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1.  The Denitrification Characteristics of Pseudomonas stutzeri SC221-M and Its Application to Water Quality Control in Grass Carp Aquaculture 
PLoS ONE  2014;9(12):e114886.
To reduce ammonium and nitrite in aquaculture water, an isolate of the denitrifying bacterium Pseudomonas stutzeri, SC221-M, was obtained. The effects of various nitrogen and carbon sources, the ratio of carbon to nitrogen and temperature on bacterial growth, denitrification rates and the expression levels of nirS and nosZ in SC221-M were studied. The following conditions were determined to be optimal for growth and denitrification in SC221-M: NaNO2 as the nitrogen source, sodium citrate as the carbon source, a carbon to nitrogen ratio range of 4–8, and a temperature range of 20–35°C. Subsequently, SC221-M and the Bacillus cereus BSC24 strain were selected to generate microbial preparations. The results showed that addition of the microbial preparations decreased various hydrochemical parameters, including total dissolved solids, ammonium, nitrite, total nitrogen and the chemical oxygen demand. Nitrogen removal rates were highest on day 9; the removal rates of BSC24, SC221-M, a mixed preparation and a 3× mixed preparation were 24.5%, 26.6%, 53.9% and 53.4%, respectively. The mixed preparation (SC221-M+BSC24) was more effective at removing nitrogen than either the SC221-M or BSC24 preparation. Roche 454 pyrosequencing and subsequent analysis indicated that the control and other groups formed separate clusters, and the microbial community structure in the water changed significantly after the addition of microbial preparations. These results indicate that the addition of microbial preparations can improve both the water quality and microbial community structure in an experimental aquaculture system. P. stutzeri strain SC221-M and its related microbial preparations are potential candidates for the regulation of water quality in commercial aquaculture systems.
doi:10.1371/journal.pone.0114886
PMCID: PMC4260960  PMID: 25489740
2.  Polyethylene Glycol-Based Hydrogels for Controlled Release of the Antimicrobial Subtilosin for Prophylaxis of Bacterial Vaginosis 
Current treatment options for bacterial vaginosis (BV) have been shown to be inadequate at preventing recurrence and do not provide protection against associated infections, such as that with HIV. This study examines the feasibility of incorporating the antimicrobial peptide subtilosin within covalently cross-linked polyethylene glycol (PEG)-based hydrogels for vaginal administration. The PEG-based hydrogels (4% and 6% [wt/vol]) provided a two-phase release of subtilosin, with an initial rapid release rate of 4.0 μg/h (0 to 12 h) followed by a slow, sustained release rate of 0.26 μg/h (12 to 120 h). The subtilosin-containing hydrogels inhibited the growth of the major BV-associated pathogen Gardnerella vaginalis with a reduction of 8 log10 CFU/ml with hydrogels containing ≥15 μg entrapped subtilosin. In addition, the growth of four common species of vaginal lactobacilli was not significantly inhibited in the presence of the subtilosin-containing hydrogels. The above findings demonstrate the potential application of vaginal subtilosin-containing hydrogels for prophylaxis of BV.
doi:10.1128/AAC.02446-14
PMCID: PMC3993243  PMID: 24566190
3.  Culturable Heavy Metal-Resistant and Plant Growth Promoting Bacteria in V-Ti Magnetite Mine Tailing Soil from Panzhihua, China 
PLoS ONE  2014;9(9):e106618.
To provide a basis for using indigenous bacteria for bioremediation of heavy metal contaminated soil, the heavy metal resistance and plant growth-promoting activity of 136 isolates from V-Ti magnetite mine tailing soil were systematically analyzed. Among the 13 identified bacterial genera, the most abundant genus was Bacillus (79 isolates) out of which 32 represented B. subtilis and 14 B. pumilus, followed by Rhizobium sp. (29 isolates) and Ochrobactrum intermedium (13 isolates). Altogether 93 isolates tolerated the highest concentration (1000 mg kg−1) of at least one of the six tested heavy metals. Five strains were tolerant against all the tested heavy metals, 71 strains tolerated 1,000 mg kg−1 cadmium whereas only one strain tolerated 1,000 mg kg−1 cobalt. Altogether 67% of the bacteria produced indoleacetic acid (IAA), a plant growth-promoting phytohormone. The concentration of IAA produced by 53 isolates was higher than 20 µg ml−1. In total 21% of the bacteria produced siderophore (5.50–167.67 µg ml−1) with two Bacillus sp. producing more than 100 µg ml−1. Eighteen isolates produced both IAA and siderophore. The results suggested that the indigenous bacteria in the soil have beneficial characteristics for remediating the contaminated mine tailing soil.
doi:10.1371/journal.pone.0106618
PMCID: PMC4154735  PMID: 25188470
4.  Novel monodisperse PEGtide dendrons: design, fabrication and evaluation of mannose receptor-mediated macrophage targeting 
Bioconjugate chemistry  2013;24(8):1332-1344.
