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1.  γ-secretase inhibitor up-regulates vascular endothelial growth factor receptor-2 and endothelial nitric oxide synthase 
Although previous studies have shown that γ-secretase inhibitors significantly suppress tumor growth via anti-angiogenesis, the mechanism involved in the regulation of tumor angiogenesis by γ-secretase inhibitors has not been clearly understood. The objective of this study was to investigate the regulation of vascular endothelial growth factor receptor (VEGFR) and endothelial nitric oxide synthase (eNOS) by a γ-secretase inhibitor in the H5V mouse microvascular endothelial cell line. H5V cells were cultured with different concentrations of the γ-secretase inhibitor DAPT for 48 h and with 100 μmol/l DAPT at different incubation times. Protein and mRNA expression of VEGFR-1, VEGFR-2, VEGFR-3 and eNOS was measured by Western blotting and real-time PCR, respectively. The VEGFR-2 kinase inhibitor was used to assess the role of VEGFR-2 in eNOS regulation. We found that the γ-secretase inhibitor DAPT increased protein and mRNA expression of VEGFR-2 and eNOS, but decreased VEGFR-1 expression and had no significant effect on VEGFR-3. Up-regulation of eNOS was blocked by the VEGFR-2 kinase inhibitor. In conclusion, the γ-secretase inhibitor enhances VEGFR-2 and eNOS expression, and the up-regulation of eNOS is dependent on an increase in VEGFR-2. Thus, we suggest that administration of the γ-secretase inhibitor be combined with disruption of eNOS or interruption of VEGF signaling, which may improve the anti-angiogenic efficacy in tumor treatments.
doi:10.3892/etm.2011.257
PMCID: PMC3440738  PMID: 22977566
angiogenesis; vascular endothelial growth factor receptor; endothelial nitric oxide synthase
2.  A New PAMPA Model Proposed on the Basis of a Synthetic Phospholipid Membrane 
PLoS ONE  2015;10(2):e0116502.
The purpose of this work was to investigate the synthetic phospholipid dependence of permeability measured by parallel artificial membrane permeability assay (PAMPA) method. Three phospholipids with hydrophobic groups of different lengths and phosphorylcholine as the hydrophilic group were concisely synthesized. Ten model drug molecules were selected because of their distinct human fraction absorbed (%FA) values and various pKa characteristics. In vitro drug permeation experiments were designed to determine the effect of the incubation time (4–20 h), pH gradient (4.6–9.32) and carbon chain length (8, 10, 12) on the drug permeability through the synthetic phospholipid membrane in the PAMPA system. The results showed that intensive and significant synthetic phospholipids dependence of permeability influenced by the length of lipid’s hydrophobic carbon chain. The effective permeability constant (Pe) of each drug increased rapidly with time, then decreased slightly after reaching the maximum; the pH gradient changed the drug permeability according to the pH-partition hypothesis for drugs with diverse pKa values; and longer hydrophobic chains in the synthetic phospholipid membrane improved the drug permeability, as observed for all test drugs at almost all incubation time points. This newly proposed PAMPA model considered the synthetic phospholipid membrane and showed good Pe-%FA correlation for the passive transport of drugs, making it a helpful supplementary method for PAMPA systems.
doi:10.1371/journal.pone.0116502
PMCID: PMC4315410  PMID: 25647086
3.  Transcriptome analysis of food habit transition from carnivory to herbivory in a typical vertebrate herbivore, grass carp Ctenopharyngodon idella 
BMC Genomics  2015;16(1):15.
Background
Although feeding behavior and food habit are ecologically and economically important properties, little is known about formation and evolution of herbivory. Grass carp (Ctenopharyngodon idella) is an ecologically appealing model of vertebrate herbivore, widely cultivated in the world as edible fish or as biological control agents for aquatic weeds. Grass carp exhibits food habit transition from carnivory to herbivory during development. However, currently little is known about the genes regulating the unique food habit transition and the formation of herbivory, and how they could achieve higher growth rates on plant materials, which have a relatively poor nutritional quality.
Results
We showed that grass carp fed with duckweed (modeling fish after food habit transition) had significantly higher relative length of gut than fish before food habit transition or those fed with chironomid larvae (fish without transition). Using transcriptome sequencing, we identified 10,184 differentially expressed genes between grass carp before and after transition in brain, liver and gut. By eliminating genes potentially involved in development (via comparing fish with or without food habit transition), we identified changes in expression of genes involved in cell proliferation and differentiation, appetite control, circadian rhythm, and digestion and metabolism between fish before and after food habit transition. Up-regulation of GHRb, Egfr, Fgf, Fgfbp1, Insra, Irs2, Jak, STAT, PKC, PI3K expression in fish fed with duckweed, consistent with faster gut growth, could promote the food habit transition. Grass carp after food habit transition had increased appetite signal in brain. Altered expressions of Per, Cry, Clock, Bmal2, Pdp, Dec and Fbxl3 might reset circadian phase of fish after food habit transition. Expression of genes involved in digestion and metabolism were significantly different between fish before and after the transition.
