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1.  Genetic Polymorphisms of SP-A, SP-B, and SP-D and Risk of Respiratory Distress Syndrome in Preterm Neonates 
Background
We examined selected polymorphisms in 3 pulmonary surfactant-associated proteins (SP) for their influence on serum SP levels and risk of respiratory distress syndrome (RDS) in preterm neonates.
Material/Methods
Premature infants from a Han population were enrolled, including 100 premature infants with RDS (case group) and 120 premature infants without RDS (control group). SNP genotyping for SP-A (+186A/G and +655C/T), SP-B (−18A/C and 1580C/T), and SP-D (Met11ThrT/C and Ala160ThrG/A) used polymerase chain reaction-restriction fragment length polymorphism. Haplotypes were calculated with Shesis software and serum SP-A/B/D levels were quantified by ELISA.
Results
Case and control groups exhibited significant differences in genotype and allele frequencies of SP-A (+186A/G, +655C/T) and SP-B (1580C/T). However, no statistically significant differences were observed in the allele and genotype frequencies of SP-B −18A/C, SP-D Met11ThrT/C, and SP-D Ala160ThrG/A. Importantly, serum SP-A and SP-B levels were reduced in RDS patients carrying SP-A (+186A/G, +655C/T) and SP-B (1580C/T) polymorphisms. AA genotype of +186A/G, SP-A level, and CC genotype of 1580C/T were independently correlated with increased RDS risk.
Conclusions
SP-A (+186A/G) and SP-B (1580C/T) polymorphisms are strongly associated with the risk of RDS in preterm infants. Notably, reduced serum SP-A levels were correlated with a high risk of RDS and may serve as novel biomarkers for RDS detection and monitoring.
doi:10.12659/MSM.898553
PMCID: PMC5207009  PMID: 28011976
Infant, Premature; Polymorphism, Single Nucleotide; Pulmonary Surfactant-Associated Proteins
2.  Bone Healing Improvements Using Hyaluronic Acid and Hydroxyapatite/Beta-Tricalcium Phosphate in Combination: An Animal Study 
BioMed Research International  2016;2016:8301624.
The purpose of this study was to investigate whether the use of HLA as an aqueous binder of hydroxyapatite/beta-tricalcium phosphate (HA-βTCP) particles can reduce the amount of bone graft needed and increase ease of handling in clinical situations. In this study, HA/βTCP was loaded in commercially available crosslinking HLA to form a novel HLA/HA-βTCP composite. Six New Zealand White rabbits (3.0–3.6 kg) were used as test subjects. Four 6 mm defects were prepared in the parietal bone. The defects were filled with the HLA/HA-βTCP composite as well as HA-βTCP particle alone. New bone formation was analyzed by micro-CT and histomorphometry. Our results indicated that even when the HA-βTCP particle numbers were reduced, the regenerative effect on bone remained when the HLA existed. The bone volume density (BV/TV ratio) of HLA/HA-βTCP samples was 1.7 times larger than that of the control sample at week 2. The new bone increasing ratio (NBIR) of HLA/HA-βTCP samples was 1.78 times higher than the control group at week 2. In conclusion, HA-βTCP powder with HLA contributed to bone healing in rabbit calvarial bone defects. The addition of HLA to bone grafts not only promoted osteoconduction but also improved handling characteristics in clinical situations.
doi:10.1155/2016/8301624
PMCID: PMC5192297  PMID: 28070520
3.  Biological Thiols-Triggered Hydrogen Sulfide Releasing Microfibers for Tissue Engineering Applications 
Acta biomaterialia  2015;27:205-213.
By electrospinning of polycaprolactone (PCL) solutions containing N-(benzoylthio)benzamide (NSHD1), a H2S donor, fibrous scaffolds with hydrogen sulfide (H2S) releasing capability (H2S-fibers) are fabricated. The resultant microfibers are capable of releasing H2S upon immersion in aqueous solution containing biological thiols under physiological conditions. The H2S release peaks of H2S-fibers appeared at 2~4 hours, while the peak of donor alone showed at 45 minutes. H2S release half-lives of H2S-fibers were 10–20 times longer than that of donor alone. Furthermore, H2S-fibers can protect cells from H2O2 induced oxidative damage by significantly decreasing the production of intracellular reactive oxygen species (ROS). Finally, we investigated the H2S-fibers application as a wound dressing in vitro. Given that H2S has a broad range of physiological functions, H2S-fibers hold great potential for various biomedical applications.
Graphical abstract
doi:10.1016/j.actbio.2015.09.010
PMCID: PMC4609630  PMID: 26363376
hydrogen sulfide; controlled release; electrospinning; NSHD1; ischemia reperfusion injury
4.  Fuzzy Decision Making Approach to Identify Optimum Enzyme Targets and Drug Dosage for Remedying Presynaptic Dopamine Deficiency 
PLoS ONE  2016;11(10):e0164589.
Model-based optimization approaches are valuable in developing new drugs for human metabolic disorders. The core objective in most optimal drug designs is positive therapeutic effects. In this study, we considered the effects of therapeutic, adverse, and target variation simultaneously. A fuzzy optimization method was applied to formulate a multiobjective drug design problem for detecting enzyme targets in the presynaptic dopamine metabolic network to remedy two types of enzymopathies caused by deficiencies of vesicular monoamine transporter 2 (VMAT2) and tyrosine hydroxylase (TH). The fuzzy membership approach transforms a two-stage drug discovery problem into a unified decision-making problem. We developed a nested hybrid differential evolution algorithm to efficiently identify a set of potential drug targets. Furthermore, we also simulated the effects of current clinical drugs for Parkinson’s disease (PD) in this model and tried to clarify the possible causes of neurotoxic and neuroprotective effects. The optimal drug design could yield 100% satisfaction grade when both therapeutic effect and the number of targets were considered in the objective. This scenario required regulating one to three and one or two enzyme targets for 50%–95% and 50%–100% VMAT2 and TH deficiencies, respectively. However, their corresponding adverse and target variation effect grades were less satisfactory. For the most severe deficiencies of VMAT2 and TH, a compromise design could be obtained when the effects of therapeutic, adverse, and target variation were simultaneously applied to the optimal drug discovery problem. Such a trade-off design followed the no free lunch theorem for optimization; that is, a more serious dopamine deficiency required more enzyme targets and lower satisfaction grade. In addition, the therapeutic effects of current clinical medications for PD could be enhanced in combination with new enzyme targets. The increase of toxic metabolites after treatment might be the cause of neurotoxic effects of some current PD medications.
