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1.  High Sensitive Sensor Fabricated by Reduced Graphene Oxide/Polyvinyl Butyral Nanofibers for Detecting Cu (II) in Water 
Graphene oxide (GO)/polyvinyl butyral (PVB) nanofibers were prepared by a simple electrospinning technique with PVB as matrix and GO as a functional nanomaterial. GO/PVB nanofibers on glassy carbon electrode (GCE) were reduced through electrochemical method to form reduced graphene oxide (RGO)/PVB nanofibers. The morphology and structure of GO/PVB nanofiber were studied by scanning election microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared (FTIR). RGO/PVB modified GCE was used for fabricating an electrochemical sensor for detecting Cu (II) in water. The analysis results showed that RGO/PVB modified GCE had good analytical results with the linear range of 0.06–2.2 μM, detection limit of 4.10 nM (S/N = 3), and the sensitivity of 103.51 μA·μM−1·cm−2.
doi:10.1155/2015/723276
PMCID: PMC4324952
2.  Computerized Detection of Lung Nodules by Means of “Virtual Dual-Energy” Radiography 
Major challenges in current computer-aided detection (CADe) schemes for nodule detection in chest radiographs (CXRs) are to detect nodules that overlap with ribs and/or clavicles and to reduce the frequent false positives (FPs) caused by ribs. Detection of such nodules by a CADe scheme is very important, because radiologists are likely to miss such subtle nodules. Our purpose in this study was to develop a CADe scheme with improved sensitivity and specificity by use of “virtual dual-energy” (VDE) CXRs where ribs and clavicles are suppressed with massive-training artificial neural networks (MTANNs). To reduce rib-induced FPs and detect nodules overlapping with ribs, we incorporated the VDE technology in our CADe scheme. The VDE technology suppressed rib and clavicle opacities in CXRs while maintaining soft-tissue opacity by use of the MTANN technique that had been trained with real dual-energy imaging. Our scheme detected nodule candidates on VDE images by use of a morphologic filtering technique. Sixty morphologic and gray-level-based features were extracted from each candidate from both original and VDE CXRs. A nonlinear support vector classifier was employed for classification of the nodule candidates. A publicly available database containing 140 nodules in 140 CXRs and 93 normal CXRs was used for testing our CADe scheme. All nodules were confirmed by computed tomography examinations, and the average size of the nodules was 17.8 mm. Thirty percent (42/140) of the nodules were rated “extremely subtle” or “very subtle” by a radiologist. The original scheme without VDE technology achieved a sensitivity of 78.6% (110/140) with 5 (1165/233) FPs per image. By use of the VDE technology, more nodules overlapping with ribs or clavicles were detected and the sensitivity was improved substantially to 85.0% (119/140) at the same FP rate in a leave-one-out cross-validation test, whereas the FP rate was reduced to 2.5 (583/233) per image at the same sensitivity level as the original CADe scheme obtained (Difference between the specificities of the original and the VDE-based CADe schemes was statistically significant). In particular, the sensitivity of our VDE-based CADe scheme for subtle nodules (66.7% = 28/42) was statistically significantly higher than that of the original CADe scheme (57.1% = 24/42). Therefore, by use of VDE technology, the sensitivity and specificity of our CADe scheme for detection of nodules, especially subtle nodules, in CXRs were improved substantially.
doi:10.1109/TBME.2012.2226583
PMCID: PMC4283823  PMID: 23193306
Chest radiography (CXR); computer-aided diagnosis (CAD); lung cancer; rib suppression; virtual dual energy (VDE)
3.  Crystallization and preliminary X-ray diffraction analysis of the Nif3-family protein MJ0927 from Methanocaldococcus jannaschii  
The hypothetical protein MJ0927 of the Nif3 family has been crystallized and X-ray diffraction data have been collected to a resolution of 2.47 Å.
MJ0927 is a member of the Nif3 family and is widely distributed across living organisms. Although several crystal structures of Nif3 proteins have been reported, structural information on archaeal Nif3 is still limited. To understand the structural differences between bacterial and archaeal Nif3 proteins, MJ0927 from Methanocaldococcus jannaschii was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.47 Å and belonged to the orthorhombic space group C222, with unit-cell parameters a = 81.21, b = 172.94, c = 147.42 Å. Determination of this structure may provide insights into the function of MJ0927.