Novel PEGtide dendrons of generation 1 through 5 (G1.0-5.0) containing alternating discrete poly(ethylene glycol) (dPEG) and amino acid/peptide moieties were designed and developed. To demonstrate their targeting utility as nanocarriers, PEGtide dendrons functionalized with mannose residues were developed and evaluated for macrophage targeting. PEGtide dendrons were synthesized using 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) protocols. The N-α-Fmoc-N-ε-(5-carboxyfluorescein)-L-lysine (Fmoc-Lys(5-FAM)-OH) and monodisperse Fmoc-dPEG6-OH were sequentially coupled to Fmoc-β-Ala-resin to obtain the resin-bound intermediate Fmoc-dPEG6-Lys(5-FAM)-β-Ala (1). G1.0 dendrons were obtained by sequentially coupling Fmoc-Lys(Fmoc)-OH, Fmoc-β-Ala-OH, and Fmoc-dPEG6-OH to (1). Dendrons of higher generation, G2.0 to 5.0, were obtained by repeating the coupling cycles used for the synthesis of G1.0. Dendrons containing eight mannose residues (G3.0-Mannose8) were developed for mannose receptor (MR)-mediated macrophage targeting by conjugating α-D-mannopyranosylphenyl isothiocyanate to G3.0 dendrons. In the present study PEGtide dendrons up to G5.0 were synthesized. The molecular weights of the dendrons determined by MALDI-TOF were in agreement with calculated values. The hydrodynamic diameters measured using dynamic light scattering (DLS) ranged from 1 to 8 nm. Cell viability in the presence of G3.0 and G3.0-Mannose8 was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and was found to be statistically indistinguishable from that of untreated cells. G3.0-Mannose8 exhibited 12-fold higher uptake than unmodified G3.0 control dendrons in MR-expressing J774.E murine macrophage-like cells. Uptake was nearly completely inhibited in the presence of 10 mg/mL mannan, a mannose analog and known MR substrate. Confocal microscopy studies demonstrated the presence of significant intracellular punctate fluorescence colocalized with a fluid endocytosis marker with little surface fluorescence in cells incubated with G3.0-Mannose8. No significant cell-associated fluorescence was observed in cells incubated with G3.0 dendrons that did not contain the targeting ligand mannose. The current studies suggest that PEGtide dendrons could be useful as nanocarriers in drug delivery and imaging applications.
doi:10.1021/bc400011v
PMCID: PMC3940669  PMID: 23808323
Dendrons; macrophage targeting; PEGtide; solid-phase peptide synthesis (SPPS)
5.  Short-Term Effects of Tillage Practices on Soil Organic Carbon Turnover Assessed by δ13C Abundance in Particle-Size Fractions of Black Soils from Northeast China 
The Scientific World Journal  2014;2014:514183.
The combination of isotope trace technique and SOC fractionation allows a better understanding of SOC dynamics. A five-year tillage experiment consisting of no-tillage (NT) and mouldboard plough (MP) was used to study the changes in particle-size SOC fractions and corresponding δ13C natural abundance to assess SOC turnover in the 0–20 cm layer of black soils under tillage practices. Compared to the initial level, total SOC tended to be stratified but showed a slight increase in the entire plough layer under short-term NT. MP had no significant impacts on SOC at any depth. Because of significant increases in coarse particulate organic carbon (POC) and decreases in fine POC, total POC did not remarkably decrease under NT and MP. A distinct increase in silt plus clay OC occurred in NT plots, but not in MP plots. However, the δ13C abundances of both coarse and fine POC increased, while those of silt plus clay OC remained almost the same under NT. The C derived from C3 plants was mainly associated with fine particles and much less with coarse particles. These results suggested that short-term NT and MP preferentially enhanced the turnover of POC, which was considerably faster than that of silt plus clay OC.
doi:10.1155/2014/514183
PMCID: PMC4138936  PMID: 25162052
6.  Does Drug-Eluting Bead Transcatheter Arterial Chemoembolization Improve the Management of Patients with Hepatocellular Carcinoma? A Meta-Analysis 
PLoS ONE  2014;9(8):e102686.
Background
Drug eluting beads (DEB) are relatively new embolic agents that allow sustained release of chemotherapeutic agents in a localized fashion to the tumor. This technique is associated with reduced systemic side effects relative to systemic chemotherapy and an increase in the dose of antineoplastic agent delivered to the lesion. The meta-analysis was undertaken to assess the effectiveness of DEB-transcatheter arterial chemoembolization (TACE) in the management of hepatocellular cancer.
Methods
We searched the Web of Science, PubMed, EBSCO, EMBASE, the Wiley Library and Google Scholar for studies on DEB-TACE in the management of hepatocellular cancer from 1979 to April 2013. The risk of bias was assessed using RevMan 5·1. Random and fixed-effects meta-analytical models were used where indicated, and between-study heterogeneity was assessed. Disease control, complications and severe complications were recorded.
Results
Five studies met the selection criteria, three RCTs and two case-control studies, published from 2010 to 2012, included 217 patients in the DEB-TACE group and 237 in the conventional-TACE group. There was no significance over disease control (OR 2.27, 95% CI 0.78–6.63) with moderate between-study heterogeneity (χ2 = 6.83, degrees of freedom [df] = 3; p<0.08; I2 = 56%). Complications in both groups were assessed and no significant difference was observed (χ2 = 6.34, degrees of freedom [df] = 4; p<0.18; I2 = 37%). Severe complications were also assessed and no significant difference was observed (χ2 = 6.47, degrees of freedom [df] = 4; p<0.17; I2 = 38%). No publication bias relating to the above outcomes was detected by funnel plot. DEB-TACE benefited disease control without an increase in complications and severe complications.
doi:10.1371/journal.pone.0102686
PMCID: PMC4118844  PMID: 25083860
7.  HPV genotypes and associated cervical cytological abnormalities in women from the Pearl River Delta region of Guangdong province, China: a cross-sectional study 
BMC Infectious Diseases  2014;14:388.