doi:10.1371/journal.pone.0164589
PMCID: PMC5063375  PMID: 27736960
5.  Impact of early-onset peritonitis on mortality and technique survival in peritoneal dialysis patients 
SpringerPlus  2016;5(1):1676.
Background
Early onset peritonitis (EOP) is not uncommon in peritoneal dialysis patients. We aimed to compare the prognosis of EOP and non-EOP peritoneal dialysis patients.
Methods
This study included subjects that underwent PD from January 1, 2004 to July 31, 2013. Patient characteristics were collected. EOP was defined as peritonitis occurring within 6 months after initiation of PD. Patient and technique survival were compared between EOP and non-EOP patients using Cox regression analyses.
Results
In total, 189 subjects were included in this study. Patients were divided into EOP (n = 55) and non-EOP groups (n = 134). There was no significant difference in the causative organisms of peritonitis between the two groups. After adjusting for age, diabetes status, serum albumin level and residual renal function, the multivariable Cox regression model revealed that EOP was an independent risk factor for patient mortality (HR 2.03, RI 1.09–3.80, p = 0.026), technique failure (HR 1.69, RI 1.12–2.87, p = 0.015) and total survival (HR 1.73, RI 1.12–2.68, p = 0.013).
Conclusions
EOP was identified as an independent risk factor for mortality and technique failure in peritoneal dialysis patients.
doi:10.1186/s40064-016-3369-9
PMCID: PMC5040655  PMID: 27733978
Early onset peritonitis; Mortality; Peritoneal dialysis
6.  The protective effect of hydrogen sulfide on systemic sclerosis associated skin and lung fibrosis in mice model 
SpringerPlus  2016;5(1):1084.
Backgroud
Systemic sclerosis (SSc) caused fibrosis can be fatal and it still lack of effective treatment. Hydrogen sulfide (H2S) appears to be an attractive therapeutic candidates. This study aimed to investigate the protective effect of H2S on SSc-associated skin and lung fibrosis.
Methods
We developed a model of SSc by subcutaneous injecting BLM to female C3H mice. The mice received daily subcutaneous injections of NaHS (56 and 112 μg/kg), an H2S donor. On days 7, 28, and 42, the mice were killed and blood samples were collected to measure the plasma H2S concentration, the skin and lung tissues was harvested for microscopic examination, immunohistochemistry and quantify biological parameters (hydroxyproline content, RT-qPCR and Western blot).
Results
In model group, the dermis of skin tissues at different time points gradually thickened, collagen deposition increased. The lung tissues presented pathological changes such as obvious inflammatory cell infiltration, increased collagen deposition and the plasma H2S concentrations points significantly decreased. Administration of NaHS markedly decreased the biomarkers of fibrosis such as α-smooth muscle actin, collagen-I, collagen-III, fibronectin, transforming growth factor-β1, Smad2/3 phosphorylation and inflammation including the marker protein of monocyte/macrophage and monocyte chemoattractant protein-1 in the lung. Compared to the low dose group, the expression in the high dose group have decreased trend, but the difference was not significant.
Conclusion
We demonstrate the beneficial effects of H2S on SSc-associated skin and lung fibrosis. H2S may be a potential therapy against this intractable disease.
doi:10.1186/s40064-016-2774-4
PMCID: PMC4947075  PMID: 27468384
Systemic sclerosis; Fibrosis; Transforming growth factor-β1; Hydrogen sulfide
7.  Fibronectin-Grafted Titanium Dental Implants: An In Vivo Study 
BioMed Research International  2016;2016:2414809.
Modification of the physiochemical properties of titanium surfaces using glow discharge plasma (GDP) and fibronectin coating has been shown to enhance the surface hydrophilicity, surface roughness, cell adhesion, migration, and proliferation. This in vivo study aimed to evaluate the bone integration efficacy of a biologically modified implant surface. Two different surface-modified implants (Ar-GDP and GDP-fib) were placed in the mandibular premolar area of six beagle dogs for 2–8 weeks. Three techniques [histologic evaluation, resonance frequency analysis (RFA), and microcomputed tomography (micro-CT) evaluation] were used to detect the implant stability and bone-implant contact. The implant stability quotient values of GDP-fib implants were significantly greater than the Ar-GDP implants at 2 and 4 weeks (P < 0.01). The bone volume/total volume ratio of GDP-fib implants was greater than the Ar-GDP implants in micro-CT evaluation. A high positive correlation was observed between RFA and micro-CT measurements. At 2 weeks, osteoblasts were seen to line the implant surface, and multinuclear osteoclasts could be seen on the surface of old parent bone. After 8 weeks, a majority of the space in the wound chamber appeared to be replaced by bone. Enhancement of the stability of biologically modified implants was proved by the results of RFA, micro-CT, and histological analysis. This enhanced stability may help fasten treatment and be clinically beneficial.
