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1.  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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1371/journal.pone.0101198
PMCID: PMC4085052  PMID: 24999807
2.  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®.
doi:10.1208/s12249-013-9997-2
PMCID: PMC3755158  PMID: 23813437
brinzolamide; liquid crystalline nanoparticles; ocular bioavailability; ocular irritation; ophthalmic delivery
3.  Genetic and physiological analysis of tolerance to acute iron toxicity in rice 
Rice  2014;7(1):8.
Background
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).
Results
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.
Conclusion
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.
doi:10.1186/s12284-014-0008-3
PMCID: PMC4052628  PMID: 24920973
Iron toxicity; Oryza sativa L.; Quantitative trait locus; Reactive oxygen species; Tolerance mechanism
4.  Late sodium current contributes to the reverse rate-dependent effect of IKr inhibition on ventricular repolarization 
Circulation  2011;123(16):1713-1720.
Background
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.
Conclusions
Endogenous late INa contributes to the RRD of IKr inhibitor-induced increases in APD and BVR and to bradycardia-related ventricular arrhythmias.
doi:10.1161/CIRCULATIONAHA.110.000661
PMCID: PMC4028960  PMID: 21482963
Late sodium current; reverse rate dependence; action potential duration; beat-to-beat variability; rabbit heart
5.  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.
doi:10.1371/journal.pone.0097637
PMCID: PMC4023978  PMID: 24836606
6.  Epidemiology, species distribution and outcome of nosocomial Candida spp. bloodstream infection in Shanghai 
BMC Infectious Diseases  2014;14:241.
Background
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.
Methods
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.
Results
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.
Conclusion
The epidemiology of candidemia in Shanghai differed from that observed in Western countries. Appropriate empiric antifungal therapy influenced the short-term survival.
doi:10.1186/1471-2334-14-241
PMCID: PMC4033490  PMID: 24886130
Candida spp; Bloodstream infection; Appropriate antifungal therapy; Survival
7.  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.
doi:10.1371/journal.pbio.1001847
PMCID: PMC4004542  PMID: 24781186
8.  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.
doi:10.1038/srep04819
PMCID: PMC4001102  PMID: 24769565
9.  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.
Background
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.
Methods
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).
Results
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).
Conclusion
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 ClinicalTrials.gov: NCT00815971.
doi:10.1186/1471-2407-14-294
PMCID: PMC4014134  PMID: 24773774
EGFR (Epidermal growth factor receptor); Plasma DNA; Erlotinib; Lung cancer
10.  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.
Background
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1371/journal.pone.0095889
PMCID: PMC3997507  PMID: 24760012
11.  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.
doi:10.1371/journal.pone.0096283
PMCID: PMC3997521  PMID: 24759784
12.  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.
Background
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.
Conclusions/Significance
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.
doi:10.1371/journal.pone.0095703
PMCID: PMC3991709  PMID: 24748022
13.  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.
doi:10.3892/ol.2014.2015
PMCID: PMC4049757  PMID: 24932233
RbAp48; murine erythroleukemia cell; sodium butyrate; erythroid differentiation
14.  New Centromere Autoantigens Identified in Systemic Sclerosis Using Centromere Protein Microarrays 
The Journal of rheumatology  2013;40(4):461-468.
Objective
To identify novel centromere protein (CENP) targets of anticentromere antibodies (ACA), and to investigate their association with clinical manifestations of systemic sclerosis (SSc).
Methods
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.
Results
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.
Conclusion
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.
doi:10.3899/jrheum.120264
PMCID: PMC3962773  PMID: 23418382
ANTICENTROMERE ANTIBODY; PROTEIN MICROARRAY; SYSTEMIC SCLEROSIS; BIOMARKERS
15.  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.
doi:10.1371/journal.pone.0089518
PMCID: PMC3934888  PMID: 24586842
16.  Drosophila Tempura, a Novel Protein Prenyltransferase α Subunit, Regulates Notch Signaling Via Rab1 and Rab11 
PLoS Biology  2014;12(1):e1001777.