Background
It is important to understand the specific HPV genotype distribution in screen-detected lesions. HPV Genotype is helpful for separating HPV-positive women at greater risk of cancer from those who can regress spontaneously and for preventing cervical cancer at early stage. The aim of this study was to investigate the high-risk HPV genotype distribution among cervical cytology abnormality in Pearl River Delta Region, Southern China
Methods
5585 HPV-infected women were screened from 77069 women in Pearl River Delta Region. Information was obtained from 3226 screened subjects through questionnaires and personal interviews. Exfoliated cervical cells were collected by doctors for HPV test with MassARRAY (Sequenom, Sandiego, CA) technique based on the matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS). The ThinPrep cytology test was performed to screen for cervical cancer. Unconditional logistic was used to determine the most common HPV carcinogenic types.
Results
Of the 3226 HPV-positive samples tested, 1744 (54.1%) with normal cervical cytology, 1482 (45.9%) with abnormal cytology. The five most common HPV types in this study were HPV16 (20.2%), HPV52 (17.1%), HPV58 (13.2%), HPV18 (9.5%), HPV6 (7.6%). Overall, HPV16 (OR = 10.5, 95% CI: 3.7 ~ 29.6), HPV33 (OR = 9.1, 95% CI: 2.8 ~ 29.2), HPV58 (OR = 6.3, 95% CI: 2.1 ~ 18.6), HPV31 (OR = 4.5, 95% CI: 1.3 ~ 15.5), multiple genotype infection (OR = 3.0, 95% CI: 1.7 ~ 14.7), especially HPV16 and HPV33, increased the risk of cytology abnormalities.
Conclusions
HPV16, HPV31, HPV33, HPV58, and multiple HPV genotype infection increased the risk of cytology abnormalities in Pearl River Delta Region and might be useful for the screening, preventing, treating, and monitoring of pre-cancer lesions in southern China.
doi:10.1186/1471-2334-14-388
PMCID: PMC4226991  PMID: 25016305
Human papillomavirus; Cytology; Cross-sectional study; Infection
8.  Curcumin attenuates angiogenesis in liver fibrosis and inhibits angiogenic properties of hepatic stellate cells 
Hepatic fibrosis is concomitant with sinusoidal pathological angiogenesis, which has been highlighted as novel therapeutic targets for the treatment of chronic liver disease. Our prior studies have demonstrated that curcumin has potent antifibrotic activity, but the mechanisms remain to be elucidated. The current work demonstrated that curcumin ameliorated fibrotic injury and sinusoidal angiogenesis in rat liver with fibrosis caused by carbon tetrachloride. Curcumin reduced the expression of a number of angiogenic markers in fibrotic liver. Experiments in vitro showed that the viability and vascularization of rat liver sinusoidal endothelial cells and rat aortic ring angiogenesis were not impaired by curcumin. These results indicated that hepatic stellate cells (HSCs) that are characterized as liver-specific pericytes could be potential target cells for curcumin. Further investigations showed that curcumin inhibited VEGF expression in HSCs associated with disrupting platelet-derived growth factor-β receptor (PDGF-βR)/ERK and mTOR pathways. HSC motility and vascularization were also suppressed by curcumin associated with blocking PDGF-βR/focal adhesion kinase/RhoA cascade. Gain- or loss-of-function analyses revealed that activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) was required for curcumin to inhibit angiogenic properties of HSCs. We concluded that curcumin attenuated sinusoidal angiogenesis in liver fibrosis possibly by targeting HSCs via a PPAR-γ activation-dependent mechanism. PPAR-γ could be a target molecule for reducing pathological angiogenesis during liver fibrosis.
doi:10.1111/jcmm.12286
PMCID: PMC4124023  PMID: 24779927
liver fibrosis; angiogenesis; hepatic stellate cell; curcumin; peroxisome proliferator-activated receptor-γ; VEGF
9.  The epidemiological characteristics of measles and difficulties of measles elimination in Hang Zhou, China 
Human Vaccines & Immunotherapeutics  2013;9(6):1296-1302.
Objective: Following the national proclaim of Measles Elimination 2012, plenty of activities for controlling the incidence had practiced in Hangzhou. However, the incidence did not decrease to low degree and remained perform as gap to the elimination target. The present study aimed to describe the epidemiological characteristics of measles, and proposed reasonable method to the target in Hangzhou. 
Method: Cases were collected by the National Notifiable Diseases Surveillance System (NNDSS) from 2004 to 2011. The descriptive epidemiology was employed to analyze characteristics of measles. 
Results: A total of 4712 confirmed cases were enrolled by the NNDSS with 7.87 per 100,000 people of incidence rate on average from 2004 to 2011. Individuals lived urban districts had higher risk of measles than counties. Infants aged <1 year observed the highest incidence rate with 239.35/100,000, and the age-specific incidence rate declined along with aged-group but reversed at adults. 52.20% of cases were floating cases and the measles vaccination was significantly different from the local cases (χ2=51.65,p <0.001). February to June was the epidemic period for measles incidence with 81.88% of cases reported in cluster. 