doi:10.1155/2016/2414809
PMCID: PMC4913050  PMID: 27366739
8.  Meta-analysis of gene–environment-wide association scans accounting for education level identifies additional loci for refractive error 
Fan, Qiao | Verhoeven, Virginie J. M. | Wojciechowski, Robert | Barathi, Veluchamy A. | Hysi, Pirro G. | Guggenheim, Jeremy A. | Höhn, René | Vitart, Veronique | Khawaja, Anthony P. | Yamashiro, Kenji | Hosseini, S Mohsen | Lehtimäki, Terho | Lu, Yi | Haller, Toomas | Xie, Jing | Delcourt, Cécile | Pirastu, Mario | Wedenoja, Juho | Gharahkhani, Puya | Venturini, Cristina | Miyake, Masahiro | Hewitt, Alex W. | Guo, Xiaobo | Mazur, Johanna | Huffman, Jenifer E. | Williams, Katie M. | Polasek, Ozren | Campbell, Harry | Rudan, Igor | Vatavuk, Zoran | Wilson, James F. | Joshi, Peter K. | McMahon, George | St Pourcain, Beate | Evans, David M. | Simpson, Claire L. | Schwantes-An, Tae-Hwi | Igo, Robert P. | Mirshahi, Alireza | Cougnard-Gregoire, Audrey | Bellenguez, Céline | Blettner, Maria | Raitakari, Olli | Kähönen, Mika | Seppala, Ilkka | Zeller, Tanja | Meitinger, Thomas | Ried, Janina S. | Gieger, Christian | Portas, Laura | van Leeuwen, Elisabeth M. | Amin, Najaf | Uitterlinden, André G. | Rivadeneira, Fernando | Hofman, Albert | Vingerling, Johannes R. | Wang, Ya Xing | Wang, Xu | Tai-Hui Boh, Eileen | Ikram, M. Kamran | Sabanayagam, Charumathi | Gupta, Preeti | Tan, Vincent | Zhou, Lei | Ho, Candice E. H. | Lim, Wan'e | Beuerman, Roger W. | Siantar, Rosalynn | Tai, E-Shyong | Vithana, Eranga | Mihailov, Evelin | Khor, Chiea-Chuen | Hayward, Caroline | Luben, Robert N. | Foster, Paul J. | Klein, Barbara E. K. | Klein, Ronald | Wong, Hoi-Suen | Mitchell, Paul | Metspalu, Andres | Aung, Tin | Young, Terri L. | He, Mingguang | Pärssinen, Olavi | van Duijn, Cornelia M. | Jin Wang, Jie | Williams, Cathy | Jonas, Jost B. | Teo, Yik-Ying | Mackey, David A. | Oexle, Konrad | Yoshimura, Nagahisa | Paterson, Andrew D. | Pfeiffer, Norbert | Wong, Tien-Yin | Baird, Paul N. | Stambolian, Dwight | Wilson, Joan E. Bailey | Cheng, Ching-Yu | Hammond, Christopher J. | Klaver, Caroline C. W. | Saw, Seang-Mei | Rahi, Jugnoo S. | Korobelnik, Jean-François | Kemp, John P. | Timpson, Nicholas J. | Smith, George Davey | Craig, Jamie E. | Burdon, Kathryn P. | Fogarty, Rhys D. | Iyengar, Sudha K. | Chew, Emily | Janmahasatian, Sarayut | Martin, Nicholas G. | MacGregor, Stuart | Xu, Liang | Schache, Maria | Nangia, Vinay | Panda-Jonas, Songhomitra | Wright, Alan F. | Fondran, Jeremy R. | Lass, Jonathan H. | Feng, Sheng | Zhao, Jing Hua | Khaw, Kay-Tee | Wareham, Nick J. | Rantanen, Taina | Kaprio, Jaakko | Pang, Chi Pui | Chen, Li Jia | Tam, Pancy O. | Jhanji, Vishal | Young, Alvin L. | Döring, Angela | Raffel, Leslie J. | Cotch, Mary-Frances | Li, Xiaohui | Yip, Shea Ping | Yap, Maurice K.H. | Biino, Ginevra | Vaccargiu, Simona | Fossarello, Maurizio | Fleck, Brian | Yazar, Seyhan | Tideman, Jan Willem L. | Tedja, Milly | Deangelis, Margaret M. | Morrison, Margaux | Farrer, Lindsay | Zhou, Xiangtian | Chen, Wei | Mizuki, Nobuhisa | Meguro, Akira | Mäkelä, Kari Matti
Nature Communications  2016;7:11008.
Myopia is the most common human eye disorder and it results from complex genetic and environmental causes. The rapidly increasing prevalence of myopia poses a major public health challenge. Here, the CREAM consortium performs a joint meta-analysis to test single-nucleotide polymorphism (SNP) main effects and SNP × education interaction effects on refractive error in 40,036 adults from 25 studies of European ancestry and 10,315 adults from 9 studies of Asian ancestry. In European ancestry individuals, we identify six novel loci (FAM150B-ACP1, LINC00340, FBN1, DIS3L-MAP2K1, ARID2-SNAT1 and SLC14A2) associated with refractive error. In Asian populations, three genome-wide significant loci AREG, GABRR1 and PDE10A also exhibit strong interactions with education (P<8.5 × 10−5), whereas the interactions are less evident in Europeans. The discovery of these loci represents an important advance in understanding how gene and environment interactions contribute to the heterogeneity of myopia.
This report by the Consortium for Refractive Error and Myopia uses gene-environment-wide interaction study (GEWIS) to identify genetic loci that affect environmental influence in myopia development, and identifies ethnic specific genetic loci that attribute to eye refractive errors.
doi:10.1038/ncomms11008
PMCID: PMC4820539  PMID: 27020472
9.  Effects of Salivary Oxidative Markers on Edentulous Patients’ Satisfaction with Prosthetic Denture Treatments: A Pilot Study 
PLoS ONE  2016;11(3):e0151605.