doi:10.1107/S1744309112049408
PMCID: PMC3539711  PMID: 23295494
Nif3; Methanocaldococcus jannaschii; metal-binding proteins
4.  Emergence of Clinical Salmonella enterica Serovar Typhimurium Isolates with Concurrent Resistance to Ciprofloxacin, Ceftriaxone, and Azithromycin 
Salmonella infection is an important public health issue for which the needs of antimicrobial treatment are increasing. A total of 546 human clinical S. enterica serovar Typhimurium isolates were recovered from patients in hospitals in China during the period of 2005 to ∼2011. Twenty percent of the isolates exhibited resistance to ciprofloxacin, and 4% were resistant to ceftriaxone. Importantly, for the first time, 12 (2%) S. Typhimurium isolates resistant to both ciprofloxacin and ceftriaxone were recovered; among these 12 isolates, two were also resistant to azithromycin, and one was resistant to all other drugs tested. The combined effects of various transferrable extended-spectrum β-lactamase determinants and a novel efflux-based ciprofloxacin resistance mechanism encoded by the mobile efflux gene oqxAB were responsible for the emergence of these extremely (highly) drug-resistant (XDR) S. Typhimurium isolates. The dissemination of resistance genes, such as those encoding ESBLs and the OqxAB pump, among Salmonella organisms will speed up the selection of XDR Salmonella, posing a huge threat to public health and Salmonella infection control.
doi:10.1128/AAC.02770-13
PMCID: PMC4068579  PMID: 24752251
5.  Clinical trial with traditional Chinese medicine intervention ''tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment'' for chronic hepatitis B-associated liver failure 
World Journal of Gastroenterology : WJG  2014;20(48):18458-18465.
AIM: To study the clinical efficacy of traditional Chinese medicine (TCM) intervention “tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” (“TTK”) for treating liver failure due to chronic hepatitis B.
METHODS: We designed the study as a randomized controlled clinical trial. Registration number of Chinese Clinical Trial Registry is ChiCTR-TRC-12002961. A total of 144 patients with liver failure due to infection with chronic hepatitis B virus were enrolled in this randomized controlled clinical study. Participants were randomly assigned to the following three groups: (1) a modern medicine control group (MMC group, 36 patients); (2) a “tonifying qi and detoxification” (“TQD”) group (72 patients); and (3) a “tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” (“TTK”) group (36 patients). Patients in the MMC group received general internal medicine treatment; patients in the “TQD” group were given a TCM formula “tonifying qi and detoxification” and general internal medicine treatment; patients in the “TTK” group were given a TCM formula of “TTK” and general internal medicine treatment. All participants were treated for 8 wk and then followed at 48 wk following their final treatment. The primary efficacy end point was the patient fatality rate in each group. Measurements of various virological and biochemical indicators served as secondary endpoints. The one-way analysis of variance and the t-test were used to compare patient outcomes in the different treatment groups.
RESULTS: At the 48-wk post-treatment time point, the patient fatality rates in the MMC, “TQD”, and “TTK” groups were 51.61%, 35.38%, and 16.67%, respectively, and the differences between groups were statistically significant (P < 0.05). However, there were no significant differences in the levels of hepatitis B virus DNA or prothrombin activity among the three groups (P > 0.05). Patients in the “TTK” group had significantly higher levels of serum total bilirubin compared to MMC subjects (339.40 μmol/L ± 270.09 μmol/L vs 176.13 μmol/L ± 185.70 μmol/L, P = 0.014). Serum albumin levels were significantly increased in both the “TQD” group and “TTK” group as compared with the MMC group (31.30 g/L ± 4.77 g/L, 30.72 g/L ± 2.89 g/L vs 28.57 g/L ± 4.56 g/L, P < 0.05). There were no significant differences in levels of alanine transaminase among the three groups (P > 0.05). Safety data showed that there was one case of stomachache in the “TQD” group and one case of gastrointestinal side effect in the “TTK” group.
CONCLUSION: Treatment with “TTK” improved the survival rates of patients with liver failure due to chronic hepatitis B. Additionally, liver tissue was regenerated and liver function was restored.
doi:10.3748/wjg.v20.i48.18458
PMCID: PMC4277987  PMID: 25561817
Clinical study; “Tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” (“TTK”); Liver regeneration; Treatment with integrated traditional and Western medicine; Chronic hepatitis B-associated liver failure
6.  The efficacy and safety of cilostazol for the secondary prevention of ischemic stroke in acute and chronic phases in Asian population- an updated meta-analysis 
BMC Neurology  2014;14(1):251.
Backgrounds
While previous meta-analysis have investigated the efficacy of cilostazol in the secondary prevention of ischemic stroke, they were criticized for their methodology, which confused the acute and chronic phases of stroke. We present a new systematic review, which differs from previous meta-analysis by distinguishing between the different phases of stroke, and includes two new randomized, controlled trials (RCTs).
Methods
All RCTs investigating the effect of cilostazol on secondary prevention of ischemic stroke were obtained. Outcomes were analyzed by Review Manager, including recurrence of cerebral infarction (ROCI), hemorrhage stroke or subarachnoid hemorrhage (HSSH), all-cause death (ACD), and modified Rankin Scale score (mRS). The Grading of Recommendations Assessment, Development and Evaluation (GRADE) assessed the quality of the evidence.
