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1.  Association between implant apex and sinus floor in posterior maxilla dental implantation: A three-dimensional finite element analysis 
The aim of the present study was to evaluate the effect of the association between the implant apex and the sinus floor in posterior maxilla dental implantation by means of three-dimensional (3D) finite element (FE) analysis. Ten 3D FE models of a posterior maxillary region with a sinus membrane and different heights of alveolar ridge with different thicknesses of sinus floor cortical bone were constructed according to anatomical data of the sinus area. Six models were constructed with the same thickness of crestal cortical bone and a 1-mm thick sinus floor cortical bone, but differing heights of alveolar ridge (between 10 and 14 mm). The four models of the second group were similar (11-mm-high alveolar ridge and 1-mm-thick crestal cortical bone) but with a changing thickness of sinus floor cortical bone (between 0.5 and 2.0 mm). The standard implant model based on the Nobel Biocare® implant system was created by computer-aided design (CAD) software and assembled into the models. The materials were assumed to be isotropic and linearly elastic. An inclined force of 129 N was applied. The maximum von Mises stress, stress distribution, implant displacement and resonance frequencies were calculated using CAD software. The von Mises stress was concentrated on the surface of the crestal cortical bone around the implant neck with the exception of that for the bicortical implantation. For immediate loading, when the implant apex broke into or through the sinus cortical bone, the maximum displacements of the implant, particularly at the implant apex, were smaller than those in the other groups. With increasing depth of the implant apex in the sinus floor cortical bone, the maximum displacements decreased and the implant axial resonance frequencies presented a linear upward tendency, but buccolingual resonance frequencies were hardly affected. This FE study on the association between implant apex and sinus floor showed that having the implant apex in contact with, piercing or breaking through the sinus floor cortical bone benefited the implant stability, particularly for immediate loading.
PMCID: PMC4316907  PMID: 25667644
finite element analysis; dental implants; stress distribution; resonance frequency; sinus floor; implant apex; cortical bone
2.  Removal of the Tag from His-tagged ILYd4, a Human CD59 Inhibitor, Significantly Improves its Physical Properties and its Activity 
Current pharmaceutical design  2012;18(27):4187-4196.
Complement dependent cytotoxicity (CDC) significantly contributes to Rituximab (RTX) and Ofatumumab (OFA) efficacies in the treatment of B-cell non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Human CD59 (hCD59) is a key complement regulatory protein that restricts the formation of the membrane attack complex and thereby inhibits CDC. hCD59 is an important determinant of the sensitivity of NHL and CLL to RTX and OFA treatment. Recently, we developed a specific and potent hCD59 inhibitor, His-tagged ILYd4, which consists of 30 amino acid sequences extending from the N-terminus of ILYd4. Our previously published results indicate that His-tagged ILYd4 can be used as a lead candidate to further develop a potential therapeutic adjuvant for RTX and OFA treatment of RTX-resistant NHL and CLL. However, these studies were conducted using ILYd4 tagged on the N-terminus with 30 additional amino acids (AA) containing 6 X His used for immobilized metal affinity chromatograph. As a further step towards the development of ILYd4-based therapeutics, we investigated the impact of the removal of this extraneous sequence on the anti-hCD59 activity. In this paper, we report the generation and characterization of tag-free ILYd4. We demonstrate that tag-free ILYd4 has over three-fold higher anti-hCD59 activities than the His-tagged ILYd4. The enhanced RTX-mediated CDC effect on B-cell malignant cells comes from tag-free ILYd4’s improved functionality and physical properties including better solubility, reduced tendency to aggregation, and greater thermal stability. Therefore, tag-free ILYd4 is a better candidate for the further development for the clinical application.
PMCID: PMC4279449  PMID: 22642361
Rituximab; complement; CD59; intermedilysin; his-tag
3.  Circulating SFRP1 promoter methylation status in gastric adenocarcinoma and esophageal square cell carcinoma 
Biomedical Reports  2014;3(1):123-127.
The secreted frizzled-related protein 1 (SFRP1) gene plays an important role in carcinogenesis of digestive system cancer. Previous studies proved that circulating DNA promoter methylation may be a suitable biomarker for cancer patients. The aim of the present study was to investigate whether the promoter methylation status of serum SFRP1 is a potential biomarker for gastric adenocarcinoma (GAC) and esophageal square cell carcinoma (ESCC). The blood samples obtained from 42 GAC and 36 ESCC patients were detected for the promoter methylation status of SFRP1 by methylation-specific polymerase chain reaction. The control group included 42 benign gastrointestinal disease volunteers (24 benign gastric disease and 18 benign esophageal disease) and 20 healthy volunteers. Serum SFRP1 methylation was evident in 30.95% (13/42) GAC patients and 38.89% (14/36) ESCC patients, which is clearly higher compared to 8.33% (2/24) in benign gastric disease, 11.11% (2/18) in benign esophageal disease and 5% (1/20) in healthy volunteers (P<0.05). The association between the serum SFRP1 promoter methylation status and the clinical pathological features were further analyzed and methylation of the SFRP1 gene was significantly associated with age >60 years in GAC patients (P=0.027). However, no correlations between the SFRP1 methylation status and other clinicopathological parameters were found. In conclusion, the SFRP1 promoter was detected frequently in the serum of GAC and ESCC patients. The detection of circulating methylated SFRP1 in the serum may be a useful biomarker for upper gastrointestinal cancer patients.
PMCID: PMC4251162  PMID: 25469261
methylation; SFRP1 gene; gastric adenocarcinoma; methylation-specific polymerase chain reaction; esophageal square cell carcinoma
4.  Ginsenoside Rb1 Prevents H2O2-Induced HUVEC Senescence by Stimulating Sirtuin-1 Pathway 
PLoS ONE  2014;9(11):e112699.