Conclusions
We suggest that the food habit transition from carnivory to herbivory in grass carp might be due to enhanced gut growth, increased appetite, resetting of circadian phase and enhanced digestion and metabolism. We also found extensive alternative splicing and novel transcript accompanying food habit transition. These differences together might account for the food habit transition and the formation of herbivory in grass carp.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1217-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-015-1217-x
PMCID: PMC4307112  PMID: 25608568
Food habit transition; Carnivory; Herbivory; Grass carp; Transcriptome sequencing
4.  Recycling Gene Carrier with High Efficiency and Low Toxicity Mediated by L-Cystine-Bridged Bis(β-cyclodextrin)s 
Scientific Reports  2014;4:7471.
Constructing safe and effective gene delivery carriers is becoming highly desirable for gene therapy. Herein, a series of supramolecular crosslinking system were prepared through host-guest binding of adamantyl-modified low molecular weight of polyethyleneimine with L-cystine-bridged bis(β-cyclodextrin)s and characterized by 1H NMR titration, electron microscopy, zeta potential, dynamic light-scattering, gel electrophoresis, flow cytometry and confocal fluorescence microscopy. The results showed that these nanometersized supramolecular crosslinking systems exhibited higher DNA transfection efficiencies and lower cytotoxicity than the commercial DNA carrier gold standard (25 kDa bPEI) for both normal cells and cancer cells, giving a very high DNA transfection efficiency up to 54% for 293T cells. Significantly, this type of supramolecular crosslinking system possesses a number of enzyme-responsive disulfide bonds, which can be cleaved by reductive enzyme to promote the DNA release but recovered by oxidative enzyme to make the carrier renewable. These results demonstrate that these supramolecular crosslinking systems can be used as promising gene carriers.
doi:10.1038/srep07471
PMCID: PMC4265772  PMID: 25503268
5.  Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles 
Objectives: Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. Methods: MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. Results: A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. Conclusions: We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection.
PMCID: PMC4307466  PMID: 25664019
Renal transplantation; acute rejection; microRNA; mRNA; transcription factor
6.  A general strategy for developing cell-permeable photo-modulatable organic fluorescent probes for live-cell super-resolution imaging 
Nature Communications  2014;5:5573.
Single-molecule localization microscopy (SMLM) achieves super-resolution imaging beyond the diffraction limit but critically relies on the use of photo-modulatable fluorescent probes. Here we report a general strategy for constructing cell-permeable photo-modulatable organic fluorescent probes for live-cell SMLM by exploiting the remarkable cytosolic delivery ability of a cell-penetrating peptide (rR)3R2. We develop photo-modulatable organic fluorescent probes consisting of a (rR)3R2 peptide coupled to a cell-impermeable organic fluorophore and a recognition unit. Our results indicate that these organic probes are not only cell permeable but can also specifically and directly label endogenous targeted proteins. Using the probes, we obtain super-resolution images of lysosomes and endogenous F-actin under physiological conditions. We resolve the dynamics of F-actin with 10 s temporal resolution in live cells and discern fine F-actin structures with diameters of ~80 nm. These results open up new avenues in the design of fluorescent probes for live-cell super-resolution imaging.
Single-molecule localization microscopy depends on the use of photo-modulatable fluorescent probes; however, many cannot be used in live-cell studies due to poor cell permeability. Pan et al. present a strategy for constructing cell-permeable probes and use it to image actin filament dynamics and lysosomes.
doi:10.1038/ncomms6573
PMCID: PMC4263135  PMID: 25410769
7.  Metabolomic approach to human brain spectroscopy identifies associations between clinical features and the frontal lobe metabolome in multiple sclerosis 
NeuroImage  2013;82:586-594.
Proton magnetic resonance spectroscopy (1H-MRS) is capable of noninvasively detecting metabolic changes that occur in the brain tissue in vivo. Its clinical utility has been limited so far, however, by analytic methods that focus on independently evaluated metabolites and require prior knowledge about which metabolites to examine. Here, we applied advanced computational methodologies from the field of metabolomics, specifically partial least squares discriminant analysis and orthogonal partial least squares, to in vivo 1H-MRS from frontal lobe white matter of 27 patients with relapsing–remitting multiple sclerosis (RRMS) and 14 healthy controls. We chose RRMS, a chronic demyelinating disorder of the central nervous system, because its complex pathology and variable disease course make the need for reliable biomarkers of disease progression more pressing. We show that in vivo MRS data, when analyzed by multivariate statistical methods, can provide reliable, distinct profiles of MRS-detectable metabolites in different patient populations. Specifically, we find that brain tissue in RRMS patients deviates significantly in its metabolic profile from that of healthy controls, even though it appears normal by standard MRI techniques. We also identify, using statistical means, the metabolic signatures of certain clinical features common in RRMS, such as disability score, cognitive impairments, and response to stress. This approach to human in vivo MRS data should promote understanding of the specific metabolic changes accompanying disease pathogenesis, and could provide biomarkers of disease progression that would be useful in clinical trials.