Results
5491 patients from six studies were included in the current study. In secondary prevention of ischemic stroke in chronic phase, cilostazol was associated with a 47% reduction in ROCI (relative risk [RR] 0.53, 95% confidence interval [CI] 0.34 to 0.81, p = 0.003), while no significant difference in HSSH and ACD compared with placebo; and 71% reduction in HSSH (RR 0.29, 95% CI 0.15 to 0.56, p = 0.0002) compared with aspirin, but not in ROCI and ACD. In the secondary prevention of ischemic stroke in acute phase, cilostazol did not show any effect in the ROCI, HSSH, ACD and mRS compared to placebo or aspirin. The quality of the evidence from chronic phase was high or moderate, and those from acute phase were moderate or low when analyzed by GRADE approach.
Conclusion
Cilostazol provided a protective effect in the secondary prevention of the chronic phase of ischemic stroke.
doi:10.1186/s12883-014-0251-7
PMCID: PMC4301843  PMID: 25527141
Acute Phase; Chronic phase; Cilostazol; Meta-analysis; Stroke
7.  Susceptibility of Human Embryonic Stem Cell-Derived Neural Cells to Japanese Encephalitis Virus Infection 
PLoS ONE  2014;9(12):e114990.
Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells, including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses, such as Japanese encephalitis virus (JEV), provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition, glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast, only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition, functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover, we discover that vimentin intermediate filament, reported as a putative neurovirulent JEV receptor, is highly expressed in NPCs and glial cells, but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally, we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection.
doi:10.1371/journal.pone.0114990
PMCID: PMC4269419  PMID: 25517725
8.  Fever of unknown origin: report of 107 cases in a university hospital 
Fever of unknown origin (FUO) is a challenging problem in clinical practice. Evaluation of patient’s characteristics may illustrate the etiologies of FUO. In present study, 107 patients with FUO hospitalized in our inpatient department between 2010 and 2011 were investigated. The median age of the patients was 48 years (15-94). The median fever duration was 8.5 weeks (3-104). The median hospital stay was 8.5 days (1-51). Etiologies of FUO were identified as follows: infectious diseases 32 (29.9%), malignancies 19 (17.8%), inflammatory rheumatic diseases 18 (16.8%), and miscellaneous diseases 15 (14.0%). In 23 (21.5%) patients, the diagnosis remained unclear. Infection group had relative shorter average fever duration and hospital stay than other groups. Shortened mean fever duration was observed in geriatric age group. In conclusion, as the most common cause of FUO in the present study, infectious cases had relative shorter average fever duration and hospital stay, and geriatric patients had shortened average fever duration as well.
PMCID: PMC4307568  PMID: 25664121
Fever of unknown origin; etiology; infection
9.  Prevalence of wearing-off and dyskinesia among the patients with Parkinson’s disease on levodopa therapy: a multi-center registry survey in mainland China 
Objective
Chronic levodopa (L-dopa) treatment in Parkinson’s disease (PD) is often associated with the development of motor complications, but the corresponding epidemiological data is rare in Chinese PD patients. The present survey was to investigate the prevalence rate of wearing-off (WO) and dyskinesia among the patients with PD in China.
Methods
From May 2012 to October 2012, a 3-step registry survey for wearing off (WO) and dyskinesia patients with PD receiving levodopa therapy was performed simultaneously at 28 movement disorders clinics in China.
Results
There were 1,558 PD patients fulfilling the inclusion criteria. Among them, 1,051 had at least one positive response of 9-item wearing off questionnaire (WOQ-9), 724 and 160 patients were finally diagnosed with WO and dyskinesia by movement disorders specialists, respectively. The overall prevalence rates of WO and dyskinesia were 46.5% (95% CI 44.0% - 48.9%) and 10.3% (95% CI 8.8% - 11.8%), respectively. The mean score of WOQ-9 for those with WO was 3.8 (SD = 1.8), with movement slowness being the most common motor symptoms and pain/aching being the most common non-motor symptoms. Better improvement of motor symptoms (n = 354, 87.8%) and long-term disease control and drug selection (n = 288, 71.5%) were the two most frequently considered factors when movement disorders specialists adjusted therapeutic strategies for patients with WO.
Conclusions
This survey provided the first multi-center epidemiological data of motor complications among PD patients on L-dopa therapy from mainland China. WO prevalence rate among Chinese PD patients was in line with, while dyskinesia prevalence rate was lower than previous reports from other Countries.