We have previously reported that Ginsenoside Rb1 may effectively prevent HUVECs from senescence, however, the detailed mechanism has not demonstrated up to now. Recent studies have shown that sirtuin-1 (Sirt1) plays an important role in the development of endothelial senescence. The purpose of this study was to explore whether Sirt1 is involved in the action of Ginsenoside Rb1 regarding protection against H2O2-induced HUVEC Senescence.
Methods and Results
Senescence induced by hydrogen peroxide (H2O2) in human umbilical vein endothelial cells (HUVECs) was examined by analyzing plasminogen activator inhibitor-1 (PAI-1) expression, cell morphology, and senescence-associated beta-galactosidase (SA-β-gal) activity. The results revealed that 42% of control-treated HUVECs were SA-β-gal positive after treatment by 60 µmol/L H2O2, however, this particular effect of H2O2 was decreased more than 2-fold (19%) in the HUVECs when pretreated with Rb1 (20 µmol/L) for 30 min. Additionally, Rb1 decreased eNOS acetylation, as well as promoted more NO production that was accompanied by an increase in Sirt1 expression. Furthermore, upon knocking down Sirt1, the effect of Rb1 on HUVEC senescence was blunted.
The present study indicated that Ginsenoside Rb1 acts through stimulating Sirt1 in order to protect against endothelial senescence and dysfunction. As such, Sirt1 appears to be of particular importance in maintaining endothelial functions and delaying vascular aging.
PMCID: PMC4227851  PMID: 25386949
5.  Fluorofenidone Offers Improved Renoprotection at Early Interventions during the Course of Diabetic Nephropathy in db/db Mice via Multiple Pathways 
PLoS ONE  2014;9(10):e111242.
Diabetic nephropathy (DN) remains the leading cause of end-stage renal disease (ESRD), a situation that is in part attributable to the lack of effective treatments. Fluorofenidone is a newly developed reagent with anti-fibrotic activity. While fluorofenidone was previously demonstrated to possess renoprotection from DN pathogenesis in db/db mice, the protective process and its underlying mechanisms have not been well studied. To characterize fluorofenidone-derived renoprotection, we treated 5, 8, or 12-week old db/db mice with daily doses of placebo, fluorofenidone, or losartan until 24 weeks of age; the time at which diabetes and DN were fully developed in placebo-treated animals. In comparison to db/db mice receiving fluorofenidone at 12-weeks old, those treated at 5-weeks had less glomerular expansion and better preservation of renal functions, judged by serum creatinine levels, albumin to creatinine ratio, and urinary albumin excretion (mg/24 hours). These benefits of early treatment were associated with significant reductions of multiple DN-promoting events, such as decreased expression of TGF-β1 and the p22phox subunit of NADPH oxidase as well as downregulated activation of protein kinase C-zeta (ζ), ERK and AKT. This improvement in renoprotection following early interventions is not a unique property of DN pathogenesis, as losartan does not apparently offer the same benefits and is not more renoprotective than fluorofenidone. Additionally, the enhanced renoprotection provided by fluorofenidone did not affect the diabetic process, as it did not alter serum levels of glycated serum proteins, glucose, triglyceride or cholesterol. Collectively, we provide evidence that fluorofenidone offers improved renoprotection at early stages of DN pathogenesis.
PMCID: PMC4210223  PMID: 25347392
6.  Mitochondrial fusion but not fission regulates larval growth and synaptic development through steroid hormone production 
eLife  2014;3:e03558.
Mitochondrial fusion and fission affect the distribution and quality control of mitochondria. We show that Marf (Mitochondrial associated regulatory factor), is required for mitochondrial fusion and transport in long axons. Moreover, loss of Marf leads to a severe depletion of mitochondria in neuromuscular junctions (NMJs). Marf mutants also fail to maintain proper synaptic transmission at NMJs upon repetitive stimulation, similar to Drp1 fission mutants. However, unlike Drp1, loss of Marf leads to NMJ morphology defects and extended larval lifespan. Marf is required to form contacts between the endoplasmic reticulum and/or lipid droplets (LDs) and for proper storage of cholesterol and ecdysone synthesis in ring glands. Interestingly, human Mitofusin-2 rescues the loss of LD but both Mitofusin-1 and Mitofusin-2 are required for steroid-hormone synthesis. Our data show that Marf and Mitofusins share an evolutionarily conserved role in mitochondrial transport, cholesterol ester storage and steroid-hormone synthesis.
eLife digest
Mitochondria are the main source of energy for cells. These vital and highly dynamic organelles continually change shape by fusing with each other and splitting apart to create new mitochondria, repairing and replacing those damaged by cell stress.
For nerve impulses to be transmitted across the gaps (called synapses) between nerve cells, mitochondria need to supply the very ends of the nerve fibers with energy. To do this, the mitochondria must be transported from the main body of the nerve cell to the tips of the nerve fibers. This may not happen if mitochondria are the wrong shape, size or damaged.
While searching for genetic mutations that disrupt nerve function in the fruit fly Drosophila, Sandoval et al. spotted mutations in a gene called Marf. Further investigations revealed that flies with mutant versions of Marf have small, round mitochondria, and their nerves cannot transmit signals to muscles when they are highly stimulated. This is because the mutant mitochondria are not easily transported along nerve fibers, and so not enough energy is supplied to the synapses. The synapses of the Marf mutants are also abnormally shaped. Sandoval et al. found that this is not because Marf is lost in the neurons themselves, but because it is lost from a hormone-producing tissue called the ring gland.