doi:10.1016/j.neuroimage.2013.05.125
PMCID: PMC3975905  PMID: 23751863
Magnetic resonance spectroscopy (MRS); Metabolomics; Relapsing–remitting multiple sclerosis; Multivariate statistics
8.  Cisplatin sensitivity is enhanced in non-small cell lung cancer cells by regulating epithelial-mesenchymal transition through inhibition of eukaryotic translation initiation factor 5A2 
BMC Pulmonary Medicine  2014;14(1):174.
Background
Epithelial-mesenchymal transition (EMT) has been believed to be related with chemotherapy resistance in non-small cell lung cancer (NSCLC). Recent studies have suggested eIF5A-2 may function as a proliferation-related oncogene in tumorigenic processes.
Methods
We used cell viability assays, western blotting, immunofluorescence, transwell-matrigel invasion assay, wound-healing assay combined with GC7 (a novel eIF5A-2 inhibitor) treatment or siRNA interference to investigate the role of eIF5A-2 playing in NSCLC chemotherapy.
Results
We found low concentrations of GC7 have little effect on NSCLC viability, but could enhance cisplatin cytotoxicity in NSCLC cells. GC7 also could reverse mesenchymal phenotype in NCI-H1299 and prevented A549 cells undergoing EMT after TGF-β1 inducement. eIF5A-2 knockdown resulted in EMT inhibition.
Conclusion
Our data indicated GC7 enhances cisplatin cytotoxicity and prevents the EMT in NSCLC cells by inhibiting eIF5A-2.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2466-14-174) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2466-14-174
PMCID: PMC4232729  PMID: 25380840
N1-guanyl-1, 7-diaminoheptane (GC7); Eukaryotic translation initiation factor 5A2 (eIF5A-2); Epithelial-mesenchymal transition (EMT); Cisplatin; Non-small cell lung cancer (NSCLC)
9.  Transcatheter Arterial Chemoembolization and Chemotherapy plus Sorafenib in a Large Hepatocellular Carcinoma with Arterioportal Shunt 
Introduction. Arterioportal shunts (APS) are sometimes encountered in patients with hepatocellular carcinoma (HCC) and associated with poor prognosis. The management of HCC with APS is a challenge so far. Case Presentations. We report here in detail a 37-year-old man who was diagnosed as an advanced HCC accompanied with severe APS and treated by two sessions of transcatheter arterial chemoembolization (TACE) and three sessions of transcatheter arterial chemotherapy (TAC) plus sorafenib therapy. The tumor shrinks were revealed continuously during 152 days after the diagnosis. Although tumor progress emerged at 209 days after the diagnosis, the patient remarkably achieved 366-day survival. Discussion. TACE plus sorafenib may be a promising treatment for advanced HCC accompanied with APS. Prospective case-control studies should be advocated to evaluate the combination of TACE, TAC, and sorafenib in the management of HCC with APS.
doi:10.1155/2014/392403
PMCID: PMC4241331  PMID: 25431715
10.  Risk Factors for Significant Wound Complications Following Wide Resection of Extremity Soft Tissue Sarcomas 
Background
Wound complications following resection of a localized soft tissue sarcoma have been associated with lower extremity location, large tumor volume, and use of preoperative radiation. Some of these wounds, however, show the potential for healing with local wound care and nonsurgical techniques. We are unaware of any published data establishing factors associated with nonhealing wounds that ultimately are treated with local or free vascularized tissue transfer.
Questions/purposes
The purpose of this study was to determine the variables associated with development of a significant wound complication defined as one that underwent a secondary procedure using local or free tissue transfer after resection of a localized soft tissue sarcoma.
Methods
Using our institution’s cancer center database, we identified 140 patients who underwent resection of a localized extremity soft tissue sarcoma at our institution between 1997 and 2010. Thirty-two patients were excluded who underwent immediate planned vascularized tissue transfer, along with 26 patients who did not receive radiation, and an additional three patients were excluded who were followed for less than 1 month. This left 79 patients, including 18 treated with postoperative external beam radiotherapy and 61 with preoperative external beam radiotherapy. Of patients receiving radiation treatment before surgery, 13 received no additional radiation treatment, 33 underwent intraoperative radiation with electrons (IOERT) to sites considered at high risk for local recurrence, and an additional 15 had perioperative brachytherapy. Univariate and multiple regression analyses were performed using frequency of local or free tissue transfer at 3 weeks or greater postoperatively owing to wound-related complications as a dependent variable.