Objectives
The purpose of this study was to assess relationships among periodontal conditions, salivary antioxidant levels, and patients’ satisfaction with their prostheses.
Methods
This study was conducted at the Division of Prosthodontics, Department of Dentistry, Taipei Medical University Hospital. The periodontal condition of patients was based on an assessment of the plaque index (PI) and gingival index (GI). The pH value, flow rate, and buffer capacity of the saliva were estimated. The salivary total antioxidant status (TAS) and superoxide dismutase (SOD) level were also determined. Patients’ satisfaction with prosthetic treatments was evaluated using the Chinese version of the short-form Oral Health Impact Profile (OHIP-14C). A multivariate regression model was used to determine whether patients’ satisfaction with prosthetic treatment was affected by their oral health status.
Results
In total, 35 edentulous patients were recruited. In the Spearman correlation analysis, salivary pH (r = -0.36, p = 0.03) and the buffer ability (r = -0.48, p<0.01) were associated with OHIP-14C scores. In the multivariate analysis, patients who had a higher GI also had a higher score of physical disabilities (β = 1.38, p = 0.04). Levels of SOD increased with the scores of psychological discomfort (β = 0.33 U/g protein, p = 0.04).
Conclusions
This study suggested that both the GI and SOD levels were associated with patients’ satisfaction with prosthetic treatments. To the best of our knowledge, this is the first study to elucidate the relationship between OHIP scores and salivary oxidative markers in edentulous patients.
doi:10.1371/journal.pone.0151605
PMCID: PMC4795643  PMID: 26986841
10.  Microarray Study of Pathway Analysis Expression Profile Associated with MicroRNA-29a with Regard to Murine Cholestatic Liver Injuries 
Accumulating evidence demonstrates that microRNA-29 (miR-29) expression is prominently decreased in patients with hepatic fibrosis, which consequently stimulates hepatic stellate cells’ (HSCs) activation. We used a cDNA microarray study to gain a more comprehensive understanding of genome-wide gene expressions by adjusting miR-29a expression in a bile duct-ligation (BDL) animal model. Methods: Using miR-29a transgenic mice and wild-type littermates and applying the BDL mouse model, we characterized the function of miR-29a with regard to cholestatic liver fibrosis. Pathway enrichment analysis and/or specific validation were performed for differentially expressed genes found within the comparisons. Results: Analysis of the microarray data identified a number of differentially expressed genes due to the miR-29a transgene, BDL, or both. Additional pathway enrichment analysis revealed that TGF-β signaling had a significantly differential activated pathway depending on the occurrence of miR-29a overexpression or the lack thereof. Furthermore, overexpression was found to elicit changes in Wnt/β-catenin after BDL. Conclusion: This study verified that an elevated miR-29a level could alleviate liver fibrosis caused by cholestasis. Furthermore, the protective effects of miR-29a correlate with the downregulation of TGF-β and associated with Wnt/β-catenin signal pathway following BDL.
doi:10.3390/ijms17030324
PMCID: PMC4813186  PMID: 26938532
miR-29a; bile duct ligation; cholestasis; liver fibrosis; TGF-β signaling pathway; Wnt signaling pathway
11.  Risk Factors for New-Onset Cardiac Valve Calcification in Patients on Maintenance Peritoneal Dialysis 
Cardiorenal Medicine  2016;6(2):150-158.
Objective
Patients with end-stage renal disease are susceptible to cardiac valve calcification (CVC) due to mineral metabolism disorders and other factors. The purpose of this study was to investigate the risk factors for new-onset CVC in patients on maintenance peritoneal dialysis (PD).
Methods
This study included patients who underwent PD catheter insertion from January 2006 to June 2013 in our Peritoneal Dialysis Center. Clinical data were collected on CVC status during echocardiography evaluations (twice) at an interval of >6 months. The data collected included intact parathyroid hormone, C-reactive protein (CRP), serum phosphorus (P), serum calcium (Ca), albumin (Alb), prealbumin and the use of five types of antihypertensive drugs, statins, active vitamin D3 and Ca tablets.
Results
In total, 194 patients — 105 (54.1%) men, average age 60.5 ± 13.0 years — were included. CVC was present in 50 (25.8%) patients during PD catheter placement. After an average PD duration of 20.9 ± 10.4 months, CVC was detected in 97 patients (50.0%). New-onset CVC was found in 62 patients (32.0%). Multivariate logistic regression analysis revealed that only serum P levels (p = 0.01, OR = 2.569), Alb levels (p = 0.04, OR = 0.935), dialysis duration (p = 0.03, OR = 1.039) and CRP levels (p = 0.02, OR = 1.031) were associated with CVC.
Conclusion
Serum P, Alb and CRP levels as well as dialysis duration are independent risk factors for CVC.
doi:10.1159/000443620
PMCID: PMC4789884  PMID: 26989400
Cardiac valve calcification; Peritoneal dialysis; Risk factor
12.  Genome-wide identification of DNA methylation provides insights into the association of gene expression in rice exposed to pesticide atrazine 
Scientific Reports  2016;6:18985.
Atrazine (ATR) is a pesticide widely used for controlling weeds for crop production. Crop contamination with ATR negatively affects crop growth and development. This study presents the first genome-wide single-base-resolution maps of DNA methylation in ATR-exposed rice. Widespread differences were identified in CG and non-CG methylation marks between the ATR-exposed and ATR-free (control) rice. Most of DNA methyltransferases, histone methyltransferases and DNA demethylase were differentially regulated by ATR. We found more genes hypermethylated than those hypomethylated in the regions of upstream, genebody and downstream under ATR exposure. A stringent group of 674 genes (p < 0.05, two-fold change) with a strong preference of differential expression in ATR-exposed rice was identified. Some of the genes were identified in a subset of loss of function mutants defective in DNA methylation/demethylation. Provision of 5-azacytidine (AZA, inhibitor of DNA methylation) promoted the rice growth and reduced ATR content. By UPLC/Q-TOF-MS/MS, 8 degraded products and 9 conjugates of ATR in AZA-treated rice were characterized. Two of them has been newly identified in this study. Our data show that ATR-induced changes in DNA methylation marks are possibly involved in an epigenetic mechanism associated with activation of specific genes responsible for ATR degradation and detoxification.
doi:10.1038/srep18985
PMCID: PMC4704053  PMID: 26739616
13.  In Vitro Analysis of Fibronectin-Modified Titanium Surfaces 
PLoS ONE  2016;11(1):e0146219.