Another problem found in flies with mutated Marf genes is that they stop developing while in their larval stage. Sandoval et al. established that this could also be related to the loss of Marf from the ring gland. The Marf protein has two different functions in the ring gland: forming and storing droplets of fatty molecules used in hormone production, and synthesising a hormone that controls when a fly larva matures into the adult fly. This suggests that the lower levels of this hormone produced by Marf mutant flies underlies their prolonged larval stages and synapse defects.
Vertebrates (animals with backbones, such as humans) have two genes that are related to the fly's Marf gene. When the human forms of these genes were introduced into mutant flies that lack a working copy of Marf, hormone production was only restored if both genes were introduced together. This indicates that these genes have separate roles in vertebrates, but that these roles are both performed by the single fly gene.
The role of Marf in tethering mitochondria in the ring gland may allow us to better understand how this process affects hormone production and how the different parts of the cell communicate.
PMCID: PMC4215535  PMID: 25313867
mitochondria transport; Charcot-Marie-Tooth type 2A; Mfn1 and Mfn2; Drp1; Opa1; lipid droplets; endoplasmic reticulum; Drosophila melanogaster
7.  Albumin-based Nanoconjugates for Targeted Delivery of Therapeutic Oligonucleotides 
Biomaterials  2013;34(32):10.1016/j.biomaterials.2013.06.066.
Nanoparticle-based delivery has become an important strategy to advance siRNA and antisense oligonucleotides into clinical reality. However, limited biodistribution of nanoparticles and the toxicity of some nanocarriers restrict the wider application of this strategy. To address these issues we aimed to construct oligonucleotide delivery systems which are non-cytotoxic and smaller than typical nanoparticles. Thus, a morpholino oligonucleotide was conjugated to a tumor-targeting RGD peptide, and then, multiple RGD-oligo conjugates were linked to a single molecule of human serum albumin via a reductively responsive linkage. The resultant nanoconjugates showed uniform and monodispersed size distribution with a diameter of 13 nm. A single nanoconjugate molecule contains 15 oligonucleotides as well as 15 targeting ligands on the surface of albumin. The nanoparticle demonstrated 61-fold enhancement in receptor-specific cellular delivery of oligonucleotides in integrin-expressing tumor cells compared to the non-targeted control nanoconjugates and were able to robustly enhance functional activity of the oligonucleotide at low nanomolar concentrations without causing cytotoxicity. Due to their small size, the targeted nanoconjugates could penetrate deeply and distribute throughout 3-D tumor spheroids, whereas the conventional nanoparticles with sizes over 300 nm could only deliver to the cells on the surface of the tumor spheroids. As a result of their greater cellular delivery, smaller size, and lack of cytotoxicity compared to conventional nanoparticles, the multivalent nanoconjugates may provide an effective tool for targeting oligonucleotides to tumors and other diseased tissues.
PMCID: PMC3810404  PMID: 23876758
Targeted delivery; Therapeutic Oligonucleotides; Albumin; Conjugation
8.  Smoking-Induced CXCL14 Expression in the Human Airway Epithelium Links Chronic Obstructive Pulmonary Disease to Lung Cancer 
CXCL14, a recently described epithelial cytokine, plays putative multiple roles in inflammation and carcinogenesis. In the context that chronic obstructive pulmonary disease (COPD) and lung cancer are both smoking-related disorders associated with airway epithelial disorder and inflammation, we hypothesized that the airway epithelium responds to cigarette smoking with altered CXCL14 gene expression, contributing to the disease-relevant phenotype. Using genome-wide microarrays with subsequent immunohistochemical analysis, the data demonstrate that the expression of CXCL14 is up-regulated in the airway epithelium of healthy smokers and further increased in COPD smokers, especially within hyperplastic/metaplastic lesions, in association with multiple genes relevant to epithelial structural integrity and cancer. In vitro experiments revealed that the expression of CXCL14 is induced in the differentiated airway epithelium by cigarette smoke extract, and that epidermal growth factor mediates CXCL14 up-regulation in the airway epithelium through its effects on the basal stem/progenitor cell population. Analyses of two independent lung cancer cohorts revealed a dramatic up-regulation of CXCL14 expression in adenocarcinoma and squamous-cell carcinoma. High expression of the COPD-associated CXCL14-correlating cluster of genes was linked in lung adenocarcinoma with poor survival. These data suggest that the smoking-induced expression of CXCL14 in the airway epithelium represents a novel potential molecular link between smoking-associated airway epithelial injury, COPD, and lung cancer.
PMCID: PMC3824052  PMID: 23597004
CXCL14; airway epithelium; smoking; COPD; lung cancer
9.  Expression quantitative trait loci infer the regulation of isoflavone accumulation in soybean (Glycine max L. Merr.) seed 
BMC Genomics  2014;15(1):680.
Mapping expression quantitative trait loci (eQTL) of targeted genes represents a powerful and widely adopted approach to identify putative regulatory variants. Linking regulation differences to specific genes might assist in the identification of networks and interactions. The objective of this study is to identify eQTL underlying expression of four gene families encoding isoflavone synthetic enzymes involved in the phenylpropanoid pathway, which are phenylalanine ammonia-lyase (PAL; EC, chalcone synthase (CHS; EC, 2-hydroxyisoflavanone synthase (IFS; EC1.14.13.136) and flavanone 3-hydroxylase (F3H; EC A population of 130 recombinant inbred lines (F5:11), derived from a cross between soybean cultivar ‘Zhongdou 27’ (high isoflavone) and ‘Jiunong 20’ (low isoflavone), and a total of 194 simple sequence repeat (SSR) markers were used in this study. Overlapped loci of eQTLs and phenotypic QTLs (pQTLs) were analyzed to identify the potential candidate genes underlying the accumulation of isoflavone in soybean seed.