Conclusion: The descriptive characteristics of measles suggested that factors included infant and adult individual, floating population, and living urban area might be relate to the elimination target. More efforts were need to ensure susceptible population had accepted qualified measles vaccination. 
doi:10.4161/hv.24032
PMCID: PMC3901820  PMID: 23732896
measles; measles elimination; immunization strategy; descriptive epidemiology
10.  Clinical outcomes of transfusion-associated iron overload in patients with refractory chronic anemia 
Background
The purpose of this study was to evaluate the clinical outcomes of transfusion-associated iron overload in patients with chronic refractory anemia.
Methods
Clinical manifestations, main organ function, results of computed tomography (CT), endocrine evaluation, and serum ferritin levels were analyzed retrospectively in 13 patients who were transfusion-dependent for more than 1 year (receiving >50 units of red blood cells) to determine the degree of iron overload and efficacy of iron-chelating therapy.
Results
Serum ferritin levels increased to 1,830–5,740 ng/mL in all patients. Ten patients had abnormal liver function. The CT Hounsfield units in the liver increased significantly in eleven patients, and were proportional to their serum ferritin levels. Skin pigmentation, liver dysfunction, and endocrine dysfunction were observed in nine patients with serum ferritin >3,500 ng/mL, eight of whom have since died. Interestingly, serum ferritin levels did not decrease significantly in nine transfusion-dependent patients who had received 15–60 days of iron-chelating therapy.
Conclusion
Transfusion-dependent patients may progress to secondary iron overload with organ impairment, which may be fatal in those who are heavily iron-overloaded. The CT Hounsfield unit is a sensitive indicator of iron overload in the liver. Iron chelation therapy should be initiated when serum ferritin is >1,000 ng/mL and continued until it is <1,000 ng/mL in transfusional iron-overloaded patients.
doi:10.2147/PPA.S56238
PMCID: PMC4003266  PMID: 24790419
anemia; aplastic; iron overload; myelodysplastic syndromes
11.  Core Functional Sequence of C-terminal GAG-binding Domain Directs Cellular Uptake of IGFBP-3-derived Peptides 
Protein and peptide letters  2014;21(2):124-131.
The current study clarifies the role of the Glycosaminoglycan (GAG)-binding domain of insulin-like growth factor binding protein-3 (IGFBP-3) in cell penetration. The cell penetration function of IGFBP-3 has been mapped to an 18-residue GAG-binding domain in the C-terminal region that mobilizes cellular uptake and nuclear localization of unrelated proteins. Uptake of KW-22, a 22-residue peptide that encompasses the 18-residue GAG-binding domain, and another IGFBP-3 peptide carrying a streptavidin protein cargo was investigated in Chinese hamster ovary (CHO) cells defective at several steps of biosynthesis of cell surface GAGs. The severity of GAG truncation was highly correlated to the impairment of uptake ranging from complete abrogation to only a partial reduction, suggesting that GAG-binding is required for uptake. The 18-residue GAG-binding domain consists of an 8-residue KK-8 basic sequence devoid of Arg and an adjacent 10-residue QR-10 sequence rich in Arg. Peptide mapping of uptake and GAG-binding activities within the KW-22 peptide showed that the 8-residue KK-8 basic peptide retained 80% of GAG-binding activity with no uptake activity while the 10-residue QR-10 peptide retained 53% of uptake activity and 18% of GAG-binding activity. This suggests that KK-8 carries out the majority of GAG-binding function while QR-10 carries out the majority of the cell entry function. To our knowledge, this is the first report of physical separation of the uptake and GAG-binding functions within a short cell penetrating peptide and may shed light on the general mechanism of uptake of Arg-rich CPPs and guide new design of Arg-rich CPP-assisted drug/gene delivery systems.
PMCID: PMC3941648  PMID: 24059751
IGFBP-3; C-terminus; GAG; Cellular Uptake; cell penetration
12.  Randomized trial of the anti-FGF23 antibody KRN23 in X-linked hypophosphatemia 
The Journal of Clinical Investigation  2014;124(4):1587-1597.
Background. X-linked hypophosphatemia (XLH) is the most common heritable form of rickets and osteomalacia. XLH-associated mutations in phosphate-regulating endopeptidase (PHEX) result in elevated serum FGF23, decreased renal phosphate reabsorption, and low serum concentrations of phosphate (inorganic phosphorus, Pi) and 1,25-dihydroxyvitamin D [1,25(OH)2D]. KRN23 is a human anti-FGF23 antibody developed as a potential treatment for XLH. Here, we have assessed the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of KRN23 following a single i.v. or s.c. dose of KRN23 in adults with XLH.
Methods. Thirty-eight XLH patients were randomized to receive a single dose of KRN23 (0.003–0.3 mg/kg i.v. or 0.1–1 mg/kg s.c.) or placebo. PK, PD, immunogenicity, safety, and tolerability were assessed for up to 50 days.