Results
Lower extremity location and vascular involvement were associated with use of delayed vascularized tissue coverage for wound-healing problems. Patients in this series who underwent preoperative external beam radiotherapy coupled with dose-escalated IOERT or chemotherapy had a similar rate of flap use compared with patients treated with postoperative radiation.
Conclusions
Patients with tumors of the lower extremity involving major neurovascular structures and for whom radiation therapy is planned should be counseled specifically because they appear to be at increased risk for use of delayed local or free vascularized tissue transfer for a nonhealing wound following resection of a localized extremity soft tissue sarcoma.
Level of Evidence
Level III, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.
doi:10.1007/s11999-013-3130-4
PMCID: PMC3792250  PMID: 23813183
11.  Usefulness of Permanent Tracheostoma in Chronic Brain Injured Patients: A Case Series 
Yonsei Medical Journal  2014;55(6):1743-1746.
Patients with severe neurological deficit, such as hypoxic ischemic injury, cerebral infarction, and traumatic brain injury, often show comatose mental status and require maintenance of long-term tracheostomy for pulmonary toileting. However, several complications, which are mostly related to the cannula, invariably occur. Permanent tracheostoma is a short, skin-lined, noncollapsing, self-sustaining opening by suturing the denuded skin lining to the margin of the tracheal stoma. This tube-free method is a useful alternative to make long-term airway without tube-related complications in chronic diseases, such as obstructive sleep apnea, and laryngeal cancer, however, it has not yet been reported in chronic brain injured patients. This case report illustrates 3 cases of vegetative patients in our rehabilitation clinic who underwent successful procedure of permanent tracheostoma. Permanent tracheostoma has some benefits associated with the free of tube-related complications, and can be considered as a useful alternative way for chronic brain injured patients with long-term tracheostomy.
doi:10.3349/ymj.2014.55.6.1743
PMCID: PMC4205718  PMID: 25323915
Permanent tracheostoma; chronic brain injury; tracheostomy; rehabilitation
12.  Lack of Negatively Charged Residues at the External Mouth of Kir2.2 Channels Enable the Voltage-Dependent Block by External Mg2+ 
PLoS ONE  2014;9(10):e111372.
Kir channels display voltage-dependent block by cytosolic cations such as Mg2+ and polyamines that causes inward rectification. In fact, cations can regulate K channel activity from both the extracellular and intracellular sides. Previous studies have provided insight into the up-regulation of Kir channel activity by extracellular K+ concentration. In contrast, extracellular Mg2+ has been found to reduce the amplitude of the single-channel current at milimolar concentrations. However, little is known about the molecular mechanism of Kir channel blockade by external Mg2+ and the relationship between the Mg2+ blockade and activity potentiation by permeant K+ ions. In this study, we applied an interactive approach between theory and experiment. Electrophysiological recordings on Kir2.2 and its mutants were performed by heterologous expression in Xenopus laevis oocytes. Our results confirmed that extracellular Mg2+ could reduce heterologously expressed WT Kir2.2 currents in a voltage dependent manner. The kinetics of inhibition and recovery of Mg2+ exhibit a 3∼4s time constant. Molecular dynamics simulation results revealed a Mg2+ binding site located at the extracellular mouth of Kir2.2 that showed voltage-dependent Mg2+ binding. The mutants, G119D, Q126E and H128D, increased the number of permeant K+ ions and reduced the voltage-dependent blockade of Kir2.2 by extracellular Mg2+.
doi:10.1371/journal.pone.0111372
PMCID: PMC4211740  PMID: 25350118
13.  Selective DNA Delivery to Tumor Cells Using an Oligoarginine-LTVSPWY Peptide 
PLoS ONE  2014;9(10):e110632.
DNA therapy for cancer requires efficient, selective and safe DNA delivery systems. Compared with other non-viral methods such as lipid or polymer-based DNA delivery vectors, peptide-based DNA delivery systems are biocompatible and biodegradable, which leads to lower immunogenicity and lower toxicity. Moreover, peptide vectors are easier to produce and their compositions easier to control because solid-phase peptide synthesis has been extensively developed. However, peptide-based systems for DNA delivery toward special tumor cells or tissues are still lacking. In this study, we constructed a non-viral 9rR-LTVSPWY peptide-based DNA delivery system and showed that it is able to efficiently and selectively transfect DNA into targeted tumor cells. This work presents a novel strategy for tumor cell-specific DNA delivery and a reference for designing more efficient DNA delivery systems targeted towards various types of cancer.
doi:10.1371/journal.pone.0110632
PMCID: PMC4206439  PMID: 25337703
14.  One Dose versus Three Weekly Doses of Benzathine Penicillin G for Patients Co-Infected with HIV and Early Syphilis: A Multicenter, Prospective Observational Study 
PLoS ONE  2014;9(10):e109667.