Thirty three eQTLs (thirteen cis-eQTLs and twenty trans-eQTLs) underlying the transcript abundance of the four gene families were identified on fifteen chromosomes. The eQTLs between Satt278-Sat_134, Sat_134-Sct_010 and Satt149-Sat_234 underlie the expression of both IFS and CHS genes. Five eQTL intervals were overlapped with pQTLs. A total of eleven candidate genes within the overlapped eQTL and pQTL were identified.
These results will be useful for the development of marker-assisted selection to breed soybean cultivars with high or low isoflavone contents and for map-based cloning of new isoflavone related genes.
PMCID: PMC4138391  PMID: 25124843
Soybean; eQTL; Isoflavone; pQTL; Candidate genes
10.  Increased Low-Frequency Oscillation Amplitude of Sensorimotor Cortex Associated with the Severity of Structural Impairment in Cervical Myelopathy 
PLoS ONE  2014;9(8):e104442.
Decreases in metabolites and increased motor-related, but decreased sensory-related activation of the sensorimotor cortex (SMC) have been observed in patients with cervical myelopathy (CM) using advanced MRI techniques. However, the nature of intrinsic neuronal activity in the SMC, and the relationship between cerebral function and structural damage of the spinal cord in patients with CM are not fully understood. The purpose of this study was to assess intrinsic neuronal activity by calculating the regional amplitude of low frequency fluctuations (ALFF) using resting-state functional MRI (rs-fMRI), and correlations with clinical and imaging indices. Nineteen patients and 19 age- and sex-matched healthy subjects underwent rs-fMRI scans. ALFF measurements were performed in the SMC, a key brain network likely to impaired or reorganized patients with CM. Compared with healthy subjects, increased amplitude of cortical low-frequency oscillations (LFO) was observed in the right precentral gyrus, right postcentral gyrus, and left supplementary motor area. Furthermore, increased z-ALFF values in the right precentral gyrus and right postcentral gyrus correlated with decreased fractional anisotropy values at the C2 level, which indicated increased intrinsic neuronal activity in the SMC corresponding to the structural impairment in the spinal cord of patients with CM. These findings suggest a complex and diverging relationship of cortical functional reorganization and distal spinal anatomical compression in patients with CM and, thus, add important information in understanding how spinal cord integrity may be a factor in the intrinsic covariance of spontaneous low-frequency fluctuations of BOLD signals involved in cortical plasticity.
PMCID: PMC4128667  PMID: 25111566
11.  Altered Inter-Subregion Connectivity of the Default Mode Network in Relapsing Remitting Multiple Sclerosis: A Functional and Structural Connectivity Study 
PLoS ONE  2014;9(7):e101198.
Background and Purpose
Little is known about the interactions between the default mode network (DMN) subregions in relapsing-remitting multiple sclerosis (RRMS). This study used diffusion tensor imaging (DTI) and resting-state functional MRI (rs-fMRI) to examine alterations of long white matter tracts in paired DMN subregions and their functional connectivity in RRMS patients.
Twenty-four RRMS patients and 24 healthy subjects participated in this study. The fiber connections derived from DTI tractography and the temporal correlation coefficient derived from rs-fMRI were combined to examine the inter-subregion structural-functional connectivity (SC-FC) within the DMN and its correlations with clinical markers.
Compared with healthy subjects, the RRMS patients showed the following: 1) significantly decreased SC and increased FC in the pair-wise subregions; 2) two significant correlations in SC-FC coupling patterns, including the positive correlation between slightly increased FC value and long white matter tract damage in the PCC/PCUN-MPFC connection, and the negative correlations between significantly increased FC values and long white matter tract damage in the PCC/PCUN-bilateral mTL connections; 3) SC alterations [log(N track) of the PCC/PCUN-left IPL, RD value of the MPFC-left IPL, FA value of the PCC/PCUN-left mTL connections] correlated with EDSS, increases in the RD value of MPFC-left IPL connection was positively correlated to the MFIS; and decreases in the FA value of PCC/PCUN-right IPL connection was negatively correlated with the PASAT; 4) decreased SC (FA value of the MPFC-left IPL, track volume of the PCC/PCUN-MPFC, and log(N track) of PCC/PCUN-left mTL connections) was positively correlated with brain atrophy.
In the connections of paired DMN subregions, we observed decreased SC and increased FC in RRMS patients. The relationship between MS-related structural abnormalities and clinical markers suggests that the disruption of this long-distance “inter-subregion” connectivity (white matter) may significantly impact the integrity of the network's function.
PMCID: PMC4085052  PMID: 24999807
12.  Ophthalmic Delivery of Brinzolamide by Liquid Crystalline Nanoparticles: In Vitro and In Vivo Evaluation 
AAPS PharmSciTech  2013;14(3):1063-1071.
Brinzolamide (BLZ) is a drug used to treat glaucoma; however, its use is restricted due to some unwanted adverse events. The goal of this study was to develop BLZ-loaded liquid crystalline nanoparticles (BLZ LCNPs) and to figure out the possibility of LCNPs as a new therapeutic system for glaucoma. BLZ LCNPs were produced by a modified emulsification method and their physicochemical aspects were estimated. In vitro release study revealed BLZ LCNPs displayed to some extent prolonged drug release behavior in contrast to that of BLZ commercial product (Azopt®). The ex vivo apparent permeability coefficient of BLZ LCNP systems demonstrated a 3.47-fold increase compared with that of Azopt®. The pharmacodynamics was checked over by calculating the percentage fall in intraocular pressure and the pharmacodynamic test showed that BLZ LCNPs had better therapeutic potential than Azopt®. Furthermore, the in vivo ophthalmic irritation was evaluated by Draize test. In conclusion, BLZ LCNPs would be a promising delivery system used for the treatment of glaucoma, with advantages such as lower doses but maintaining the effectiveness, better ocular bioavailability, and patient compliance compared with Azopt®.