Background
Glow discharge plasma (GDP) procedure is an effective method for grafting various proteins, including albumin, type I collagen, and fibronectin, onto a titanium surface. However, the behavior and impact of titanium (Ti) surface modification is yet to be unraveled.
Purpose
The purpose of this study is to evaluate and analyze the biological properties of fibronectin-grafted Ti surfaces treated by GDP.
Materials and Methods
Grade II Ti discs were initially cleaned and autoclaved to obtain original specimens. Subsequently, the specimens were GDP treated and grafted with fibronectin to form Ar-GDP (Argon GDP treatment only) and GDP-fib (fibronectin coating following GDP treatment) groups. Blood coagulation test and MG-63 cell culture were performed to evaluate the biological effects on the specimen.
Results
There was no significant difference between Ar-GDP and GDP-fib groups in blood compatibility analysis. While in the MTT test, cellular proliferation was benefited from the presence of fibronectin coating. The numbers of cells on Ar-GDP and GDP-fib specimens were greater than those in the original specimens after 24 h of culturing.
Conclusions
GDP treatment combined with fibronectin grafting favored MG-63 cell adhesion, migration, and proliferation on titanium surfaces, which could be attributed to the improved surface properties.
doi:10.1371/journal.pone.0146219
PMCID: PMC4711664  PMID: 26731536
14.  Combining ‘Bottom-Up’ and ‘Top-Down’ Methods to Assess Ethnic Difference in Clearance: Bitopertin as an Example 
Clinical Pharmacokinetics  2015;55:823-832.
Background and Objectives
We propose a strategy for studying ethnopharmacology by conducting sequential physiologically based pharmacokinetic (PBPK) prediction (a ‘bottom-up’ approach) and population pharmacokinetic (popPK) confirmation (a ‘top-down’ approach), or in reverse order, depending on whether the purpose is ethnic effect assessment for a new molecular entity under development or a tool for ethnic sensitivity prediction for a given pathway. The strategy is exemplified with bitopertin.
Methods
A PBPK model was built using Simcyp® to simulate the pharmacokinetics of bitopertin and to predict the ethnic sensitivity in clearance, given pharmacokinetic data in just one ethnicity. Subsequently, a popPK model was built using NONMEM® to assess the effect of ethnicity on clearance, using human data from multiple ethnic groups. A comparison was made to confirm the PBPK-based ethnic sensitivity prediction, using the results of the popPK analysis.
Results
PBPK modelling predicted that the bitopertin geometric mean clearance values after 20 mg oral administration in Caucasians would be 1.32-fold and 1.27-fold higher than the values in Chinese and Japanese, respectively. The ratios of typical clearance in Caucasians to the values in Chinese and Japanese estimated by popPK analysis were 1.20 and 1.17, respectively. The popPK analysis results were similar to the PBPK modelling results.
Conclusion
As a general framework, we propose that PBPK modelling should be considered to predict ethnic sensitivity of pharmacokinetics prior to any human data and/or with data in only one ethnicity. In some cases, this will be sufficient to guide initial dose selection in different ethnicities. After clinical trials in different ethnicities, popPK analysis can be used to confirm ethnic differences and to support dose justification and labelling. PBPK modelling prediction and popPK analysis confirmation can complement each other to assess ethnic differences in pharmacokinetics at different drug development stages.
Electronic supplementary material
The online version of this article (doi:10.1007/s40262-015-0356-1) contains supplementary material, which is available to authorized users.
doi:10.1007/s40262-015-0356-1
PMCID: PMC4916198  PMID: 26715215
15.  Development and Testing of X-Ray Imaging-Enhanced Poly-L-Lactide Bone Screws 
PLoS ONE  2015;10(10):e0140354.
Nanosized iron oxide particles exhibit osteogenic and radiopaque properties. Thus, iron oxide (Fe3O4) nanoparticles were incorporated into a biodegradable polymer (poly-L-lactic acid, PLLA) to fabricate a composite bone screw. This multifunctional, 3D printable bone screw was detectable on X-ray examination. In this study, mechanical tests including three-point bending and ultimate tensile strength were conducted to evaluate the optimal ratio of iron oxide nanoparticles in the PLLA composite. Both injection molding and 3D printing techniques were used to fabricate the PLLA bone screws with and without the iron oxide nanoparticles. The fabricated screws were implanted into the femoral condyles of New Zealand White rabbits. Bone blocks containing the PLLA screws were resected 2 and 4 weeks after surgery. Histologic examination of the surrounding bone and the radiopacity of the iron-oxide-containing PLLA screws were evaluated. Our results indicated that addition of iron oxide nanoparticles at 30% significantly decreased the ultimate tensile stress properties of the PLLA screws. The screws with 20% iron oxide exhibited strong radiopacity compared to the screws fabricated without the iron oxide nanoparticles. Four weeks after surgery, the average bone volume of the iron oxide PLLA composite screws was significantly greater than that of PLLA screws without iron oxide. These findings suggested that biodegradable and X-ray detectable PLLA bone screws can be produced by incorporation of 20% iron oxide nanoparticles. Furthermore, these screws had significantly greater osteogenic capability than the PLLA screws without iron oxide.
doi:10.1371/journal.pone.0140354
PMCID: PMC4605620  PMID: 26466309
16.  Activation of Mir-29a in Activated Hepatic Stellate Cells Modulates Its Profibrogenic Phenotype through Inhibition of Histone Deacetylases 4 
PLoS ONE  2015;10(8):e0136453.