Electronic supplementary material
The online version of this article (doi:10.1186/2047-9158-3-26) contains supplementary material, which is available to authorized users.
doi:10.1186/2047-9158-3-26
PMCID: PMC4323338  PMID: 25671102
Parkinson’s disease; Wearing-off; Dyskinesia; Epidemiology
10.  P2X7 receptor antagonism inhibits p38 MAPK activation and ameliorates neuronal apoptosis after subarachnoid hemorrhage in the rat 
Critical care medicine  2013;41(12):10.1097/CCM.0b013e31829a8246.
Objective
Brilliant blue G (BBG), a selective P2X7 receptor (P2X7R) antagonist, exhibits neuroprotective properties. This study examined whether BBG treatment ameliorates early brain injury (EBI) after experimental subarachnoid hemorrhage (SAH), specifically via inhibiting p38 mitogen-activated protein kinase (MAPK)-related proapoptotic pathways.
Design
Controlled in vivo laboratory study.
Setting
Animal research laboratory.
Subjects
One hundred-fifty four adult male Sprague-Dawley rats weighing 280–320g.
Interventions
SAH was induced in rats by endovascular perforation. Experiment 1 implemented sham-operated rats (sham) and SAH animals, which received vehicle (SAH+vehicle), BBG (SAH+BBG) or BBG plus BzATP (SAH+BBG+BzATP). The animals were intraperitoneally treated with BBG (30mg/kg) at 30 minutes after SAH. BzATP (50μg/rat), a P2X7R agonist, was intracerebroventricularly administered. Experiment 2 implemented sham-operated rats (sham) and SAH animals, which received vehicle (SAH+vehicle), scramble small interfering RNA (siRNA) (SAH+scramble siRNA) or P2X7R siRNA (SAH+P2X7R siRNA). SAH grading, neurobehavioral score and brain edema were evaluated at 24 and 72 hours after surgery. The expression of phosphorylated p38 MAPK, phosphorylated extracellular signal-regulated kinases (ERKs), phosphorylated c-Jun N-terminal kinases (JNKs), P2X7R, Bcl-2 and cleaved caspase-3 in the left cerebral hemisphere were determined by Western blot. Neuronal apoptosis was examined by double immunofluorescence staining using P2X7R, terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end-labeling (TUNEL) and NeuN.
Measurements and main results
BBG significantly improved neurobehavioral function and ameliorated brain water content at 24 and 72 hours after SAH. BzATP reversed these treatment effects. BBG attenuated neuronal apoptosis in the subcortex, which was associated with decreased expression of phosphorylated p38 MAPK and cleaved caspase-3, and an increased expression of Bcl-2 in the left cerebral hemisphere. The beneficial effects of P2X7R siRNA were also mediated by a p38 MAPK pathway.
Conclusions
Inhibition of P2X7R by BBG or P2X7R siRNA can prevent EBI via p38 MAPK after SAH.
doi:10.1097/CCM.0b013e31829a8246
PMCID: PMC3841260  PMID: 23963136
apoptosis; subarachnoid hemorrhage; early brain injury; P2X7 receptor; p38 mitogen-activated protein kinase; brilliant blue G
11.  A Simple, Rapid and Sensitive FRET Assay for Botulinum Neurotoxin Serotype B Detection 
PLoS ONE  2014;9(12):e114124.
Botulinum neurotoxins (BoNTs), the most potent naturally-occurring neurotoxins known to humans, comprise seven distinct serotypes (BoNT/A-G), each of which exhibits unique substrate specificity. Many methods have been developed for BoNT detection, in particular for BoNT/A, with various complexity and sensitivity, while substrate based FRET assay is considered as the most widely used approach due to its simplicity and sensitivity. In this study, we designed a vesicle-associated membrane protein 2 (VAMP2) based FRET assay based on the understanding of the VAMP2 and light chain/B (LC/B) interactions in our previous studies. The current design constituted the shortest peptide, VAMP2 (63–85), with FRET dyes (EDAN and Dabcyl) labelled at position 76 and 85, respectively, which showed minimal effect on VAMP2 substrate catalysis by LC/B and therefore enhanced the sensitivity of the assay. The FRET peptide, designated as FVP-B, was specific to LC/B, with a detection sensitivity as low as ∼20 pM in 2 h. Importantly, FVP-B showed the potential to be scaled up and used in high throughput screening of LC/B inhibitor. The currently developed FRET assay is one of the most economic and rapid FRET assays for LC/B detection.
doi:10.1371/journal.pone.0114124
PMCID: PMC4250190  PMID: 25437190
12.  An Elevated Peripheral Blood Lymphocyte-to-Monocyte Ratio Predicts Favorable Response and Prognosis in Locally Advanced Breast Cancer following Neoadjuvant Chemotherapy 
PLoS ONE  2014;9(11):e111886.