Background
One dose of benzathine penicillin G (BPG) has been recommended for HIV-infected patients with early syphilis (primary, secondary, and early latent syphilis) in the sexually transmitted diseases treatment guidelines, but clinical data to support such a recommendation are limited.
Methods
We prospectively observed the serological response to 1 or 3 weekly doses of BPG in HIV-infected adults who sought treatment of early syphilis at 8 hospitals around Taiwan. Rapid plasma reagin (RPR) titers were followed every 3–6 months after treatment. The serological response was defined as a 4-fold or greater decline in RPR titers at 12 months of treatment. The missing values were treated by following the last-observed-carried-forward principle. We hypothesized that 1 dose was non-inferior to 3 weekly doses of BPG with the non-inferiority margin for the difference of serological response set to 10%.
Results
Between 2007 and 2012, 573 patients completed at least 12 months of follow-up: 295 (51.5%) receiving 1 dose of BPG (1-dose group) and 278 (48.5%) 3 doses (3-dose group). Overall, 198 patients (67.1%; 95% confidence interval [CI], 61.4–72.5%) in the 1-dose group achieved serological response at 12 months, as did 208 patients (74.8%; 95% CI, 69.3–79.8%) in the 3-dose group (one-sided 95% CI of the difference, 15.1%). In the multivariate analysis, secondary syphilis (adjusted odds ratio [AOR], 1.90; 95% CI 1.17–3.09), RPR titer ≥32 (AOR, 1.93; 95% CI, 1.38–2.69), and 3 doses of BPG (AOR, 1.68; 95% CI, 1.20–2.36) were independently associated with a serological response. The time to the first episode of treatment failure was 1184 (standard deviation [SD], 70.5) and 1436 (SD, 80.0) days for 1- and 3-dose group, respectively.
Conclusions
Single-dose BPG resulted in a higher serological failure rate and shorter time to treatment failure than 3 weekly doses of BPG in the treatment of early syphilis in HIV-infected patients.
doi:10.1371/journal.pone.0109667
PMCID: PMC4186862  PMID: 25286091
15.  Effect of Pioglitazone on In-Stent Restenosis after Coronary Drug-Eluting Stent Implantation: A Meta-Analysis of Randomized Controlled Trials 
PLoS ONE  2014;9(10):e109614.
Background
In-stent restenosis (ISR) remains a common life-threatening complication and some studies have shown that pioglitazone can reduce the incidence of ISR in patients with drug-eluting stents (DES) implantation. We conducted a meta-analysis to assess the effect of pioglitazone in preventing ISR after DES implantation.
Methods
Randomized controlled trials (RCTs) investigating the effects of pioglitazone for ISR after DES implantation were identified by systematic searches of multiple online databases and manual searches of related reference lists of identified trials through May 2014. The primary endpoint was the rate of ISR. Secondary endpoints included minimum lumen diameter, percentage stenosis of stented vessels, late loss, in-stent neointimal volume, target vessel revascularization (TVR), target lesion revascularization, myocardial infarction, stent thrombosis and death.
Results
Five studies, comprising 255 pioglitazone-treated patients and 245 controls, were identified in the current meta-analysis. Pioglitazone did not significantly reduce the rate of ISR (P = 0.20) with low heterogeneity (I2 = 13.3%, P = 0.32). For the secondary outcomes, pioglitazone did not substantially affect the pooled estimates of these endpoints except late loss (P = 0.01) and TVR (P = 0.04).
Conclusions
The limited evidence indicates that pioglitazone does not demonstrate markedly beneficial effect in patients subjected to coronary DES implantation. However, the results should be interpreted with care given the small sample size. Further large-scale RCTs are needed.
doi:10.1371/journal.pone.0109614
PMCID: PMC4184901  PMID: 25279761
16.  Validation of a Pretransplant Risk Score for New-Onset Diabetes After Kidney Transplantation 
Diabetes Care  2013;36(10):2881-2886.
OBJECTIVE
Identification of patients at high risk for new-onset diabetes after kidney transplantation (NODAT) will facilitate clinical trials for its prevention.
RESEARCH DESIGN AND METHODS
We previously described a pretransplant predictive risk model for NODAT using seven pretransplant variables (age, planned use of maintenance corticosteroids, prescription for gout medicine, BMI, fasting glucose, fasting triglycerides, and family history of diabetes). We have now applied the initial model to a cohort of 474 transplant recipients from another center for validation. We performed two analyses in the validation cohort. The first was a standard model with variables derived from the original study. The second was a summary score model, in which the sum of dichotomized variables (all the variables dichotomized at clinically relevant cut points) was used to categorize, individuals into low (0–1), intermediate (2, 3), or high (4–7) risk groups. We also conducted a combined database analyses, merging the initial and validation cohorts (n = 792) to obtain better estimates for a prediction equation.