Results. KRN23 significantly increased the maximum renal tubular threshold for phosphate reabsorption (TmP/GFR), serum Pi, and 1,25(OH)2D compared with that of placebo (P < 0.01). The maximum serum Pi concentration occurred later following s.c. dosing (8–15 days) compared with that seen with i.v. dosing (0.5–4 days). The effect duration was dose related and persisted longer in patients who received s.c. administration. Changes from baseline in TmP/GFR, serum Pi, and serum 1,25(OH)2D correlated with serum KRN23 concentrations. The mean t1/2 of KRN23 was 8–12 days after i.v. administration and 13–19 days after s.c. administration. Patients did not exhibit increased nephrocalcinosis or develop hypercalciuria, hypercalcemia, anti-KRN23 antibodies, or elevated serum parathyroid hormone (PTH) or creatinine.
Conclusion. KRN23 increased TmP/GFR, serum Pi, and serum 1,25(OH)2D. The positive effect of KR23 on serum Pi and its favorable safety profile suggest utility for KRN23 in XLH patients.
Trial registration. Clinicaltrials.gov NCT00830674.
Funding. Kyowa Hakko Kirin Pharma, Inc.
doi:10.1172/JCI72829
PMCID: PMC3973088  PMID: 24569459
13.  Clinical characteristics of congenital cervical atresia based on anatomy and ultrasound: a retrospective study of 32 cases 
Background
To explore the clinical characteristics of congenital cervical atresia.
Methods
This retrospective analysis included 32 cases of congenital cervical atresia treated from March 1984 to September 2010. The anatomic location, ultrasonic features, surgical treatments, and outcomes were recorded.
Results
Based on clinical characteristics observed during preoperative ultrasound and intraoperative exploration, congenital cervical atresia was divided into four types. Type I (n?=?22/32, 68.8%) is incomplete cervical atresia. Type II (n?=?5/32, 15.6%) defines a short and solid cervix with a round end; the structure lacked uterosacral and cardinal ligament attachments to the lower uterine body. Type III (n?=?2/32, 6.3%) is complete cervical atresia, in which the lowest region of the uterus exhibited a long and solid cervix. Type IV (n?=?3/32, 9.4%) defines the absence of a uterine isthmus, in which no internal os was detected, and a blind lumen was found under the uterus.
Conclusions
Observations of clinical characteristics of congenital cervical atresia based on the anatomy and ultrasound may inform diagnosis and treatment strategy.
doi:10.1186/2047-783X-19-10
PMCID: PMC3996070  PMID: 24555664
cervicovaginal operation; congenital cervical atresia; Müllerian duct anomaly
14.  Screening candidate anticancer drugs for brain tumor chemotherapy: Pharmacokinetic-driven approach for a series of (E)-N-(substituted aryl)-3-(substituted phenyl)propenamide analogues 
Investigational new drugs  2012;30(6):2263-2273.
Summary
A pharmacokinetic [PK]-driven screening process was implemented to select new agents for brain tumor chemotherapy from a series of low molecular weight anticancer agents [ON27x] that consisted of 141 compounds. The screening procedures involved a combination of in silico, in vitro and in vivo mouse studies that were cast into a pipeline of tier 1 and tier 2 failures that resulted in a final investigation of 2 analogues in brain tumor-bearing mice. Tier 1 failures included agents with a molecular weight of > 450 Da, a predicted log P (log P) of either < 2 or > 3.5, and a cytotoxicity IC50 value of > 2 uM. Next, 18 compounds underwent cassette dosing studies in normal mice that identified compounds with high systemic clearance, and low blood-brain barrier [BBB] penetration. These indices along with a derived parameter, referred to as the brain exposure index, comprised tier 2 failures that led to the administration of 2 compounds [ON27570, ON27740] as single agents [discrete dosing] to mice bearing intracerebral tumors. Comparison of ON27570’s resultant PK parameters to those obtained in the cassette dosing format suggested a drug-drug interaction most likely at the level of BBB transport, and prompted the use of the in vitro MDCK-MDR1 transport model to help assess the nature of the discrepancy. Overall, the approach was able to identify candidate compounds with suitable PK characteristics yet further revisions to the method, such as the use of in vitro metabolism and transport assays, may improve the PK-directed approach to identify efficacious agents for brain tumor chemotherapy.
doi:10.1007/s10637-012-9806-x
PMCID: PMC3387347  PMID: 22383114
Pharmacokinetics; Drug development; Brain tumor; CNS; Preclinical
15.  Integrated Pharmacokinetic-Driven Approach to Screen Candidate Anticancer Drugs for Brain Tumor Chemotherapy 
The AAPS Journal  2012;15(1):250-257.