Purpose
Neoadjuvant chemotherapy (NCT) is a standard treatment option for locally advanced breast cancer. However, the lack of an efficient method to predict treatment response and patient prognosis hampers the clinical evaluation of patient eligibility for NCT. An elevated lymphocyte-to-monocyte ratio (LMR) has been reported to be associated with a favorable prognosis for certain hematologic malignancies and for nasopharyngeal carcinoma; however, this association has not been investigated in breast cancer. The purpose of this study was to evaluate whether pre-NCT LMR analysis could predict the prognosis of patients with locally advanced breast cancer.
Methods
A retrospective cohort of 542 locally advanced breast cancer patients (T3/T4 and/or N2/N3 disease) receiving NCT followed by radical surgery was recruited between May 2002 and August 2011 at the Fudan University Shanghai Cancer Center. Counts for pre-NCT peripheral absolute lymphocytes and monocytes were obtained and used to calculate the LMR.
Results
Univariate and multivariate analysis revealed that higher LMR levels (≥4.25) were significantly associated with favorable DFS (P = 0.009 and P = 0.011, respectively). Additionally, univariate analysis revealed that a higher lymphocyte count (≥1.5×109/L) showed borderline significance for improved DFS (P = 0.054), while a lower monocyte count (<0.4×109/L) was associated with a significantly better DFS (P = 0.010).
Conclusions
An elevated pre-NCT peripheral LMR level was a significantly favorable factor for locally advanced breast cancer patient prognosis. This easily obtained variable may serve as a valuable marker to predict the outcomes of locally advanced breast cancer.
doi:10.1371/journal.pone.0111886
PMCID: PMC4221197  PMID: 25372468
13.  Curcumin could reduce the monomer of TTR with Tyr114Cys mutation via autophagy in cell model of familial amyloid polyneuropathy 
Transthyretin (TTR) familial amyloid polyneuropathy (FAP) is an autosomal dominant inherited neurodegenerative disorder caused by various mutations in the transthyretin gene. We aimed to identify the mechanisms underlying TTR FAP with Tyr114Cys (Y114C) mutation. Our study showed that TTR Y114C mutation led to an increase in monomeric TTR and impaired autophagy. Treatment with curcumin resulted in a significant decrease of monomeric TTR by recovering autophagy. Our research suggests that impairment of autophagy might be involved in the pathogenesis of TTR FAP with Y114C mutation, and curcumin might be a potential therapeutic approach for TTR FAP.
doi:10.2147/DDDT.S70866
PMCID: PMC4222630  PMID: 25382970
curcumin; familial amyloid polyneuropathy; transthyretin; autophagy
14.  Development of Two Barthel Index-Based Supplementary Scales for Patients with Stroke 
PLoS ONE  2014;9(10):e110494.
Background
The Barthel Index (BI) assesses actual performance of activities of daily living (ADL). However, comprehensive assessment of ADL functions should include two other constructs: self-perceived difficulty and ability.
Objective
The aims of this study were to develop two BI-based Supplementary Scales (BI-SS), namely, the Self-perceived Difficulty Scale and the Ability Scale, and to examine the construct validity of the BI-SS in patients with stroke.
Method
The BI-SS was first developed by consultation with experts and then tested on patients to confirm the clarity and feasibility of administration. A total of 306 participants participated in the construct validity study. Construct validity was investigated using Mokken scale analysis and analyzing associations between scales. The agreement between each pair of the scales’ scores was further examined.
Results
The Self-perceived Difficulty Scale consisted of 10 items, and the Ability Scale included 8 items (excluding both bladder and bowel control items). Items in each individual scale were unidimensional (H≥0.5). The scores of the Self-perceived Difficulty and Ability Scales were highly correlated with those of the BI (rho = 0.78 and 0.90, respectively). The scores of the two BI-SS scales and BI were significantly different from each other (p<.001). These results indicate that both BI-SS scales assessed unique constructs.
Conclusions
The BI-SS had overall good construct validity in patients with stroke. The BI-SS can be used as supplementary scales for the BI to comprehensively assess patients’ ADL functions in order to identify patients’ difficulties in performing ADL tasks, plan intervention strategies, and assess outcomes.
doi:10.1371/journal.pone.0110494
PMCID: PMC4203801  PMID: 25329051
15.  The deviation of growth model for transparent conductive graphene 
Nanoscale Research Letters  2014;9(1):581.