RESULTS
Although the frequency of several risk factors differed significantly between the two cohorts, the models performed similarly in each cohort. Using the summary score model, incidences of NODAT in low-risk, medium-risk, and high-risk groups in the initial cohort were 12, 29, and 56%, and in the validation cohort incidences were 11, 29, and 51%.
CONCLUSIONS
A pretransplant model for NODAT, including many type 2 diabetes risk factors, predicted NODAT in the validation cohort.
doi:10.2337/dc13-0428
PMCID: PMC3781551  PMID: 24009296
18.  Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3* 
The Journal of Biological Chemistry  2014;289(44):30625-30634.
Background: The role of the unspliced XBP1 remains unclear.
Results: Disturbed flow concomitantly up-regulates XBP1u and HDAC3, which form a complex with Akt1 and mTOR, leading to Nrf2-mediated HO-1 expression.
Conclusion: XBP1u and HDAC3 synergistically exert a protective effect on disturbed flow-induced oxidative stress via regulation of HO-1 expression.
Significance: This study provides new insights into the physiological roles of XBP1u and HDAC3.
It is well known that atherosclerosis occurs geographically at branch points where disturbed flow predisposes to the development of plaque via triggering of oxidative stress and inflammatory reactions. In this study, we found that disturbed flow activated anti-oxidative reactions via up-regulating heme oxygenase 1 (HO-1) in an X-box-binding protein 1 (XBP1) and histone deacetylase 3 (HDAC3)-dependent manner. Disturbed flow concomitantly up-regulated the unspliced XBP1 (XBP1u) and HDAC3 in a VEGF receptor and PI3K/Akt-dependent manner. The presence of XBP1 was essential for the up-regulation of HDAC3 protein. Overexpression of XBP1u and/or HDAC3 activated Akt1 phosphorylation, Nrf2 protein stabilization and nuclear translocation, and HO-1 expression. Knockdown of XBP1u decreased the basal level and disturbed flow-induced Akt1 phosphorylation, Nrf2 stabilization, and HO-1 expression. Knockdown of HDAC3 ablated XBP1u-mediated effects. The mammalian target of rapamycin complex 2 (mTORC2) inhibitor, AZD2014, ablated XBP1u or HDAC3 or disturbed flow-mediated Akt1 phosphorylation, Nrf2 nuclear translocation, and HO-1 expression. Neither actinomycin D nor cycloheximide affected disturbed flow-induced up-regulation of Nrf2 protein. Knockdown of Nrf2 abolished XBP1u or HDAC3 or disturbed flow-induced HO-1 up-regulation. Co-immunoprecipitation assays demonstrated that XBP1u physically bound to HDAC3 and Akt1. The region of amino acids 201 to 323 of the HDAC3 protein was responsible for the binding to XBP1u. Double immunofluorescence staining revealed that the interactions between Akt1 and mTORC2, Akt1 and HDAC3, Akt1 and XBP1u, HDAC3, and XBP1u occurred in the cytosol. Thus, we demonstrate that XBP1u and HDAC3 exert a protective effect on disturbed flow-induced oxidative stress via up-regulation of mTORC2-dependent Akt1 phosphorylation and Nrf2-mediated HO-1 expression.
doi:10.1074/jbc.M114.571984
PMCID: PMC4215241  PMID: 25190803
Cell Signaling; Endothelial Cell; Histone Deacetylase (HDAC); Oxidative Stress; Shear Stress
19.  An NMR Metabolomic Study on the Effect of Alendronate in Ovariectomized Mice 
PLoS ONE  2014;9(9):e106559.
Alendronate sodium (Fosamax) is most widely used for the prevention and treatment of osteoporosis. It is a type of anti-resorptive agent that reduces the risk of fractures by changing bone turnover and bone mineral density. We investigated the effect of Fosamax on a mouse model of osteoporosis. Twenty-seven female C57BL/6JNarl mice were divided into three groups: sham, ovariectomized (OVX) and OVX + Fosamax (Fosamax). After 23 weeks, bone density of femurs was analyzed using microcomputed tomography (micro-CT), and serum was analyzed for osteoblast and osteoclast activity, as well as metabolites using nuclear magnetic resonance (NMR) spectroscopy. Fosamax increased bone mineral density and cortical bone thickness, and decreased osteoblast activity slightly. Fosamax did not significantly change osteoclast activity. Serum metabolomics revealed that Fosamax had profound effects on overall metabolism, as significantly higher concentrations of metabolites associated with energy metabolism (including TCA-cycle intermediates and glucose), 3-hydroxybutyrate, taurine, allantoin, acetate, and ethanol, as well as lower concentrations of aspartate were observed in the Fosamax-treated mice compared with the OVX mice. These results suggest that alendronate may work by increasing bone density through altered metabolic activity.
doi:10.1371/journal.pone.0106559
PMCID: PMC4153652  PMID: 25184758
20.  Structure and domain organization of Drosophila Tudor 
Cell Research  2014;24(9):1146-1149.
doi:10.1038/cr.2014.63
PMCID: PMC4152741  PMID: 24810300
21.  Distance from Construction Site and Risk for Coccidioidomycosis, Arizona, USA 
Emerging Infectious Diseases  2014;20(9):1464-1471.