Background
Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis and that its downregulation influences the activation of hepatic stellate cells (HSCs). In addition, inhibition of the activity of histone deacetylases 4 (HDAC4) has been shown to strongly reduce HSC activation in the context of liver fibrosis.
Objectives
In this study, we examined whether miR-29a was involved in the regulation of HDAC4 and modulation of the profibrogenic phenotype in HSCs.
Methods
We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates to clarify the role of miR-29a in cholestatic liver fibrosis, using the bile duct-ligation (BDL) mouse model. Primary HSCs from both mice were treated with a miR-29a mimic and antisense inhibitor in order to analyze changes in profibrogenic gene expression and HSC activation using real-time quantitative RT-PCR, immunofluorescence staining, western blotting, and cell proliferation and migration assays.
Results
After BDL, overexpression of miR-29a decreased collagen-1α1, HDAC4 and activated HSC markers of glial fibrillary acidic protein expression in miR-29aTg mice compared to wild-type littermates. Overexpression of miR-29a and HDAC4 RNA-interference decreased the expression of fibrotic genes, HDAC4 signaling, and HSC migration and proliferation. In contrast, knockdown of miR-29a with an antisense inhibitor increased HDAC4 function, restored HSC migration, and accelerated HSC proliferation.
Conclusions
Our results indicate that miR-29a ameliorates cholestatic liver fibrosis after BDL, at least partially, by modulating the profibrogenic phenotype of HSCs through inhibition of HDAC4 function.
doi:10.1371/journal.pone.0136453
PMCID: PMC4549141  PMID: 26305546
17.  Virus Nanoparticles Mediated Osteogenic Differentiation of Bone Derived Mesenchymal Stem Cells 
Advanced Science  2015;2(10):1500026.
There are few methodologies that allow manipulating a biomaterial surface at nanometer scale, which controllably influence different cellular functions. In this study, virus nanoparticles with different structural features are selected to prepare 2D substrates with defined nanoscale topographies and the cellular responses are investigated. It is demonstrated that the viral nanoparticle based substrates could accelerate and enhance osteogenesis of bone derived mesenchymal stem cells as indicated by the upregulation of osteogenic markers, including bone morphogenetic protein‐2, osteocalcin, and osteopontin, at both gene and protein expression levels. Moreover, alkaline phosphatase activity and calcium mineralization, both indicators for a ­successful bone formation, are also increased in cells grown on these nanoscale possessed substrates. These discoveries and developments present a new paradigm for nanoscale engineering of a biomaterial surface.
doi:10.1002/advs.201500026
PMCID: PMC5115314  PMID: 27980904
biomaterials; mesenchymal stem cells; nanotopography; osteogenesis; virus nanoparticles
18.  Effects of Computer Navigation versus Conventional Total Knee Arthroplasty on Endothelial Damage Marker Levels: A Prospective Comparative Study 
PLoS ONE  2015;10(5):e0126663.
Total knee arthroplasty (TKA) inevitably perturbs the femoral medullary canal, which increases blood loss or morbidities associated with marrow embolization postoperatively. Computer navigation TKA reportedly minimizes medullary disturbance to alleviate perioperative blood loss. We performed a prospective comparative study, enrolling 87 patients with osteoarthritic knees from March 2011 to December 2011 in our hospital. The patients were separated into two groups, according to the surgeon they visited. Fifty-four patients underwent computer navigation TKAs and 33 had conventional TKAs. Levels of cell adhesion molecules (CAMs), including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and platelet endothelial cellular adhesion molecule-1 (PECAM-1) in sera and hemovac drainage were measured by ELISA before and 24 hours after the surgery. We showed that patients receiving computer navigation TKAs had less blood loss and lower CAMs in serum and hemovac drainage after the operation. Less postoperative elevation of serum ICAM-1 (p=0.022) and PECAM-1 (p=0.003) from the preoperative baseline after the surgery was also noted. This study provides molecular evidence for the differential extent in vascular injury between conventional and navigation TKAs and sheds light on the possible benefits of computer navigation TKAs.
Trial Registration
ClinicalTrials.gov NCT02206321
doi:10.1371/journal.pone.0126663
PMCID: PMC4425488  PMID: 25955252
19.  Association between serum leptin levels and peritoneal dialysis: A meta-analysis 
There is limited information available with regard to the association between serum leptin levels, or other adipokines, and serum lipid levels and insulin sensitivity in patients undergoing peritoneal dialysis (PD). Thus, the aim of the present study was to perform a meta-analysis investigating this association. Potential relevant studies were identified through searching the following databases: MEDLINE, Science Citation Index, Cochrane Library, PubMed, Embase, CINAHL, Chinese Biomedical, Chinese Journal Full-Text and Weipu Journal. Statistical analyses were calculated using version 12.0 STATA software. In total, 21 case-control studies comprising 1,187 subjects (574 patients and 613 controls) were collected for the meta-analysis. The results identified a statistically significant difference in the serum levels of leptin when comparing the PD patients with the healthy controls [controls vs. cases, standardized mean difference (SMD), 2.09; 95% confidence interval (CI), 1.58–2.59; P<0.001]. Furthermore, ethnicity-subgroup analysis indicated that the PD patients of Asian and Caucasian populations were associated with increased serum levels of leptin (Asian population, SMD, 2.05; 95% CI, 1.48–2.62; P<0.001; Caucasian population, SMD, 2.19; 95% CI, 1.19–3.18; P<0.001). Therefore, serum leptin levels may be used as a prognostic marker for PD.
doi:10.3892/etm.2015.2441
PMCID: PMC4486802  PMID: 26170953
leptin; serum; peritoneal dialysis; meta-analysis
20.  MicroRNA-185-5p mediates regulation of SREBP2 expression by hepatitis C virus core protein 
AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus (HCV) core protein in HepG2 cells.