The goal of the study was to develop an effective screening strategy to select new agents for brain tumor chemotherapy from a series of low molecular weight anticancer agents [ON123x] by the combined use of in silico, in vitro cytotoxicity, and in vitro ADME profiling studies. The results of these studies were cast into a pipeline of tier 1 and tier 2 procedures that resulted in the identification of ON123300 as the lead compound. Of the 154 ON123xx compounds, 13 met tier 1 screening criteria based on physicochemical properties [i.e., MW < 450 Da, predicted log P between 2 and 3.5] and in vitro glioma cell cytotoxicity [i.e., IC50 < 10 μM] and were further tested in tier 2 assays. The tier 2 profiling studies consisted of metabolic stability, MDCK-MDR1 cell permeability and plasma and brain protein binding that were combined to globally assess whether favorable pharmacokinetic properties and brain penetration could be achieved in vivo. In vivo cassette dosing studies were conducted in mice for 12 compounds that permitted examination of in vitro/in vivo relationships that confirmed the suitability of the in vitro assays. A parameter derived from the in vitro assays accurately predicted the extent of drug accumulation in the brain based on the area under the drug concentration–time curve in brain measured in the cassette dosing study (r2 = 0.920). Overall, the current studies demonstrated the value of an integrated pharmacokinetic-driven approach to identify potentially efficacious agents for brain tumor chemotherapy.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-012-9428-4) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-012-9428-4
PMCID: PMC3535101  PMID: 23180160
brain tumor; CNS; drug development; pharmacokinetics; preclinical
16.  Phytochemical Compositions and Antioxidant and Anti-Inflammatory Activities of Crude Extracts from Ficus pandurata H. (Moraceae) 
Background. Ficus pandurata H. (Moraceae) is widely used in traditional Chinese medicine as a healthy food condiment or a medicine for treatment of various diseases including inflammation. Objective. The purpose of the present study is to investigate the phytochemical compositions and antioxidant and anti-inflammatory activities of crude water (FPW) and ethanolic extracts (FPE) from Ficus pandurata H. Methods. Phytochemical compositions were identified by a high-performance liquid chromatography-electrospray ionization-mass spectrometry method (HPLC-ESI-MS). The antioxidant activities were evaluated by diphenylpicrylhydrazyl (DPPH) and hydroxyl radical assays, and the anti-inflammatory activities were evaluated by paw edema and levels of inflammatory mediator TNF-α and PGE2 in monosodium urate (MSU) crystal-induced rats. Results. Six compounds were identified by HPLC-MS method, and abundance of phenolics was found in FPE. The FPE showed concentration-dependent-significant scavenging of DPPH and hydroxyl radicals with IC50 values 118.4 and 192.9 μg/mL, respectively. The FPE treatment significantly inhibited the paw edema and the production of TNF-α and PGE2 in MSU crystal-induced rats. Conclusion. The FPE exerted stronger antioxidant and anti-inflammatory activities which may be attributed to its high phenolic content.
doi:10.1155/2013/215036
PMCID: PMC3804050  PMID: 24191163
17.  Upregulated MicroRNA-155 Expression in Peripheral Blood Mononuclear Cells and Fibroblast-Like Synoviocytes in Rheumatoid Arthritis 
Objective. This study was to screen for the miRNAs differently expressed in peripheral blood mononuclear cells (PBMC) of RA, to further identify the expression of miR-155 in RA PBMC and fibroblast-like synoviocytes (FLS), and to evaluate the function of miR-155 in RA-FLS. Methods. Microarray was used to screen for differentially expressed miRNAs in RA PBMC. miR-155 expression in PBMC and FLS of RA were identified by real-time PCR. Enforced overexpression and downexpression of miR-155 were used to investigate the function of miR-155 in RA-FLS. Expression of IKBKE which was previously identified as the actual target of miR-155 was examined by Western blot and real-time PCR in RA-FLS. Results. miR-155 levels were increased in both PBMC and FLS of RA and could be induced by TNF-α. Upregulation of miR-155 decreased MMP-3 levels and suppressed proliferation and invasion of RA-FLS. Inverse relationship between the expressions of miR-155 and the MMPs production-related protein IKBKE was found. Conclusion. An inflammatory milieu may alter miRNA expression profiles in rheumatoid arthritis. miR-155 is upregulated in RA-FLS, and it may be a protective factor against the inflammatory effect in part by attenuating expression of IKBKE.
doi:10.1155/2013/296139
PMCID: PMC3789322  PMID: 24151514
18.  The Fas/Fas Ligand Death Receptor Pathway Contributes to Phenylalanine-Induced Apoptosis in Cortical Neurons 
PLoS ONE  2013;8(8):e71553.
Phenylketonuria (PKU), an autosomal recessive disorder of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene, leads to childhood mental retardation by exposing neurons to cytotoxic levels of phenylalanine (Phe). A recent study showed that the mitochondria-mediated (intrinsic) apoptotic pathway is involved in Phe-induced apoptosis in cultured cortical neurons, but it is not known if the death receptor (extrinsic) apoptotic pathway and endoplasmic reticulum (ER) stress-associated apoptosis also contribute to neurodegeneration in PKU. To answer this question, we used specific inhibitors to block each apoptotic pathway in cortical neurons under neurotoxic levels of Phe. The caspase-8 inhibitor Z-IETD-FMK strongly attenuated apoptosis in Phe-treated neurons (0.9 mM, 18 h), suggesting involvement of the Fas receptor (FasR)-mediated cell death receptor pathway in Phe toxicity. In addition, Phe significantly increased cell surface Fas expression and formation of the Fas/FasL complex. Blocking Fas/FasL signaling using an anti-Fas antibody markedly inhibited apoptosis caused by Phe. In contrast, blocking the ER stress-induced cell death pathway with salubrinal had no effect on apoptosis in Phe-treated cortical neurons. These experiments demonstrate that the Fas death receptor pathway contributes to Phe-induced apoptosis and suggest that inhibition of the death receptor pathway may be a novel target for neuroprotection in PKU patients.
doi:10.1371/journal.pone.0071553
PMCID: PMC3737091  PMID: 23940767
19.  MicroRNA-326 Functions as a Tumor Suppressor in Glioma by Targeting the Nin One Binding Protein (NOB1) 
PLoS ONE  2013;8(7):e68469.