Working adjacent to a project involving excavation of desert soil did not increase the risk for infection.
Coccidioides spp. fungi, which are present in soil in the southwestern United States, can become airborne when the soil is disrupted, and humans who inhale the spores can become infected. In 2012, our institution in Maricopa County, Arizona, USA, began a building project requiring extensive excavation of soil. One year after construction began, we compared the acquisition of coccidioidomycosis in employees working adjacent to the construction site (campus A) with that of employees working 13 miles away (campus B). Initial testing indicated prior occult coccidioidal infection in 20 (11.4%) of 176 campus A employees and in 19 (13.6%) of 140 campus B employees (p = 0.55). At the 1-year follow-up, 3 (2.5%) of 120 employees from campus A and 8 (8.9%) of 90 from campus B had flow cytometric evidence of new coccidioidal infection (p = 0.04). The rate of coccidioidal acquisition differed significantly between campuses, but was not higher on the campus with construction.
doi:10.3201/eid2009.131588
PMCID: PMC4178394  PMID: 25148473
Coccidioides; coccidioidomycosis; fungal infection; fungi; Arizona; construction site
22.  Wnts Enhance Neurotrophin-Induced Neuronal Differentiation in Adult Bone-Marrow-Derived Mesenchymal Stem Cells via Canonical and Noncanonical Signaling Pathways 
PLoS ONE  2014;9(8):e104937.
Wnts were previously shown to regulate the neurogenesis of neural stem or progenitor cells. Here, we explored the underlying molecular mechanisms through which Wnt signaling regulates neurotrophins (NTs) in the NT-induced neuronal differentiation of human mesenchymal stem cells (hMSCs). NTs can increase the expression of Wnt1 and Wnt7a in hMSCs. However, only Wnt7a enables the expression of synapsin-1, a synaptic marker in mature neurons, to be induced and triggers the formation of cholinergic and dopaminergic neurons. Human recombinant (hr)Wnt7a and general neuron makers were positively correlated in a dose- and time-dependent manner. In addition, the expression of synaptic markers and neurites was induced by Wnt7a and lithium, a glycogen synthase kinase-3β inhibitor, in the NT-induced hMSCs via the canonical/β-catenin pathway, but was inhibited by Wnt inhibitors and frizzled-5 (Frz5) blocking antibodies. In addition, hrWnt7a triggered the formation of cholinergic and dopaminergic neurons via the non-canonical/c-jun N-terminal kinase (JNK) pathway, and the formation of these neurons was inhibited by a JNK inhibitor and Frz9 blocking antibodies. In conclusion, hrWnt7a enhances the synthesis of synapse and facilitates neuronal differentiation in hMSCS through various Frz receptors. These mechanisms may be employed widely in the transdifferentiation of other adult stem cells.
doi:10.1371/journal.pone.0104937
PMCID: PMC4149376  PMID: 25170755
23.  Jarisch-Herxheimer reaction among HIV-positive patients with early syphilis: azithromycin versus benzathine penicillin G therapy 
Introduction
The Jarisch-Herxheimer reaction, a febrile inflammatory reaction that often occurs after the first dose of chemotherapy in spirochetal diseases, may result in deleterious effects to patients with neurosyphilis and to pregnant women. A single 2-g oral dose of azithromycin is an alternative treatment to benzathine penicillin G for early syphilis in areas with low macrolide resistance. With its potential anti-inflammatory activity, the impact of azithromycin on the incidence of the Jarisch-Herxheimer reaction in HIV-positive patients with early syphilis has rarely been investigated.
Methods
In HIV-positive patients with early syphilis, the Jarisch-Herxheimer reaction was prospectively investigated using the same data collection form in 119 patients who received benzathine penicillin G between 2007 and 2009 and 198 who received azithromycin between 2012 and 2013, when shortage of benzathine penicillin G occurred in Taiwan. Between 2012 and 2013, polymerase chain reaction (PCR) assay was performed to detect Treponema pallidum DNA in clinical specimens, and PCR restriction fragment length polymorphism of the 23S ribosomal RNA was performed to detect point mutations (2058G or A2059G) that are associated with macrolide resistance.