A forward genetic screen in Drosophila looking for Notch signaling regulators identifies Tempura, a new and non-redundant protein prenyltransferase of Rab proteins.
Vesicular trafficking plays a key role in tuning the activity of Notch signaling. Here, we describe a novel and conserved Rab geranylgeranyltransferase (RabGGT)-α–like subunit that is required for Notch signaling-mediated lateral inhibition and cell fate determination of external sensory organs. This protein is encoded by tempura, and its loss affects the secretion of Scabrous and Delta, two proteins required for proper Notch signaling. We show that Tempura forms a heretofore uncharacterized RabGGT complex that geranylgeranylates Rab1 and Rab11. This geranylgeranylation is required for their proper subcellular localization. A partial dysfunction of Rab1 affects Scabrous and Delta in the secretory pathway. In addition, a partial loss Rab11 affects trafficking of Delta. In summary, Tempura functions as a new geranylgeranyltransferase that regulates the subcellular localization of Rab1 and Rab11, which in turn regulate trafficking of Scabrous and Delta, thereby affecting Notch signaling.
Author Summary
Notch signaling is an evolutionarily conserved signaling pathway that regulates many developmental processes. Abnormal Notch signaling activity can lead to numerous diseases and developmental defects. To better understand the regulation of this pathway, we performed a forward genetic screen for Notch signaling components that have not been previously identified in Drosophila. Here, we report the identification of an evolutionarily conserved protein, Tempura, which is required for Notch signaling-mediated lateral inhibition and cell fate determination of external sensory organs. We show that loss of tempura leads to mistrafficking of Delta and Scabrous, two important Notch signaling components. In addition, Rab1 and Rab11, two major coordinators of vesicular trafficking, are mislocalizaed in tempura mutants. We further show that Tempura functions as a subunit of a previously uncharacterized lipid modification complex to geranylgeranylate (a type of prenylation) Rab1 and Rab11. This post-translational modification is shown to be required for the proper subcellular localization and function of these Rabs. We find that dysfunction of Rab1 causes an accumulation of Delta and Scabrous in the secretory pathway and dysfunction of Rab11 further interferes with the trafficking of Delta. In addition to the known Rab geranylgeranyltransferse, our data indicate the presence of another functionally nonredundant Rab geranylgeranyltransferse, Tempura.
doi:10.1371/journal.pbio.1001777
PMCID: PMC3904817  PMID: 24492843
17.  MicroRNA-21 expression is associated with overall survival in patients with glioma 
Diagnostic Pathology  2013;8:200.
Background
MicroRNA-21 has been proved to be associated with glioma proliferation and invasion; thus, we sought to clarify the clinical value of miR-21 expression in glioma tissues with WHO grade I to IV.
Methods
One hundred and fifty-two pairs of human gliomas and non-neoplastic brain tissues were evaluated using real-time PCR. The association of miR-21 expression with clinicopathological factors or the prognosis of glioma patients was also analyzed. In this study, survival analysis was performed using the Kaplan-Meier method and Cox’s proportional hazards model.
Results
MiR-21 was more greatly expressed in glioma tissues compared to the corresponding non-neoplastic brain tissues (P < 0.001). This observed high miR-21 expression was significantly associated with high pathological grades and the Karnofsky performance score of glioma patients. In addition, overall patient survival for those with low miR-21 expression was significantly longer than those patients with high miR-21 expression (P < 0.001). Moreover, multivariate Cox regression analysis indicated that miR-21 might be an independent prognostic marker for glioma patient survival.
Conclusions
Our data show that miR-21 may be a candidate independent marker for gliomas, especially those with high pathological grades, and this could also be a potential therapeutic target for molecular glioma therapy.
Virtual slide
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1445749171109834.
doi:10.1186/1746-1596-8-200
PMCID: PMC3933412  PMID: 24326156
Glioma; MiR-21; Overall survival
18.  Exoprotein Production Correlates with Morphotype Changes of Nonmotile Shewanella oneidensis Mutants 
Journal of Bacteriology  2013;195(7):1463-1474.