Malignant glioma is the most common type of primary brain tumor in adults, characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. Alterations in the activity of the 26S proteasome have been associated with malignant glioma cells, although the specific defects have not been identified. Recently, microRNA-326 (miR-326) was shown to play an important role in glioblastoma and breast cancer, but the underlying molecular mechanisms remain unclear. In the present study, the human Nin one binding protein (NOB1) was identified as a direct target of miR-326 and a potential oncogene in human glioma. Similar to NOB1 silencing by shRNA, overexpression of miR-326 in human glioma cell lines (A172 and U373) caused cell cycle arrest at the G1 phase, delayed cell proliferation and enhanced apoptosis. MiR-326 inhibited colony formation in soft agar and decreased growth of a xenograft tumor model, suggesting that miR-326 and NOB1 are required for tumorigenesis in vitro and in vivo. Furthermore, these processes were shown to involve the MAPK pathway. NOB1 overexpression in human glioma samples was detected by Affymetrix array analysis, and NOB1 mRNA and protein levels were shown to be increased in high-grade glioma compared to low-grade glioma and normal brain tissue. Furthermore, high levels of NOB1 were associated with unfavorable prognosis of glioma patients. Taken together, these results indicate that miR-326 and NOB1 may play an important role in the development of glioma.
doi:10.1371/journal.pone.0068469
PMCID: PMC3711818  PMID: 23869222
20.  Downregulation of MicroRNA-130a Contributes to Endothelial Progenitor Cell Dysfunction in Diabetic Patients via Its Target Runx3 
PLoS ONE  2013;8(7):e68611.
Dysfunction of endothelial progenitor cells (EPCs) contributes to diabetic vascular disease. MicroRNAs (miRs) have emerged as key regulators of diverse cellular processes including angiogenesis. We recently reported that miR-126, miR-130a, miR-21, miR-27a, and miR-27b were downregulated in EPCs from type II diabetes mellitus (DM) patients, and downregulation of miR-126 impairs EPC function. The present study further explored whether dysregulated miR-130a were also related to EPC dysfunction. EPCs were cultured from peripheral blood mononuclear cells of diabetic patients and healthy controls. Assays on EPC function (proliferation, migration, differentiation, apoptosis, and colony and tubule formation) were performed. Bioinformatics analyses were used to identify the potential targets of miR-130a in EPCs. Gene expression of miR-103a and Runx3 was measured by real-time PCR, and protein expression of Runx3, extracellular signal-regulated kinase (ERK), vascular endothelial growth factor (VEGF) and Akt was measured by Western blotting. Runx3 promoter activity was measured by luciferase reporter assay. A miR-130a inhibitor or mimic and lentiviral vectors expressing miR-130a, or Runx3, or a short hairpin RNA targeting Runx3 were transfected into EPCs to manipulate miR-130a and Runx3 levels. MiR-130a was decreased in EPCs from DM patients. Anti-miR-130a inhibited whereas miR-130a overexpression promoted EPC function. miR-130a negatively regulated Runx3 (mRNA, protein and promoter activity) in EPCs. Knockdown of Runx3 expression enhanced EPC function. MiR-130a also upregulated protein expression of ERK/VEGF and Akt in EPCs. In conclusion, miR-130a plays an important role in maintaining normal EPC function, and decreased miR-130a in EPCs from DM contributes to impaired EPC function, likely via its target Runx3 and through ERK/VEGF and Akt pathways.
doi:10.1371/journal.pone.0068611
PMCID: PMC3709913  PMID: 23874686
21.  miR-26a and its Target CKS2 Modulate Cell Growth and Tumorigenesis of Papillary Thyroid Carcinoma 
PLoS ONE  2013;8(7):e67591.
Background
While many studies have shown that levels of miR-26a are lower in papillary thyroid carcinoma (PTC), the role and mechanism of miR-26a in PTC are unclear.
Method
We used database searches to select potential mRNA targets of miR-26a. Anti-miR-26a, miR-26a mimic, siRNA for CKS2 and their effects on cell growth, cell-cycle distribution and colony formation were evaluated. We also evaluate the over-expressed miR-26a in TPC-1 cells in severe combined immune-deficient mice. We used luciferase reporter assays, real-time PCR and western blot analysis to measure the expression and activity of miR-26a, CKS2, and related factors such as cyclin B1, cyclin A, cdk1, bcl-xl and Akt. Finally, we measured the relationship between the levels of miR-26a and CKS2 in PTC and normal thyroid tissues.
Results
Relative to normal thyroid tissues, miR-26a is consistently down-regulated in TPC specimens, and CKS2 was identified as a potential target. Up-regulated miR-26a expression or down-regulated CKS2 expression in TPC-1 and CGTH W3 cells lines caused G2 phase-arrest. Decreased miR-26a expression or increased CKS2 expression could have inverse function on PTC cell lines. CyclinB1, cyclinA, bcl-xl and AKt are indirectly regulated by miR-26a in a CKS2-dependent manner. Finally, CKS2 is overexpressed in PTC specimens relative to normal thyroid tissue, and a significant inverse correlation exists between miR-26a and CKS2 expression in clinical PTC specimens.
Conclusion
Our data indicate that miR-26a functions as a growth-suppressive miRNA in PTC, and that its suppressive effects are mediated mainly by repressing CKS2 expression.
doi:10.1371/journal.pone.0067591
PMCID: PMC3702500  PMID: 23861775
22.  National Park Development in China: Conservation or Commercialization? 
Ambio  2011;41(3):247-261.