Results
The overall incidence of the Jarisch-Herxheimer reaction was significantly lower in patients receiving azithromycin than those receiving benzathine penicillin G (14.1% vs. 56.3%, p<0.001). The risk increased with higher rapid plasma reagin (RPR) titres (adjusted odds ratio [AOR] per 1-log2 increase, 1.21; confidence interval [CI], 1.04–1.41), but decreased with prior penicillin therapy for syphilis (AOR, 0.37; 95% CI, 0.19–0.71) and azithromycin treatment (AOR, 0.15; 95% CI, 0.08–0.29). During the study period, 310 specimens were obtained from 198 patients with syphilis for PCR assays, from whom T. pallidum was identified in 76 patients, one of whom (1.3%) was found to be infected with T. pallidum harbouring the macrolide resistance mutation (A2058G). In subgroup analyses confined to the 75 patients infected with T. pallidum lacking resistance mutation, a statistically significantly lower risk for the Jarisch-Herxheimer reaction following azithromycin treatment was noted.
Conclusions
Treatment with azithromycin was associated with a lower risk for the Jarisch-Herxheimer reaction than that with benzathine penicillin G in HIV-positive patients with early syphilis. Previous benzathine penicillin G therapy for syphilis decreased the risk, whereas higher RPR titres increased the risk, for the reaction.
doi:10.7448/IAS.17.1.18993
PMCID: PMC4150017  PMID: 25174641
sexually transmitted diseases; spirochetal disease; macrolides; macrolide resistance; immunomodulation
24.  Large Sample Area and Size Are Needed for Forest Soil Seed Bank Studies to Ensure Low Discrepancy with Standing Vegetation 
PLoS ONE  2014;9(8):e105235.
A large number of small-sized samples invariably shows that woody species are absent from forest soil seed banks, leading to a large discrepancy with the seedling bank on the forest floor. We ask: 1) Does this conventional sampling strategy limit the detection of seeds of woody species? 2) Are large sample areas and sample sizes needed for higher recovery of seeds of woody species? We collected 100 samples that were 10 cm (length) ×10 cm (width) ×10 cm (depth), referred to as larger number of small-sized samples (LNSS) in a 1 ha forest plot, and placed them to germinate in a greenhouse, and collected 30 samples that were 1 m×1 m×10 cm, referred to as small number of large-sized samples (SNLS) and placed them (10 each) in a nearby secondary forest, shrub land and grass land. Only 15.7% of woody plant species of the forest stand were detected by the 100 LNSS, contrasting with 22.9%, 37.3% and 20.5% woody plant species being detected by SNLS in the secondary forest, shrub land and grassland, respectively. The increased number of species vs. sampled areas confirmed power-law relationships for forest stand, the LNSS and SNLS at all three recipient sites. Our results, although based on one forest, indicate that conventional LNSS did not yield a high percentage of detection for woody species, but SNLS strategy yielded a higher percentage of detection for woody species in the seed bank if samples were exposed to a better field germination environment. A 4 m2 minimum sample area derived from power equations is larger than the sampled area in most studies in the literature. Increased sample size also is needed to obtain an increased sample area if the number of samples is to remain relatively low.
doi:10.1371/journal.pone.0105235
PMCID: PMC4139333  PMID: 25140738
25.  Optimal beam arrangement for pulmonary ventilation image-guided intensity-modulated radiotherapy for lung cancer 
Background
The principal aim of this study was to evaluate the feasibility of incorporating four-dimensional (4D)-computed tomography (CT)-based functional information into treatment planning and to evaluate the potential benefits of individualized beam setups to better protect lung functionality in patients with non-small cell lung cancer (NSCLC).
Methods
Peak-exhale and peak-inhale CT scans were carried out in 16 patients with NSCLC treated with intensity-modulated radiotherapy (IMRT). 4D-CT-based ventilation information was generated from the two sets of CT images using deformable image registration. Four kinds of IMRT plans were generated for each patient: two anatomic plans without incorporation of ventilation information, and two functional plans with ventilation information, using either five equally spaced beams (FESB) or five manually optimized beams (FMOB). The dosimetric parameters of the plans were compared in terms of target and normal tissue structures, with special focus on dose delivered to total lung and functional lung.
Results
In both the anatomic and functional plans, the percentages of both the functional and total lung regions irradiated at V5, V10, and V20 (percentage volume irradiated to >5, >10 and >20 Gy, respectively) were significantly lower for FMOB compared with FESB (P < 0.05), but there was no significant difference for V30 (P > 0.05). Compared with FESB, a greater degree of sparing of the functional lung was achieved in functional IMRT plans with optimal beam arrangement, without compromising target volume coverage or the irradiated volume of organs at risk, such as the spinal cord, esophagus, and heart.
Conclusions
Pulmonary ventilation image-guided IMRT planning with further optimization of beam arrangements improves the preservation of functional lung in patients with NSCLC.
doi:10.1186/1748-717X-9-184
PMCID: PMC4141960  PMID: 25127899
Intensity-modulated radiotherapy; Pulmonary ventilation; Four-dimensional computed tomography; Plan optimization

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