PMCID: PMC3755158  PMID: 23813437
brinzolamide; liquid crystalline nanoparticles; ocular bioavailability; ocular irritation; ophthalmic delivery
13.  Genetic and physiological analysis of tolerance to acute iron toxicity in rice 
Rice  2014;7(1):8.
Fe toxicity occurs in lowland rice production due to excess ferrous iron (Fe2+) formation in reduced soils. To contribute to the breeding for tolerance to Fe toxicity in rice, we determined quantitative trait loci (QTL) by screening two different bi-parental mapping populations under iron pulse stresses (1,000 mg L−1 = 17.9 mM Fe2+ for 5 days) in hydroponic solution, followed by experiments with selected lines to determine whether QTLs were associated with iron exclusion (i.e. root based mechanisms), or iron inclusion (i.e. shoot-based mechanisms).
In an IR29/Pokkali F8 recombinant inbred population, 7 QTLs were detected for leaf bronzing score on chromosome 1, 2, 4, 7 and 12, respectively, individually explaining 9.2-18.7% of the phenotypic variation. Two tolerant recombinant inbred lines carrying putative QTLs were selected for further experiments. Based on Fe uptake into the shoot, the dominant tolerance mechanism of the tolerant line FL510 was determined to be exclusion with its root architecture being conducive to air transport and thus the ability to oxidize Fe2+ in rhizosphere. In line FL483, the iron tolerance was related mainly to shoot-based mechanisms (tolerant inclusion mechanism). In a Nipponbare/Kasalath/Nipponbare backcross inbred population, 3 QTLs were mapped on chromosomes 1, 3 and 8, respectively. These QTLs explained 11.6-18.6% of the total phenotypic variation. The effect of QTLs on chromosome 1 and 3 were confirmed by using chromosome segment substitution lines (SL), carrying Kasalath introgressions in the genetic background on Nipponbare. The Fe uptake in shoots of substitution lines suggests that the effect of the QTL on chromosome 1 was associated with shoot tolerance while the QTL on chromosome 3 was associated with iron exclusion.
Tolerance of certain genotypes were classified into shoot- and root- based mechanisms. Comparing our findings with previously reported QTLs for iron toxicity tolerance, we identified co-localization for some QTLs in both pluse and chronic stresses, especially on chromosome 1.
PMCID: PMC4052628  PMID: 24920973
Iron toxicity; Oryza sativa L.; Quantitative trait locus; Reactive oxygen species; Tolerance mechanism
14.  Late sodium current contributes to the reverse rate-dependent effect of IKr inhibition on ventricular repolarization 
Circulation  2011;123(16):1713-1720.
The reverse rate dependence (RRD) of actions of IKr-blocking drugs to increase action potential duration (APD) and beat-to-beat variability (BVR) of APD is proarrhythmic. Therefore we determined if inhibition of endogenous, physiological late Na+ current (late INa) attenuates the RRD and proarrhythmic effect of IKr inhibition.
Methods and Results
Duration of the monophasic action potential (MAPD) was measured from female rabbit hearts paced at cycle lengths from 400 to 2000 ms and BVR was calculated. In the absence of drug, MAPD90 and BVR increased as the cycle length was increased from 400 to 2000 ms (n=36 and 26, p<0.01). Both E-4031 (20 nmol/L) and d-sotalol (10 μmol/L) increased MAPD90 and BVR at all stimulation rates and the increase was greater at slower than at faster pacing rates (n=19 and 11, 12 and 7, respectively, p<0.01). TTX (1 μmol/L) significantly attenuated the RRD of MAPD90, reduced BVR, (p<0.01), and abolished torsade de pointes (TdP) in 5 of 6 hearts treated with either 20 nmol/L E-4031 or 10 μmol/L d-sotalol. Endogenous late INa in cardiomyocytes stimulated at cycle lengths from 500 to 4000 ms was greater at slower than at faster stimulation rates, and rapidly decreased during the first several beats at faster but not at slower rates (p<0.01, n=8). In a computational model, simulated RRD of APD caused by E-4031 and d-sotalol was attenuated when late INa was inhibited.
Endogenous late INa contributes to the RRD of IKr inhibitor-induced increases in APD and BVR and to bradycardia-related ventricular arrhythmias.
PMCID: PMC4028960  PMID: 21482963
Late sodium current; reverse rate dependence; action potential duration; beat-to-beat variability; rabbit heart
15.  Transgenic Mice Convert Carbohydrates to Essential Fatty Acids 
PLoS ONE  2014;9(5):e97637.
Transgenic mice (named “Omega mice”) were engineered to carry both optimized fat-1 and fat-2 genes from the roundworm Caenorhabditis elegans and are capable of producing essential omega-6 and omega-3 fatty acids from saturated fats or carbohydrates. When maintained on a high-saturated fat diet lacking essential fatty acids or a high-carbohydrate, no-fat diet, the Omega mice exhibit high tissue levels of both omega-6 and omega-3 fatty acids, with a ratio of ∼1∶1. This study thus presents an innovative technology for the production of both omega-6 and omega-3 essential fatty acids, as well as a new animal model for understanding the true impact of fat on human health.
PMCID: PMC4023978  PMID: 24836606
16.  Epidemiology, species distribution and outcome of nosocomial Candida spp. bloodstream infection in Shanghai 
BMC Infectious Diseases  2014;14:241.
Yeasts, mostly Candida, are important causes of bloodstream infections (BSI), responsible for significant mortality and morbidity among hospitalized patients. The epidemiology and species distribution vary from different regions. The goals of this study were to report the current epidemiology of Candida BSI in a Shanghai Teaching Hospital and estimate the impact of appropriate antifungal therapy on the outcome.