The rapid development of parks and ecotourism in China has attracted worldwide attention, not only for the beauty of the landscape that the parks are protecting but also for their abundant and often unique biodiversity. However, in some areas, the development of ecotourism has actually led to the degradation of local ecological, economic, and social systems. Using National Forest Parks for demonstration, this article analyzes the current political, institutional, legal, environmental, and economic issues concerning National Parks in China, and examines their potential future development. Although the intention of National Park systems in China is to raise environmental quality, and to protect biodiversity and social livelihoods, their success has varied. Future success will be measured by their capacity to reduce poverty, to promote long-term rehabilitation of wildlife habitats, and to simultaneously protect Chinese culture and biodiversity.
doi:10.1007/s13280-011-0194-9
PMCID: PMC3357853  PMID: 22038320
National Parks; Conservation; Natural resources; Degradation; Ecotourism; China
23.  The Angiotensin II Type 1 Receptor C-Terminal Lys Residues Interact with Tubulin and Modulate Receptor Export Trafficking 
PLoS ONE  2013;8(2):e57805.
The physiological and pathological functions of angiotensin II are largely mediated through activating the cell surface angiotensin II type 1 receptor (AT1R). However, the molecular mechanisms underlying the transport of newly synthesized AT1R from the endoplasmic reticulum (ER) to the cell surface remain poorly defined. Here we demonstrated that the C-terminus (CT) of AT1R directly and strongly bound to tubulin and the binding domains were mapped to two consecutive Lys residues at positions 310 and 311 in the CT membrane-proximal region of AT1R and the acidic CT of tubulin, suggestive of essentially ionic interactions between AT1R and tubulin. Furthermore, mutation to disrupt tubulin binding dramatically inhibited the cell surface expression of AT1R, arrested AT1R in the ER, and attenuated AT1R-mediated signaling measured as ERK1/2 activation. These data demonstrate for the first time that specific Lys residues in the CT juxtamembrane region regulate the processing of AT1R through interacting with tubulin. These data also suggest an important role of the microtubule network in the cell surface transport of AT1R.
doi:10.1371/journal.pone.0057805
PMCID: PMC3581488  PMID: 23451270
24.  Double Cohesion in Structural DNA Nanotechnology 
Organic & biomolecular chemistry  2006;4(18):3414-3419.
Double cohesion has proved to be a useful tool to assemble robust 2D arrays of large tiles. Here we present a variety of examples showing the utility of this approach. We apply this principle to the 3 types of 2D lattice sections of arrays whose individual tiles are inherently 3 dimensional, because they contain three vectors that span 3-space. This application includes motifs which are based on the tensegrity triangle, the six-helix bundle motif and on three skewed triple crossover molecules. All of these designs have the potential to form 3 dimensional structures if all three directions of propagation are allowed. If one direction is blunted, 2D arrays form, and all 3 combinations are presented here. In addition, a large parallelogram array that was not attainable previously using single duplex cohesion was also constructed using double cohesion. For comparison, arrays which use another type of double cohesion, double paranemic (PX) cohesion are also presented. Double cohesion of sticky ends proved to be the more effective tool to assemble large motifs into arrays.
doi:10.1039/b605212f
PMCID: PMC3491902  PMID: 17036134
DNA nanotechnology; 2D DNA lattices; robust DNA nanostructures
25.  High Bone Sialoprotein (BSP) Expression Correlates with Increased Tumor Grade and Predicts a Poorer Prognosis of High-Grade Glioma Patients 
PLoS ONE  2012;7(10):e48415.
Objectives
To investigate the expression and prognostic value of bone sialoprotein (BSP) in glioma patients.
Methods
We determined the expression of BSP using real-time RT-PCR and immunohistochemistry in tissue microarrays containing 15 normal brain and 270 glioma samples. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test. Univariate and multivariate analyses were performed by the stepwise forward Cox regression model.
Results
Both BSP mRNA and protein levels were significantly elevated in high-grade glioma tissues compared with those of normal brain and low-grade glioma tissues, and BSP expression positively correlated with tumor grade (P<0.001). Univariate and multivariate analysis showed high BSP expression was an independent prognostic factor for a shorter progression-free survival (PFS) and overall survival (OS) in both grade III and grade IV glioma patients [hazard ratio (HR) = 2.549 and 3.154 for grade III glioma, and HR = 1.637 and 1.574 for grade IV glioma, respectively]. Patients with low BSP expression had a significantly longer median OS and PFS than those with high BSP expression. Small extent of resection and lineage of astrocyte served as independent risk factors of both shorter PFS and OS in grade III glioma patients; GBM patients without O6-methylguanine (O6-meG) DNA methyltransferase (MGMT) methylation and Karnofsky performance score (KPS) less than 70 points were related to poor prognosis. Lack of radiotherapy related to shorter OS but not affect PFS in both grade III and grade IV glioma patients.
Conclusion
High BSP expression occurs in a significant subset of high-grade glioma patients and predicts a poorer outcome. The study identifies a potentially useful molecular marker for the categorization and targeted therapy of gliomas.
doi:10.1371/journal.pone.0048415
PMCID: PMC3485236  PMID: 23119009

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