METHODS: HCV genotype 1b core protein was cloned and expressed in HepG2 cells. The cholesterol content was determined after transfection. The expression of sterol regulatory element binding protein 2 (SREBP2) and the rate-limiting enzyme in cholesterol synthesis (HMGCR) was measured by quantitative real-time PCR and immunoblotting after transfection. The effects of core protein on the SREBP2 promoter and 3’-untranslated region were analyzed by luciferase assay. We used different target predictive algorithms, microRNA (miRNA) mimics/inhibitors, and site-directed mutation to identify a putative target of a particular miRNA.
RESULTS: HCV core protein expression in HepG2 cells increased the total intracellular cholesterol level (4.05 ± 0.17 vs 6.47 ± 0.68, P = 0.001), and this increase corresponded to an increase in SREBP2 and HMGCR mRNA levels (P = 0.009 and 0.037, respectively) and protein expression. The molecular mechanism study revealed that the HCV core protein increased the expression of SREBP2 by enhancing its promoter activity (P = 0.004). In addition, miR-185-5p expression was tightly regulated by the HCV core protein (P = 0.041). Moreover, overexpression of miR-185-5p repressed the SREBP2 mRNA level (P = 0.022) and protein expression. In contrast, inhibition of miR-185-5p caused upregulation of SREBP2 protein expression. miR-185-5p was involved in the regulation of SREBP2 expression by HCV core protein.
CONCLUSION: HCV core protein disturbs the cholesterol homeostasis in HepG2 cells via the SREBP2 pathway; miR-185-5p is involved in the regulation of SREBP2 by the core protein.
doi:10.3748/wjg.v21.i15.4517
PMCID: PMC4402298  PMID: 25914460
Cholesterol; Hepatitis C virus core protein; miR-185-5p; Steatosis; Sterol response element binding proteins
21.  Nasal high flow clears anatomical dead space in upper airway models 
Journal of Applied Physiology  2015;118(12):1525-1532.
Recent studies showed that nasal high flow (NHF) with or without supplemental oxygen can assist ventilation of patients with chronic respiratory and sleep disorders. The hypothesis of this study was to test whether NHF can clear dead space in two different models of the upper nasal airways. The first was a simple tube model consisting of a nozzle to simulate the nasal valve area, connected to a cylindrical tube to simulate the nasal cavity. The second was a more complex anatomically representative upper airway model, constructed from segmented CT-scan images of a healthy volunteer. After filling the models with tracer gases, NHF was delivered at rates of 15, 30, and 45 l/min. The tracer gas clearance was determined using dynamic infrared CO2 spectroscopy and 81mKr-gas radioactive gamma camera imaging. There was a similar tracer-gas clearance characteristic in the tube model and the upper airway model: clearance half-times were below 1.0 s and decreased with increasing NHF rates. For both models, the anterior compartments demonstrated faster clearance levels (half-times < 0.5 s) and the posterior sections showed slower clearance (half-times < 1.0 s). Both imaging methods showed similar flow-dependent tracer-gas clearance in the models. For the anatomically based model, there was complete tracer-gas removal from the nasal cavities within 1.0 s. The level of clearance in the nasal cavities increased by 1.8 ml/s for every 1.0 l/min increase in the rate of NHF. The study has demonstrated the fast-occurring clearance of nasal cavities by NHF therapy, which is capable of reducing of dead space rebreathing.
doi:10.1152/japplphysiol.00934.2014
PMCID: PMC4482836  PMID: 25882385
nasal high flow; insufflation; upper airways; dead space; carbon dioxide; krypton
22.  Gluconeogenesis, lipogenesis, and HBV replication are commonly regulated by PGC-1α-dependent pathway 
Oncotarget  2015;6(10):7788-7803.
PGC-1α, a major metabolic regulator of gluconeogenesis and lipogenesis, is strongly induced to coactivate Hepatitis B virus (HBV) gene expression in the liver of fasting mice. We found that 8-Br-cAMP and glucocorticoids synergistically induce PGC-1α and its downstream targets, including PEPCK and G6Pase. Also, HBV core promoter activity was synergistically enhanced by 8-Br-cAMP and glucocorticoids. Graptopetalum paraguayense (GP), a herbal medicine, is commonly used in Taiwan to treat liver disorders. Partially purified fraction of GP (named HH-F3) suppressed 8-Br-cAMP/glucocorticoid-induced G6Pase, PEPCK and PGC-1α expression and suppressed HBV core promoter activity. HH-F3 blocked HBV core promoter activity via inhibition of PGC-1α expression. Ectopically expressed PGC-1α rescued HH-F3-inhibited HBV surface antigen expression, HBV mRNA production, core protein levels, and HBV replication. HH-F3 also inhibited fatty acid synthase (FASN) expression and decreased lipid accumulation by down-regulating PGC-1α. Thus, HH-F3 can inhibit HBV replication, gluconeogenesis and lipogenesis by down-regulating PGC-1α. Our study indicates that targeting PGC-1α may be a therapeutic strategy for treatment of HBV infections. HH-F3 may have potential use for the treatment of chronic hepatitis B patients with associated metabolic syndrome.
PMCID: PMC4480716  PMID: 25762623
Graptopetalum paraguayense; HBV; Gluconeogenesis; Lipogenesis; PGC-1α
23.  Charcoal Reflectance Reveals Early Holocene Boreal Deciduous Forests Burned at High Intensities 
PLoS ONE  2015;10(4):e0120835.