An approximate growth model was employed to predict the time required to grow a graphene film by chemical vapor deposition (CVD). Monolayer graphene films were synthesized on Cu foil at various hydrogen flow rates from 10 to 50 sccm. The sheet resistance of the graphene film was 310Ω/□ and the optical transmittance was 97.7%. The Raman intensity ratio of the G-peak to the 2D peak of the graphene film was as high as ~4 when the hydrogen flow rate was 30 sccm. The fitting curve obtained by the deviation equation of growth model closely matches the data. We believe that under the same conditions and with the same setup, the presented growth model can help manufacturers and academics to predict graphene growth time more accurately.
doi:10.1186/1556-276X-9-581
PMCID: PMC4214604  PMID: 25364316
Graphene; Transparent conductive electrode; Chemical vapor deposition
16.  Inhibition of autophagy potentiates the efficacy of Gli inhibitor GANT-61 in MYCN-amplified neuroblastoma cells 
BMC Cancer  2014;14(1):768.
Background
Aberrant Hedgehog (Hh) signaling is often associated with neuroblastoma (NB), a childhood malignancy with varying clinical outcomes due to different molecular characteristics. Inhibition of Hh signaling with small molecule inhibitors, particularly with GANT-61, significantly suppresses NB growth. However, NB with MYCN amplification is less sensitive to GANT-61 than those without MYCN amplification.
Methods
Autophagic process was examined in two MYCN amplified and two MYCN non-amplified NB cells treated with GANT-61. Subsequently, chemical and genetic approaches were applied with GANT-61 together to evaluate the role of autophagy in GANT-61 induced cell death.
Results
Here we show that GANT-61 enhanced autophagy in MYCN amplified NB cells. Both an autophagic inhibitor 3-methyladenine (3-MA) and genetic disruption of ATG5 or ATG7 expression suppressed GANT-61 induced autophagy and significantly increased apoptotic cell death, whereas pre-treatment with an apoptotic inhibitor, Z-VAD-FMK, rescued GANT-61 induced cell death and had no effect on the autophagic process. In the other hand, GANT-61 barely induced autophagy in MYCN non-amplified NB cells, but overexpression of MYCN in MYCN non-amplified NB cells recapitulated GANT-61 induced autophagy seen in MYCN amplified NB cells, suggesting that the level of GANT-61 induced autophagy in NB cells is related to MYCN expression level in cells.
Conclusion
Aberrant Hh signaling activation as an oncogenic driver in NB renders inhibition of Hh signaling an effective measure to suppress NB growth. However, our data suggest that enhanced autophagy concomitant with Hh signaling inhibition acts as a pro-survival factor to maintain cell viability, which reduces GANT-61 efficacy. Besides, MYCN amplification is likely involved in the induction of the pro-survival autophagy. Overall, simultaneous inhibition of both Hh signaling and autophagy could be a better way to treat MYCN amplified NB.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-768) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2407-14-768
PMCID: PMC4210511  PMID: 25323222
Neuroblastoma; GANT-61; MYCN amplification; Autophagy
17.  PMQR genes oqxAB and aac(6′)Ib-cr accelerate the development of fluoroquinolone resistance in Salmonella typhimurium 
Emergence of multidrug-resistant Salmonella typhimurium strains, especially the ACSSuT and nalidixic acid R types, has significantly compromised the effectiveness of current strategies to control Salmonella infections, resulting in increased morbidity and mortality. Clinical S. typhimurium isolates recovered in Hong Kong during the period of 2005–2011 were increasingly resistant to ciprofloxacin (CIP) and antibiotics of the ACSSuT group. Our data revealed that oqxAB and aac(6′)Ib-cr were encoded on plasmids of various sizes and the presence of these two elements together with a single gyrA mutation in S. typhimurium were sufficient to mediate resistance to CIP. Acquisition of the oqxAB and aac(6′)Ib-cr encoding plasmids by S. typhimurium caused a fourfold increase in CIP minimal inhibitory concentration. Furthermore, the presence of oqxAB and aac(6′)Ib-cr in Salmonella dramatically increased the mutation prevention concentration of CIP which may due to mutational changes in the drug target genes. In conclusion, possession of oqxAB and aac(6′)Ib-cr encoding plasmid facilitate the selection of CIP resistant S. typhimurium, thereby causing a remarkable increase of CIP resistance among clinical Salmonella strains in Hong Kong.
doi:10.3389/fmicb.2014.00521
PMCID: PMC4183184  PMID: 25324840
S. typhimurium; ciprofloxacin resistance; ACSSuT R type; oqxAB; aac(6′)Ib-cr
18.  P2X7R/cryopyrin inflammasome axis inhibition reduces neuroinflammation after SAH 
Neurobiology of disease  2013;58:296-307.
