Zuo, Xian-Bo | Dong, Zi-Ming | Wang, Lu-Wen | Fan, Xue-Pin | Wang, Jin | Zhou, Qi | Ma, Guo-Shun | Zhang, Qin-Xian | Liu, Hai | Jian, Xin-Ying | Lian, Sin-Yong | Wang, Jin-Sheng | Chang, Fu-Bao | Lu, Chang-Dong | Miao, Jian-Jun | Chen, Zhi-Guo | Wang, Ran | Guo, Ming | Fan, Zeng-Lin | Tao, Ping | Liu, Tai-Jing | Wei, Jin-Chang | Kong, Qing-Peng | Fan, Lei | Wang, Xian-Zeng | Gao, Fu-Sheng | Wang, Tian-Yun | Xie, Dong | Wang, Li | Chen, Shu-Qing | Yang, Wan-Cai | Hong, Jun-Yan | Wang, Liang | Qiu, Song-Liang | Goldstein, Alisa M. | Yuan, Zhi-Qing | Chanock, Stephen J. | Zhang, Xue-Jun | Taylor, Philip R. | Wang, Li-Dong
Human Molecular Genetics  2012;21(9):2132-2141.
Genome-wide association studies have identified susceptibility loci for esophageal squamous cell carcinoma (ESCC). We conducted a meta-analysis of all single-nucleotide polymorphisms (SNPs) that showed nominally significant P-values in two previously published genome-wide scans that included a total of 2961 ESCC cases and 3400 controls. The meta-analysis revealed five SNPs at 2q33 with P< 5 × 10−8, and the strongest signal was rs13016963, with a combined odds ratio (95% confidence interval) of 1.29 (1.19–1.40) and P= 7.63 × 10−10. An imputation analysis of 4304 SNPs at 2q33 suggested a single association signal, and the strongest imputed SNP associations were similar to those from the genotyped SNPs. We conducted an ancestral recombination graph analysis with 53 SNPs to identify one or more haplotypes that harbor the variants directly responsible for the detected association signal. This showed that the five SNPs exist in a single haplotype along with 45 imputed SNPs in strong linkage disequilibrium, and the strongest candidate was rs10201587, one of the genotyped SNPs. Our meta-analysis found genome-wide significant SNPs at 2q33 that map to the CASP8/ALS2CR12/TRAK2 gene region. Variants in CASP8 have been extensively studied across a spectrum of cancers with mixed results. The locus we identified appears to be distinct from the widely studied rs3834129 and rs1045485 SNPs in CASP8. Future studies of esophageal and other cancers should focus on comprehensive sequencing of this 2q33 locus and functional analysis of rs13016963 and rs10201587 and other strongly correlated variants.
PMCID: PMC3315211  PMID: 22323360
19.  A Study of the Blue-Light-Dependent Phosphorylation, Degradation, and Photobody Formation of Arabidopsis CRY2 
Molecular Plant  2012;5(3):200-207.
Arabidopsis cryptochrome 2 (CRY2) is a blue-light receptor mediating blue-light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation. CRY2 is a constitutive nuclear protein that undergoes blue-light-dependent phosphorylation, ubiquitination, photobody formation, and degradation in the nucleus, but the relationship between these blue-light-dependent events remains unclear. It has been proposed that CRY2 phosphorylation triggers a conformational change responsible for the subsequent ubiquitination and photobody formation, leading to CRY2 function and/or degradation. We tested this hypothesis by a structure-function study, using mutant CRY2–GFP fusion proteins expressed in transgenic Arabidopsis. We show that changes of lysine residues of the NLS (Nuclear Localization Signal) sequence of CRY2 to arginine residues partially impair the nuclear importation of the CRY2K541R and CRY2K554/5R mutant proteins, resulting in reduced phosphorylation, physiological activities, and degradation in response to blue light. In contrast to the wild-type CRY2 protein that forms photobodies exclusively in the nucleus, the CRY2K541R and CRY2K554/5R mutant proteins form protein bodies in both the nucleus and cytosol in response to blue light. These results suggest that photoexcited CRY2 molecules can aggregate to form photobody-like structure without the nucleus-dependent protein modifications or the association with the nuclear CRY2-interacting proteins. Taken together, the observation that CRY2 forms photobodies markedly faster than CRY2 phosphorylation in response to blue light, we hypothesize that the photoexcited cryptochromes form oligomers, preceding other biochemical changes of CRY2, to facilitate photobody formation, signal amplification, and propagation, as well as desensitization by degradation.