Wildfire size, frequency, and severity are increasing in the Alaskan boreal forest in response to climate warming. One of the potential impacts of this changing fire regime is the alteration of successional trajectories, from black spruce to mixed stands dominated by aspen, a vegetation composition not experienced since the early Holocene. Such changes in vegetation composition may consequently alter the intensity of fires, influencing fire feedbacks to the ecosystem. Paleorecords document past wildfire-vegetation dynamics and as such, are imperative for our understanding of how these ecosystems will respond to future climate warming. For the first time, we have used reflectance measurements of macroscopic charcoal particles (>180μm) from an Alaskan lake-sediment record to estimate ancient charring temperatures (termed pyrolysis intensity). We demonstrate that pyrolysis intensity increased markedly from an interval of birch tundra 11 ky ago (mean 1.52%Ro; 485°C), to the expansion of trees on the landscape ∼10.5 ky ago, remaining high to the present (mean 3.54%Ro; 640°C) irrespective of stand composition. Despite differing flammabilities and adaptations to fire, the highest pyrolysis intensities derive from two intervals with distinct vegetation compositions. 1) the expansion of mixed aspen and spruce woodland at 10 cal. kyr BP, and 2) the establishment of black spruce, and the modern boreal forest at 4 cal. kyr BP. Based on our analysis, we infer that predicted expansion of deciduous trees into the boreal forest in the future could lead to high intensity, but low severity fires, potentially moderating future climate-fire feedbacks.
doi:10.1371/journal.pone.0120835
PMCID: PMC4390342  PMID: 25853712
24.  HOXB7 Promotes Malignant Progression by Activating the TGFβ Signaling Pathway 
Cancer research  2014;75(4):709-719.
Overexpression of HOXB7 in breast cancer cells induces an epithelial–mesenchymal transition and promotes tumor progression and lung metastasis. However, the underlying mechanisms for HOXB7-induced aggressive phenotypes in breast cancer remain largely unknown. Here, we report that phosphorylation of SMAD3 was detected in a higher percentage in primary mammary tumor tissues from double-transgenic MMTV-Hoxb7/Her2 mice than tumors from single-transgenic Her2/neu mice, suggesting activation of TGFβ/SMAD3 signaling by HOXB7 in breast tumor tissues. As predicted, TGFβ2 was high in four MMTV-Hoxb7/Her2 transgenic mouse tumor cell lines and two breast cancer cell lines transfected with HOXB7, whereas TGFβ2 was low in HOXB7-depleted cells. HOXB7 directly bound to and activated the TGFβ2 promoter in luciferase and chromatin immunoprecipitation assays. Increased migration and invasion as a result of HOXB7 overexpression in breast cancer cells were reversed by knockdown of TGFβ2 or pharmacologic inhibition of TGFβ signaling. Furthermore, knockdown of TGFβ2 in HOXB7-overexpressing MDA-MB-231 breast cancer cells dramatically inhibited metastasis to the lung. Interestingly, HOXB7 overexpression also induced tumor-associated macrophage (TAM) recruitment and acquisition of an M2 tumor-promoting phenotype. TGFβ2 mediated HOXB7-induced activation of macrophages, suggesting that TAMs may contribute to HOXB7-promoted tumor metastasis. Providing clinical relevance to these findings, by real-time PCR analysis, there was a strong correlation between HOXB7 and TGFβ2 expression in primary breast carcinomas. Taken together, our results suggest that HOXB7 promotes tumor progression in a cell-autonomous and non–cell-autonomous manner through activation of the TGFβ signaling pathway.
doi:10.1158/0008-5472.CAN-14-3100
PMCID: PMC4352303  PMID: 25542862
25.  Upregulation of TLRs and IL-6 as a Marker in Human Colorectal Cancer 
Toll-like receptors (TLRs) not only form an important part of the innate immune system but also serve to activate the adaptive immune system in response to cancer. Real-time PCR; immunohistochemical stain and Western blotting analyses were performed to clarify molecular alterations in colorectal cancer (CRC) patients. We identified Toll-like receptor 1 (TLR1), TLR2, TLR4 and TLR8 gene expression levels and downstream gene, i.e., interleukin-6 (IL-6), IL-8, interferon-α (IFN-α) and myeloid differentiation primary-response protein-88 (MyD88), expression levels in CRC patients and in cancer cell lines. CRC tissues have higher TLR1, TLR2, TLR4, TLR8, IL-6 and IL-8 gene expression levels than do the normal colon mucosa (p < 0.05). TLR2 expression varied in different cell types (mucosa and lymphocytes). There was no difference in the MyD88 and IFN-α gene expression levels between cancerous and normal colon mucosa. CRC patients had higher levels of IL-6 (p = 0.002) and IL-8 (p = 0.038) expression than healthy volunteers did; and higher IL-6 and IL-8 expression was also found to signify a higher risk of recurrence. CL075 (3M002) treatments can reduce the production of IL-8 in different cancer cell lines. The signaling pathway of TLRs in cancer tissue is different from that in normal cells; and is MyD88-independent. Higher expression levels of TLR1, TLR2, TLR 4 and TLR 8 mRNA were related to upregulation inflammatory cytokines IL-6 and IL-8 gene expression in tissue and to the upregulation of IL-6 in blood. The concentration of IL-6 in serum can be used as an indicator of the possibility of CRC recurrence. Treatment with 3M002 can reduce IL-6 production in vitro and may prevent CRC recurrence. Our findings provide evidence that TLR1, TLR2, TLR4 and TLR8 gene expression induce downstream IL-6 and IL-8 gene expression; detection of these expression levels can serve as a CRC marker.
doi:10.3390/ijms16010159
PMCID: PMC4307241  PMID: 25547486
Toll-like receptors; real-time PCR; immunohistochemical stain; Western blotting; colorectal cancer

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