Neuroinflammation contributes to the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Cytotoxic events following SAH, such as extracellular accumulation of adenosine triphosphate (ATP), may activate the P2X purinoceptor 7 (P2X7R)/cryopyrin inflammasome axis, thus inducing the proinflammatory cytokines IL-1β/IL-18 secretion. We therefore hypothesized that inhibition of P2X7R/cryopyrin inflammasome axis would ameliorate neuroinflammation after SAH. In the present study, SAH was induced by the endovascular perforation in rats. Small interfering RNAs (siRNAs) of P2X7R or cryopyrin were administered intracerebroventricularly 24 hours before SAH. Brilliant Blue G (BBG), a non-competitive antagonist of P2X7R, was administered intraperitoneally 30 minutes following SAH. Post-assessments including SAH severity score, neurobehavioral test, brain water content, Western blot and immunofluorescence, were performed. Administration of P2X7R and cryopyrin siRNA as well as pharmacologic blockade of P2X7R by BBG ameliorated neurological deficits and brain edema at 24 hours following SAH. Inhibition of P2X7R/cryopyrin inflammasome axis suppressed caspase-1 activation, which subsequently decreased maturation of IL-1β/IL-18. To investigate the link between P2X7R and cryopyrin inflammasome in vivo, Benzoylbenzoyl-ATP (BzATP), a P2X7R agonist, was given to lipopolysaccharide (LPS) primed naive rats with scramble or cryopyrin siRNAs. In LPS-primed naïve rats, BzATP induced caspase-1 activation and mature IL-1β release was neutralized by cryopyrin siRNA. Thus, the P2X7R/cryopyrin inflammasome axis may contribute to neuroinflammation via activation of caspase-1 and thereafter mature IL-1β/IL-18 production following SAH. Therapeutic interventions targeting P2X7R/cryopyrin pathway may be a novel approach to ameliorate EBI following SAH.
doi:10.1016/j.nbd.2013.06.011
PMCID: PMC3771387  PMID: 23816751
subarachnoid hemorrhage; early brain injury; cryopyrin; P2X purinoceptor 7; inflammation; edema
19.  Expression, purification, crystallization and preliminary X-ray analysis of the RecQ helicase catalytic core from Deinococcus radiodurans  
The DrRecQ helicase catalytic core has been crystallized to a resolution of 2.9 Å. The determination of its structure will lead to structural and functional insight into the DNA repair mechanism.
The RecQ proteins are a highly conserved group of DNA helicases which play crucial roles in the maintenance of genome stability. DrRecQ from the radioresistant bacterium Deinococcus radiodurans is a special member of the RecQ family because it contains three Helicase-and-RNase-D-C-terminal (HRDC) domains at the C-terminus. The helicase catalytic core is essential for ATPase and DNA-unwinding activities. In this work, the helicase catalytic core of DrRecQ was expressed in Escherichia coli, purified and crystallized. Crystals were obtained using the sitting-drop vapour diffusion method and X-ray diffraction data were collected to 2.9 Å resolution. The crystals belong to space group P212121, with unit-cell parameters a = 84.75, b = 95.61, c = 183.83 Å.
doi:10.1107/S1744309112037517
PMCID: PMC3490466  PMID: 23027755
RecQ; helicase; Deinococcus radiodurans
20.  Expression, purification, crystallization and preliminary X-ray analysis of ribitol-5-phosphate cytidylyltransferase from Bacillus subtilis  
The B. subtilis ribitol-5-phosphate cytidylyltransferase enzyme TarI was crystallized; determination of its structure will lead to structural and functional insight into the biosynthesis of wall teichoic acids.
TarI is a ribitol-5-phosphate cytidylyltransferase that catalyzes the formation of CDP-ribitol, which is involved in the biosynthesis of wall teichoic acids, from CTP and ribitol 5-phosphate. TarI from Bacillus subtilis (BsTarI) was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.78 Å and belonged to the monoclinic space group C2, with unit-cell parameters a = 103.74, b = 60.97, c = 91.80 Å, β = 113.48°. The initial structural model indicated that the crystals of BsTarI contained a dimer in the asymmetric unit.
doi:10.1107/S1744309112035142
PMCID: PMC3497978  PMID: 23027746
ribitol-5-phosphate cytidylyltransferase; wall teichoic acids; Bacillus subtilis; TarI
21.  Prevention of Immune Nephritis by the Small Molecular Weight Immunomodulator Iguratimod in MRL/lpr Mice 
PLoS ONE  2014;9(10):e108273.
Objective
This study was performed to investigate the therapeutic effects of iguratimod in a lupus mouse model.
Methods
Female MRL/lpr mice were treated with iguratimod, vehicle solution or cyclophosphamide. Proteinuria was monitored and kidney injury was blindly scored by a renal pathologist. Serum anti-double-stranded DNA antibodies were monitored by radioimmunoassay. Kidney IgG and CD20 were stained by immunohistochemistry. Splenic lymphocyte phenotypes were analyzed by flow cytometry. BAFF, IL-17A, IL-6, and IL-21 levels in serum and splenic lymphocytes were detected by ELISA or quantitative PCR.