PMCID: PMC3355346  PMID: 22311776
protein phosphorylation; signal transduction; fluorescence imaging; protein degradation; photobody
20.  Phenotypic Heterogeneity of Genomically-Diverse Isolates of Streptococcus mutans 
PLoS ONE  2013;8(4):e61358.
High coverage, whole genome shotgun (WGS) sequencing of 57 geographically- and genetically-diverse isolates of Streptococcus mutans from individuals of known dental caries status was recently completed. Of the 57 sequenced strains, fifteen isolates, were selected based primarily on differences in gene content and phenotypic characteristics known to affect virulence and compared with the reference strain UA159. A high degree of variability in these properties was observed between strains, with a broad spectrum of sensitivities to low pH, oxidative stress (air and paraquat) and exposure to competence stimulating peptide (CSP). Significant differences in autolytic behavior and in biofilm development in glucose or sucrose were also observed. Natural genetic competence varied among isolates, and this was correlated to the presence or absence of competence genes, comCDE and comX, and to bacteriocins. In general strains that lacked the ability to become competent possessed fewer genes for bacteriocins and immunity proteins or contained polymorphic variants of these genes. WGS sequence analysis of the pan-genome revealed, for the first time, components of a Type VII secretion system in several S. mutans strains, as well as two putative ORFs that encode possible collagen binding proteins located upstream of the cnm gene, which is associated with host cell invasiveness. The virulence of these particular strains was assessed in a wax-worm model. This is the first study to combine a comprehensive analysis of key virulence-related phenotypes with extensive genomic analysis of a pathogen that evolved closely with humans. Our analysis highlights the phenotypic diversity of S. mutans isolates and indicates that the species has evolved a variety of adaptive strategies to persist in the human oral cavity and, when conditions are favorable, to initiate disease.
PMCID: PMC3628994  PMID: 23613838
21.  Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity 
mAbs  2013;5(3):418-431.
Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies.
PMCID: PMC4169035  PMID: 23567210
IgG1; IgG1λ; Lc; antibody; disulfide bond; lambda light chain; light chain; serine; stability and serine deletion
22.  Gene Regulation by CcpA and Catabolite Repression Explored by RNA-Seq in Streptococcus mutans 
PLoS ONE  2013;8(3):e60465.
A bacterial transcriptome of the primary etiological agent of human dental caries, Streptococcus mutans, is described here using deep RNA sequencing. Differential expression profiles of the transcriptome in the context of carbohydrate source, and of the presence or absence of the catabolite control protein CcpA, revealed good agreement with previously-published DNA microarrays. In addition, RNA-seq considerably expanded the repertoire of DNA sequences that showed statistically-significant changes in expression as a function of the presence of CcpA and growth carbohydrate. Novel mRNAs and small RNAs were identified, some of which were differentially expressed in conditions tested in this study, suggesting that the function of the S. mutans CcpA protein and the influence of carbohydrate sources has a more substantial impact on gene regulation than previously appreciated. Likewise, the data reveal that the mechanisms underlying prioritization of carbohydrate utilization are more diverse than what is currently understood. Collectively, this study demonstrates the validity of RNA-seq as a potentially more-powerful alternative to DNA microarrays in studying gene regulation in S. mutans because of the capacity of this approach to yield a more precise landscape of transcriptomic changes in response to specific mutations and growth conditions.
PMCID: PMC3610829  PMID: 23555977
23.  Two Gene Clusters Coordinate Galactose and Lactose Metabolism in Streptococcus gordonii 
Applied and Environmental Microbiology  2012;78(16):5597-5605.