From January 2008 to December 2012, all consecutive patients who developed Candida BSI at Ruijin University Hospital were enrolled. Underlying diseases, clinical severity, species distribution, antifungal therapy and its impact on the outcome were analyzed.
A total of 121 episodes of Candida BSI were identified, with an incidence of 0.32 episodes/1,000 admissions (0.21 in 2008 and 0.42 in 2012) The proportion of candidemia caused by non-albicans species (62.8%), including C. parapsilosis (19.8%), C. tropicalis (14.9%), C. glabrata (7.4%), C. guilliermondii (5.8%), C. sake (5.0%) was higher than that of candidemia caused by C. albicans (37.2%). The overall crude 28-day mortality was 28.1% and significantly reduced with appropriate empiric antifungal therapy administered within 5 days (P = 0.006). Advanced age (OR 1.04; P = 0.014), neutropenia < 500/mm3 (OR 17.44; P < 0.001) were independent risk factors for 28-day mortality, while appropriate empiric antifungal therapy (OR 0.369; P = 0.035) was protective against 28-day mortality.
The epidemiology of candidemia in Shanghai differed from that observed in Western countries. Appropriate empiric antifungal therapy influenced the short-term survival.
PMCID: PMC4033490  PMID: 24886130
Candida spp; Bloodstream infection; Appropriate antifungal therapy; Survival
17.  The Retromer Complex Is Required for Rhodopsin Recycling and Its Loss Leads to Photoreceptor Degeneration 
PLoS Biology  2014;12(4):e1001847.
Rhodopsin recycling via the retromer, rather than degradation through lysosomes, can alleviate light-induced photoreceptor degeneration in Drosophila.
Rhodopsin mistrafficking can cause photoreceptor (PR) degeneration. Upon light exposure, activated rhodopsin 1 (Rh1) in Drosophila PRs is internalized via endocytosis and degraded in lysosomes. Whether internalized Rh1 can be recycled is unknown. Here, we show that the retromer complex is expressed in PRs where it is required for recycling endocytosed Rh1 upon light stimulation. In the absence of subunits of the retromer, Rh1 is processed in the endolysosomal pathway, leading to a dramatic increase in late endosomes, lysosomes, and light-dependent PR degeneration. Reducing Rh1 endocytosis or Rh1 levels in retromer mutants alleviates PR degeneration. In addition, increasing retromer abundance suppresses degenerative phenotypes of mutations that affect the endolysosomal system. Finally, expressing human Vps26 suppresses PR degeneration in Vps26 mutant PRs. We propose that the retromer plays a conserved role in recycling rhodopsins to maintain PR function and integrity.
Author Summary
Upon light exposure, rhodopsins—light-sensing proteins in the eye—trigger visual transduction signaling to activate fly photoreceptor cells. After activation, rhodopsins can be internalized from the cell surface into endosomes and then degraded in lysosomes. This mechanism prevents constant activation of the visual transduction pathway, thereby maintaining the function and integrity of photoreceptor cells. It is not known, however, whether these internalized rhodopsins can be recycled. Here, we show that the retromer, an evolutionarily conserved protein complex, is required for the recycling of rhodopsins. We find that loss of key retromer subunits (Vps35 or Vps26) causes rhodopsin mislocalization in the photoreceptors and severe light-induced photoreceptor degeneration. Conversely, gain of retromer subunits can alleviate photoreceptor degeneration in some contexts. Human retromer components can stand in for depleted fruit fly retromer, suggesting that this complex plays a role in recycling light sensors in both vertebrate and invertebrate photoreceptors.
PMCID: PMC4004542  PMID: 24781186
18.  Transittability of complex networks and its applications to regulatory biomolecular networks 
Scientific Reports  2014;4:4819.
We have often observed unexpected state transitions of complex systems. We are thus interested in how to steer a complex system from an unexpected state to a desired state. Here we introduce the concept of transittability of complex networks, and derive a new sufficient and necessary condition for state transittability which can be efficiently verified. We define the steering kernel as a minimal set of steering nodes to which control signals must directly be applied for transition between two specific states of a network, and propose a graph-theoretic algorithm to identify the steering kernel of a network for transition between two specific states. We applied our algorithm to 27 real complex networks, finding that sizes of steering kernels required for transittability are much less than those for complete controllability. Furthermore, applications to regulatory biomolecular networks not only validated our method but also identified the steering kernel for their phenotype transitions.
PMCID: PMC4001102  PMID: 24769565
19.  Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays 
BMC Cancer  2014;14:294.
Lung cancer patients with mutations in the epidermal growth factor receptor (EGFR) are primary candidates for EGFR-targeted therapy. Reliable analyses of such mutations have previously been possible only in tumour tissue. Here, we demonstrate that mutations can be detected in plasma samples with allele-specific PCR assays.
Pairs of the diagnostic biopsy and plasma obtained just prior to start of erlotinib treatment were collected from 199 patients with adenocarcinoma of non-small-cell lung cancer. DNA from both sample types was isolated and examined for the presence of mutations in exons 18–21 of the EGFR gene, employing the cobas® EGFR Tissue Test and cobas® EGFR Blood Test (in development, Roche Molecular Systems, Inc., CA, USA).
Test results were obtained in all 199 (100%) plasma samples and 196/199 (98%) of the biopsies. EGFR-activating mutations were identified in 24/199 (12%) plasma samples and 28/196 (14%) biopsy samples, and 17/196 (9%) matched pairs contained the same mutation. Six EGFR mutations were present only in plasma samples but not in the biopsy samples. The overall concordance of the EGFR gene mutations detected in plasma and biopsy tissue was 179/196 (91%) (kappa value: 0.621).