We report a previously undescribed mechanism for the rugose morphotype in Shewanella oneidensis, a research model for investigating redox transformations of environmental contaminants. Bacteria may form smooth or rugose colonies on agar plates. In general, conversion from the smooth to rugose colony morphotype is attributed to increased production of exopolysaccharide (EPS). In this work, we discovered that aflagellate S. oneidensis mutants grew into rugose colonies, whereas those with nonfunctional flagellar filaments remained smooth. EPS production was not altered in either case, but mutants with the rugose morphotype showed significantly reduced exoprotein secretion. The idea that exoproteins at a reduced level correlate with rugosity gained support from smooth suppressor strains of an aflagellate rugose fliD (encoding the capping protein) mutant, which restored the exoprotein level to the levels of the wild-type and mutant strains with a smooth morphotype. Further analyses revealed that SO1072 (a putative GlcNAc-binding protein) was one of the highly upregulated exoproteins in these suppressor strains. Most intriguingly, this study identified a compensatory mechanism of SO1072 to flagellins possibly mediated by bis-(3′-5′)-cyclic dimeric GMP.
doi:10.1128/JB.02187-12
PMCID: PMC3624527  PMID: 23335418
20.  The C8ORF38 homologue Sicily is a cytosolic chaperone for a mitochondrial complex I subunit 
The Journal of Cell Biology  2013;200(6):807-820.
Sicily, which was identified in a screen for proteins involved in neurodegeneration, interacts with cytosolic Hsp90 to chaperone the complex I subunit ND42, before its mitochondrial import.
Mitochondrial complex I (CI) is an essential component in energy production through oxidative phosphorylation. Most CI subunits are encoded by nuclear genes, translated in the cytoplasm, and imported into mitochondria. Upon entry, they are embedded into the mitochondrial inner membrane. How these membrane-associated proteins cope with the hydrophilic cytoplasmic environment before import is unknown. In a forward genetic screen to identify genes that cause neurodegeneration, we identified sicily, the Drosophila melanogaster homologue of human C8ORF38, the loss of which causes Leigh syndrome. We show that in the cytoplasm, Sicily preprotein interacts with cytosolic Hsp90 to chaperone the CI subunit, ND42, before mitochondrial import. Loss of Sicily leads to loss of CI proteins and preproteins in both mitochondria and cytoplasm, respectively, and causes a CI deficiency and neurodegeneration. Our data indicate that cytosolic chaperones are required for the subcellular transport of ND42.
doi:10.1083/jcb.201208033
PMCID: PMC3601355  PMID: 23509070
21.  Surveillance of the prevalence, antibiotic susceptibility, and genotypic characterization of invasive candidiasis in a teaching hospital in China between 2006 to 2011 
BMC Infectious Diseases  2013;13:353.
Background
Invasive candidiasis is an important nosocomial infection associated with high mortality among immunosuppressive or critically ill patients. We described the incidence of invasive candidiasis in our hospital over 6 years and showed the antifungal susceptibility and genotypes of the isolated yeast.
Method
The yeast species were isolated on CHROMagar Candida medium and identified using an yeast identification card, followed by analysis of the D1/D2 domain of 26S rDNA. The susceptibilities of the isolates to flucytosine, amphotericin B, fluconazole, itraconazole, and voriconazole were tested using the ATB FUNGUS 3 system, and that to caspofungin was tested using E-test strips. C. albicans was genotyped using single-strand conformation polymorphism of CAI (Candida albicans I) microsatellite DNA combined with GeneScan data.
Results
From January 2006 to December 2011, a total of 259 isolates of invasive Candida spp. were obtained from 253 patients, among them 6 patients had multiple positive samples. Ninety-one stains were from blood and 168 from sterile fluids, accounting for 6.07% of all pathogens isolated in our hospital. Most of these strains were C. albicans (41.29% in blood/59.06% in sterile body fluids), followed by C. tropicalis (18.06%/25.72%), C. parapsilosis (17.42%/5.43%), C. glabrata (11.61%/3.99%) and other Candida spp. (11.61%/5.80%). Most Candida spp. were isolated from the ICU. The new species-specific CLSI candida MIC breakpoints were applied to these date. Resistance to fluconazole occurred in 6.6% of C. albicans isolates, 10.6% of C. tropicalis isolates and 15.0% of C. glabrata isolates. For the 136 C. albicans isolates, 54 CAI patterns were recognized. The C. albicans strains from blood or sterile body fluids showed no predominant CAI genotypes. C. albicans isolates from different samples from the same patient had the same genotype.