Streptococcus gordonii is an early colonizer of the human oral cavity and an abundant constituent of oral biofilms. Two tandemly arranged gene clusters, designated lac and gal, were identified in the S. gordonii DL1 genome, which encode genes of the tagatose pathway (lacABCD) and sugar phosphotransferase system (PTS) enzyme II permeases. Genes encoding a predicted phospho-β-galactosidase (LacG), a DeoR family transcriptional regulator (LacR), and a transcriptional antiterminator (LacT) were also present in the clusters. Growth and PTS assays supported that the permease designated EIILac transports lactose and galactose, whereas EIIGal transports galactose. The expression of the gene for EIIGal was markedly upregulated in cells growing on galactose. Using promoter-cat fusions, a role for LacR in the regulation of the expressions of both gene clusters was demonstrated, and the gal cluster was also shown to be sensitive to repression by CcpA. The deletion of lacT caused an inability to grow on lactose, apparently because of its role in the regulation of the expression of the genes for EIILac, but had little effect on galactose utilization. S. gordonii maintained a selective advantage over Streptococcus mutans in a mixed-species competition assay, associated with its possession of a high-affinity galactose PTS, although S. mutans could persist better at low pHs. Collectively, these results support the concept that the galactose and lactose systems of S. gordonii are subject to complex regulation and that a high-affinity galactose PTS may be advantageous when S. gordonii is competing against the caries pathogen S. mutans in oral biofilms.
PMCID: PMC3406145  PMID: 22660715
24.  Transmission and Maintenance Cycle of Bartonella quintana among Rhesus Macaques, China 
Emerging Infectious Diseases  2013;19(2):297-300.
We detected Bartonella quintana in 48.6% of captive rhesus macaques from an animal facility in Beijing, China. Prevalence of infection increased over the period of observation. Our findings suggest that macaques may serve as reservoir hosts for B. quintana and that Pedicinus obtusus lice might act as efficient vectors.
PMCID: PMC3563275  PMID: 23347418
Bartonella quintana; rhesus macaques; reservoir host; lice; transmission; China; vector-borne infections; Bartonella
25.  Establishment, characterization and immortalization of a fibroblast cell line from the Chinese red belly toad Bombina maxima skin 
Cytotechnology  2011;64(1):95-105.
The skin of the amphibian Bombina maxima is rich in biologically active proteins and peptides, most of which have mammalian analogues. The physiological functions of most of the mammalian analogues are still unknown. Thus, Bombina maxima skin may be a promising model to reveal the physiological role of these proteins and peptides because of their large capacity for secretion. To investigate the physiological role of these proteins and peptides in vitro, a fibroblast cell line was successfully established from Bombina maxima tadpole skin. The cell line grew to form a monolayer with cells of a uniform shape and abundant rough endoplasmic reticulum, which are typical characteristics of fibroblasts. Further identification at a molecular level revealed that they strongly expressed the fibroblast marker protein vimentin. The chromosome number of these cells is 2n = 28, and most of them were diploid. Growth property analysis showed that they grew well for 14 passages. However, cells showed decreased proliferative ability after passage 15. Thus, we tried to immortalize the cells through the overexpression of SV40 T antigen. After selecting by G418, cells stably expressed SV40 large T antigen and showed enhanced proliferative ability and increased telomerase activity. Signal transduction analysis revealed functional p42 mitogen-activated protein (MAP) kinase in immortalized Bombina maxima dermal fibroblasts. Primary fibroblast cells and the immortalized fibroblast cells from Bombina maxima cultured in the present study can be used to investigate the physiological role of Bombina maxima skin-secreted proteins and peptides. In addition, the methods for primary cell culturing and cell immortalization will be useful for culturing and immortalizing cells from other types of amphibians.
PMCID: PMC3261454  PMID: 21948116
Bombina maxima; Fibroblast; Immortalization; Skin; Vimentin

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