Mutational analysis of the EGFR gene in plasma samples is feasible with allele-specific PCR assays and represents a non-invasive supplement to biopsy analysis.
Trial registration
M-20080012 from March 10, 2008 and reported to NCT00815971.
PMCID: PMC4014134  PMID: 24773774
EGFR (Epidermal growth factor receptor); Plasma DNA; Erlotinib; Lung cancer
20.  Positive Lymph Node Metastasis Has a Marked Impact on the Long-Term Survival of Patients with Hepatocellular Carcinoma with Extrahepatic Metastasis 
PLoS ONE  2014;9(4):e95889.
The prognosis of hepatocellular carcinoma (HCC) patients with extrahepatic metastasis is extremely poor. However, what is the main risk factor for survival remains unclear for these patients. We aimed to find out the relative frequency, incidence and locations of extrahepatic metastases and the risk factors of long-term survival of the patients.
132 HCC patients with extrahepatic metastasis diagnosed by 18F-FDG PET/CT and conventional workup were enrolled into this study. The incidence and locations of extrahepatic metastases were summarized, and the related risk factors of overall survival were analyzed.
The most frequent extrahepatic metastatic sites were lymph nodes in 72 (54.5%), bone in 33 (25.0%) and lung in 28 (21.2%) patients. On univariate analysis, prothrombin time, Child-Pugh grade, portal/hepatic vein invasion and lymph node metastasis were independent risk factors of overall survival. On multivariate analysis, lymph node metastasis was the only independent risk factor of overall survival. The cumulative survival rates at 1- and 3-years after diagnosis of extrahepatic metastasis of HCC were 34.4% and 9.3%, respectively. The median survival time was 7 months (range 1 ∼38 months). The median survival time for patients with or without lymph node metastasis were 5 months (range 1∼38 months) and 12 months (range 1∼30 months), respectively (P = 0.036).
This study showed lymph nodes to be the most frequent site of extrahepatic metastases for primary HCC. Lymph node metastasis was the main risk factor of overall survival in patients with HCC with extrahepatic metastasis.
PMCID: PMC3997507  PMID: 24760012
21.  Opposing Effects of PI3K/Akt and Smad-Dependent Signaling Pathways in NAG-1-Induced Glioblastoma Cell Apoptosis 
PLoS ONE  2014;9(4):e96283.
Nonsteroidal anti-inflammatory drug (NSAID) activated gene-1 (NAG-1) is a divergent member of the transforming growth factor-beta (TGF-β) superfamily. NAG-1 plays remarkable multifunctional roles in controlling diverse physiological and pathological processes including cancer. Like other TGF-β family members, NAG-1 can play dual roles during cancer development and progression by negatively or positively modulating cancer cell behaviors. In glioblastoma brain tumors, NAG-1 appears to act as a tumor suppressor gene; however, the precise underlying mechanisms have not been well elucidated. In the present study, we discovered that overexpression of NAG-1 induced apoptosis in U87 MG, U118 MG, U251 MG, and T98G cell lines via the intrinsic mitochondrial pathway, but not in A172 and LN-229 cell lines. NAG-1 could induce the phosphorylation of PI3K/Akt and Smad2/3 in all six tested glioblastoma cell lines, except Smad3 phosphorylation in A172 and LN-229 cell lines. In fact, Smad3 expression and its phosphorylation were almost undetectable in A172 and LN-229 cells. The PI3K inhibitors promoted NAG-1-induced glioblastoma cell apoptosis, while siRNAs to Smad2 and Smad3 decreased the apoptosis rate. NAG-1 also stimulated the direct interaction between Akt and Smad3 in glioblastoma cells. Elevating the level of Smad3 restored the sensitivity to NAG-1-induced apoptosis in A172 and LN-229 cells. In conclusion, our results suggest that PI3K/Akt and Smad-dependent signaling pathways display opposing effects in NAG-1-induced glioblastoma cell apoptosis.
PMCID: PMC3997521  PMID: 24759784
22.  Different Relationships between Temporal Phylogenetic Turnover and Phylogenetic Similarity and in Two Forests Were Detected by a New Null Model 
PLoS ONE  2014;9(4):e95703.
Ecologists have been monitoring community dynamics with the purpose of understanding the rates and causes of community change. However, there is a lack of monitoring of community dynamics from the perspective of phylogeny.
Methods/Principle Findings
We attempted to understand temporal phylogenetic turnover in a 50 ha tropical forest (Barro Colorado Island, BCI) and a 20 ha subtropical forest (Dinghushan in southern China, DHS). To obtain temporal phylogenetic turnover under random conditions, two null models were used. The first shuffled names of species that are widely used in community phylogenetic analyses. The second simulated demographic processes with careful consideration on the variation in dispersal ability among species and the variations in mortality both among species and among size classes. With the two models, we tested the relationships between temporal phylogenetic turnover and phylogenetic similarity at different spatial scales in the two forests. Results were more consistent with previous findings using the second null model suggesting that the second null model is more appropriate for our purposes. With the second null model, a significantly positive relationship was detected between phylogenetic turnover and phylogenetic similarity in BCI at a 10 m×10 m scale, potentially indicating phylogenetic density dependence. This relationship in DHS was significantly negative at three of five spatial scales. This could indicate abiotic filtering processes for community assembly. Using variation partitioning, we found phylogenetic similarity contributed to variation in temporal phylogenetic turnover in the DHS plot but not in BCI plot.
The mechanisms for community assembly in BCI and DHS vary from phylogenetic perspective. Only the second null model detected this difference indicating the importance of choosing a proper null model.