Conclusions
Invasive candidiasis has been commonly encountered in our hospital in the past 6 years, with increasing frequency of non-C. albicans. Resistance to fluconazole was highly predictive of resistance to voriconazole. CAI SSCP genotyping showed that all C. albicans strains were polymorphic. Invasive candidiasis were commonly endogenous infection.
doi:10.1186/1471-2334-13-353
PMCID: PMC3733982  PMID: 23899244
Invasive Candida; Epidemiology; Antifungal susceptibility; Genotyping
22.  Association between Polymorphisms of Complement Pathway Genes and Age-Related Macular Degeneration in a Chinese Population 
Purpose.
We assessed the association between complement pathway genes and age-related macular degeneration (AMD) in a Chinese population.
Methods.
In a case-control study, 165 AMD patients and 216 unrelated controls were recruited from two hospitals in central China. We selected and genotyped six single nucleotide polymorphisms (SNPs) of four complement pathway genes, including rs800292 and rs1410996 of complement H (CFH), rs9332739 of complement 2 (C2), rs4151667 of complement factor B (CFB), and rs2241394 and rs2230199 of complement 3 (C3). The associations between SNPs and AMD, adjusted by age and sex, were assessed by using logistic regression models and haplotype association analysis.
Results.
In our study, two SNPs of CFH and their haplotypes were associated significantly with AMD, and the adjusted odd ratios (ORs) were 2.45 (95% confidence interval [CI] 1.25–4.79) for rs800292 (genotype GG versus AA), 2.49 (95% CI 1.24–5.00) for rs1410996 (genotype TT versus CC), and 4.45 (95% CI 2.32–8.55) for haplotype block of rs800292–rs1410996 (haplotype G–C versus A–C), respectively. The haplotype of C2/CFB also was associated significantly with AMD, and the adjusted OR was 8.86 (95% CI 1.88–41.69) for the haplotype block of rs9332739–rs4151667 (haplotype G–A versus G–T), though no relationship was found in genotype association analysis of the two SNPs of C2/CFB. With the sample size of our study, no relationship was found for AMD and the two SNPs of C3.
Conclusions.
Gene variants in CFH and C2/CFB contribute to AMD in the Chinese population.
The association between age-related macular degeneration (AMD) and six single-nucleotide polymorphisms (SNPs) at 4 complement pathway genes, including rs800292 (CFH), rs1410996 (CFH), rs4151667 (CFB), rs9332739 (C2), rs2241394 (C3), and rs2230199 (C3), were studied in a Chinese population.
doi:10.1167/iovs.12-10453
PMCID: PMC3544419  PMID: 23233260
23.  A Mutation in EGF Repeat-8 of Notch Discriminates Between Serrate/Jagged and Delta Family Ligands 
Science (New York, N.Y.)  2012;338(6111):1229-1232.
Notch signaling affects many developmental and cellular processes and has been implicated in congenital disorders, stroke, and numerous cancers. The Notch receptor binds its ligands Delta and Serrate and is able to discriminate between them in different contexts. However, the specific domains in Notch responsible for this selectivity are poorly defined. Through genetic screens in Drosophila, we isolated a mutation, Notchjigsaw, that affects Serrate- but not Delta-dependent signaling. Notchjigsaw carries a missense mutation in epidermal growth factor repeat-8 (EGFr-8) and is defective in Serrate binding. A homologous point mutation in mammalian Notch2 also exhibits defects in signaling of a mammalian Serrate homolog, Jagged1. Hence, an evolutionarily conserved valine in EGFr-8 is essential for ligand selectivity and provides a molecular handle to study numerous Notch-dependent signaling events.
doi:10.1126/science.1228745
PMCID: PMC3663443  PMID: 23197537
24.  Decrease of 5-Hydroxymethylcytosine Is Associated with Progression of Hepatocellular Carcinoma through Downregulation of TET1 
PLoS ONE  2013;8(5):e62828.