PMCID: PMC3991709  PMID: 24748022
23.  Involvement of RbAp48 in erythroid differentiation of murine erythroleukemia cells induced by sodium butyrate 
Oncology Letters  2014;7(6):1785-1789.
Normal mammalian terminal erythroid differentiation is a precisely regulated process during which the progenitor cells execute particular programs to form a mature erythrocytic phenotype. In the present study, it was found that RbAp48, a histone-binding protein associated with retinoblastoma protein, was upregulated during terminal erythroid maturation in vivo and in vitro. This indicated that RbAp48, at least in part, participated in the regulation of murine erythropoiesis. Following sodium butyrate (SB) induction, murine erythroleukemia (MEL) cells began to re-enter erythroid differentiation and the ratio of differentiated cells reached ~80% at 72 h. The erythroid maturation-related mRNA expression of α-globin, β-globin and glycophorin A (GPA) was increased markedly, which indicated that SB induced MEL differentiation. During MEL differentiation, the RbAp48 level showed a 1.5-fold increase at 72 h, and the globin transcription factor (GATA)-1 level was also upregulated in the early stage of differentiation. By contrast, the c-Myc level was gradually downregulated in MEL differentiation. Using an immunofluorescence assay, the results of the study directly showed that the average fluorescence intensity of RbAp48 in each cell reached an almost 1.7-fold increase at 72 and 96 h. This was consistent with the western blot results of RbAp48 during MEL differentiation. In addition, reduced expression of RbAp48 by RNA inference decreased SB-induced MEL differentiation by ~20%, indicating that a high level of RbAp48 was essential for MEL differentiation. Taken together, these results established a functional link between RbAp48 and erythroid differentiation.
PMCID: PMC4049757  PMID: 24932233
RbAp48; murine erythroleukemia cell; sodium butyrate; erythroid differentiation
24.  New Centromere Autoantigens Identified in Systemic Sclerosis Using Centromere Protein Microarrays 
The Journal of rheumatology  2013;40(4):461-468.
To identify novel centromere protein (CENP) targets of anticentromere antibodies (ACA), and to investigate their association with clinical manifestations of systemic sclerosis (SSc).
A CENP-focused protein microarray was fabricated by spotting 14 purified CENP. These microarrays were individually incubated with 35 ACA-positive SSc sera and 20 ACA-negative healthy control samples. Newly identified CENP autoantigens with high sensitivities were selected for validation and characterization.
Statistical analysis revealed 11 CENP are potential target antigens of ACA in patients with SSc. Of them, 5 [CENP-P, CENP-Q, CENP-M (isoform I), CENP-J, and CENP-T] are novel, among which CENP-P and CENP-Q showed high sensitivities in ACA-positive SSc sera of 34.3% and 28.6%, respectively. Subsequently, 186 SSc sera (35 ACA-positives and 151 negatives), 69 ACA-positive sera from other various autoimmune diseases (primary Sjögren syndrome, systemic lupus erythematosus, rheumatoid arthritis, and primary biliary cirrhosis), and 31 healthy sera were assayed for the presence of anti-CENP-P and -Q autoantibodies by ELISA followed by Western blotting analysis. CENP-P and -Q autoantibodies were detected in ACA-positive sera of various disease groups; among them, SSc showed the highest detection rate. Anti-CENP-P was also found in 9 of the 151 ACA-negative sera. Analyses of the correlation with clinical information showed anti-CENP-P-positive patients had higher levels of IgG, IgA, and erythrocyte sedimentation rate among the ACA-positive cohort and were more vulnerable to renal disease in the ACA-negative patients with SSc. Regardless of ACA status, anti-CENP-P or Q-negative patients seem to be predominantly affected by interstitial lung disease.
CENP-P and CENP-Q were identified as novel ACA autoantigens by CENP microarray assays followed by validation of ELISA and Western blotting. Both of them have prognostic utility for interstitial lung disease. CENP-P was associated with renal disease in an ACA-negative cohort.
PMCID: PMC3962773  PMID: 23418382
25.  Clinical Validation of a PCR Assay for the Detection of EGFR Mutations in Non–Small-Cell Lung Cancer: Retrospective Testing of Specimens from the EURTAC Trial 
PLoS ONE  2014;9(2):e89518.
The EURTAC trial demonstrated that the tyrosine kinase inhibitor (TKI) erlotinib was superior to chemotherapy as first-line therapy for advanced non-small cell lung cancers (NSCLC) that harbor EGFR activating mutations in a predominantly Caucasian population. Based on EURTAC and several Asian trials, anti-EGFR TKIs are standard of care for EGFR mutation-positive NSCLC. We sought to validate a rapid multiplex EGFR mutation assay as a companion diagnostic assay to select patients for this therapy. Samples from the EURTAC trial were prospectively screened for EGFR mutations using a combination of laboratory-developed tests (LDTs), and tested retrospectively with the cobas EGFR mutation test (EGFR PCR test). The EGFR PCR test results were compared to the original LDT results and to Sanger sequencing, using a subset of specimens from patients screened for the trial. Residual tissue was available from 487 (47%) of the 1044 patients screened for the trial. The EGFR PCR test showed high concordance with LDT results with a 96.3% overall agreement. The clinical outcome of patients who were EGFR-mutation detected by the EGFR PCR test was very similar to the entire EURTAC cohort. The concordance between the EGFR PCR test and Sanger sequencing was 90.6%. In 78.9% of the discordant samples, the EGFR PCR test result was confirmed by a sensitive deep sequencing assay. This retrospective study demonstrates the clinical utility of the EGFR PCR test in the accurate selection of patients for anti-EGFR TKI therapy. The EGFR PCR test demonstrated improved performance relative to Sanger sequencing.
PMCID: PMC3934888  PMID: 24586842

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