DNA methylation is an important epigenetic modification and is frequently altered in cancer. Convert of 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5 hmC) by ten-eleven translocation (TET) family enzymes plays important biological functions in embryonic stem cells, development, aging and disease. Recent reports showed that level of 5 hmC was altered in various types of cancers. However, the change of 5 hmC level in hepatocellular carcinoma (HCC) and association with clinical outcome were not well defined. Here, we reported that level of 5 hmC was decreased in HCC tissues, as compared with non-tumor tissues. Clincopathological analysis showed the decreased level of 5 hmC in HCC was associated with tumor size, AFP level and poor overall survival. We also found that the decreased level of 5 hmC in non-tumor tissues was associated with tumor recurrence in the first year after surgical resection. In an animal model with carcinogen DEN-induced HCC, we found that the level of 5 hmC was gradually decreased in the livers during the period of induction. There was further reduction of 5 hmC in tumor tissues when tumors were developed. In contrast, level of 5 mC was increased in HCC tissues and the increased 5 mC level was associated with capsular invasion, vascular thrombosis, tumor recurrence and overall survival. Furthermore, our data showed that expression of TET1, but not TET2 and TET3, was downregulated in HCC. Taken together, our data indicated 5 hmC may be served as a prognostic marker for HCC and the decreased expression of TET1 is likely one of the mechanisms underlying 5 hmC loss in HCC.
doi:10.1371/journal.pone.0062828
PMCID: PMC3650038  PMID: 23671639
25.  Analytic performance studies and clinical reproducibility of a real-time PCR assay for the detection of epidermal growth factor receptor gene mutations in formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer 
BMC Cancer  2013;13:210.
Background
Epidermal growth factor receptor (EGFR) gene mutations identify patients with non-small cell lung cancer (NSCLC) who have a high likelihood of benefiting from treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be technically challenging, resulting in longer turn-around-time, with limited sensitivity for low levels of mutations. This manuscript details the technical performance verification studies and external clinical reproducibility studies of the cobas EGFR Mutation Test, a rapid multiplex real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21.
Methods
The assay’s limit of detection was determined using 25 formalin-fixed paraffin-embedded tissue (FFPET)-derived and plasmid DNA blends. Assay performance for a panel of 201 specimens was compared against Sanger sequencing with resolution of discordant specimens by quantitative massively parallel pyrosequencing (MPP). Internal and external reproducibility was assessed using specimens tested in duplicate by different operators, using different reagent lots, instruments and at different sites. The effects on the performance of the cobas EGFR test of endogenous substances and nine therapeutic drugs were evaluated in ten FFPET specimens. Other tests included an evaluation of the effects of necrosis, micro-organisms and homologous DNA sequences on assay performance, and the inclusivity of the assay for less frequent mutations.
Results
A >95% hit rate was obtained in blends with >5% mutant alleles, as determined by MPP analysis, at a total DNA input of 150 ng. The overall percent agreement between Sanger sequencing and the cobas test was 96.7% (negative percent agreement 97.5%; positive percent agreement 95.8%). Assay repeatability was 98% when tested with two operators, instruments, and reagent lots. In the external reproducibility study, the agreement was > 99% across all sites, all operators and all reagent lots for 11/12 tumors tested. Test performance was not compromised by endogenous substances, therapeutic drugs, necrosis up to 85%, and common micro-organisms. All of the assessed less common mutations except one (exon 19 deletion mutation 2236_2248 > AGAC) were detected at a similar DNA input level as that for the corresponding predominant mutation.
Conclusion
The cobas EGFR Mutation Test is a sensitive, accurate, rapid, and reproducible assay.
doi:10.1186/1471-2407-13-210
PMCID: PMC3660201  PMID: 23621958
EGFR mutation testing; Molecular diagnostics; Companion diagnostics; Non-small cell lung cancer; Analytical validation; Reproducibility

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