Results
Compared with the vehicle-treated controls, MRL/lpr mice treated with iguratimod showed less protenuria, less acute pathological lesions and no chronic changes in the kidneys. There were significant differences in glomerular injury and vasculitis scores, as well as in the semi-quantitave analysis of immune complex deposition between the two groups. Disease activity markers in sera (anti-dsDNA antibodies and immunoglobulin levels) were reduced and hypocomplementemia was attenuated. Lymphocyte expression of BAFF, IL-6, IL-17A and IL-21 was decreased. The abnormal splenic B220+ T cell and plasma cell populations in MRL/lpr mice were reduced by iguratimod treatment, with recovery of the total B cell population and inhibition of B cell infiltration of the kidney tissue. The dosage of iguratimod used in this study showed no significant cytotoxic effects in vivo and no overt side-effects were observed.
Conclusion
Iguratimod ameliorates immune nephritis in MRL/lpr mice via a non-antiproliferative mechanism. Our data suggest a potential therapeutic role of iguratimod in lupus.
doi:10.1371/journal.pone.0108273
PMCID: PMC4182720  PMID: 25271634
22.  MicroRNA-27a promotes proliferation and suppresses apoptosis by targeting PLK2 in laryngeal carcinoma 
BMC Cancer  2014;14(1):678.
Background
miRNA-27a has been confirmed as an important regulator in carcinogenesis and other pathological processes. Whether and how it plays a role in the laryngeal carcinoma is unknown.
Methods
Mature miRNA-27a expression in laryngeal cancer was detected by qRT-PCR. Gain-of-function studies using mature miR-27a were performed to investigate cell proliferation and apoptosis in the Hep2 cells. In silico database analysis and luciferase reporter assay were applied to predict and validate the direct target, respectively. Loss-of-function assays were performed to investigate the functional significance of the miR-27a target gene. qRT-PCR and Western blot were used to evaluate mRNA and protein levels of the target, respectively.
Results
miR-27a was significantly up-regulated in the laryngeal tumor tissues compared to the adjacent non-tumor tissues. In silico database analysis result revealed that PLK2 is a potential target of miR-27a. luciferase reporter assay result showed the direct inhibition of miR-27a on PLK2-3′UTR. In the cases with miR-27a up-regulation, PLK2 protein expression level was significantly lower in cancer tissues than that in the adjacent non-tumor tissues, which showed a negative correlation with miR-27a expression level. Both miR-27a and knockdown of PLK2 caused the increase of the cell viability and colony formation and inhibition of the late apoptosis in the Hep2 cell lines. Moreover, miR-27a but not PLK2 also repressed the early apoptosis in the Hep2 cells. Additionally, no alteration of the Hep2 cell cycle induced by miR-27a was detected.
Conclusions
miR-27a acts as an oncogene in laryngeal squamous cell carcinoma through down-regulation of PLK2 and may provide a novel clue into the potential mechanism of LSCC oncogenesis or serve as a useful biomarker in diagnosis and therapy in laryngeal cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-678) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2407-14-678
PMCID: PMC4177177  PMID: 25239093
Laryngeal squamous cell carcinoma; miR-27a; PLK2; Apoptosis; Proliferation
23.  Plasmonic Structure Enhanced Exciton Generation at the Interface between the Perovskite Absorber and Copper Nanoparticles 
The Scientific World Journal  2014;2014:128414.
The refractive index and extinction coefficient of a triiodide perovskite absorber (TPA) were obtained by fitting the transmittance spectra of TPA/PEDOT:PSS/ITO/glass using the transfer matrix method. Cu nanoplasmonic structures were designed to enhance the exciton generation in the TPA and to simultaneously reduce the film thickness of the TPA. Excitons were effectively generated at the interface between TPA and Cu nanoparticles, as observed through the 3D finite-difference time-domain method. The exciton distribution is advantageous for the exciton dissociation and carrier transport.
doi:10.1155/2014/128414
PMCID: PMC4176912  PMID: 25295290
24.  Selection of target mutation in rat gastrointestinal tract E. coli by minute dosage of enrofloxacin 
It has been suggested that bacterial resistance is selected within a mutation selection window of antibiotics. More recent studies showed that even extremely low concentration of antibiotic could select resistant bacteria in vitro. Yet little is known about the exact antibiotic concentration range that can effectively select for resistant organisms in animal gastrointestinal (GI) tract. In this study, the effect of different dosages of enrofloxacin on resistance and mutation development in rat GI tract E. coli was investigated by determining the number of resistant E. coli recoverable from rat fecal samples. Our data showed that high dose antibiotic treatment could effectively eliminate E. coli with single gyrA mutation in the early course of treatment, yet the eradication effects diminished upon prolonged treatment. Therapeutic and sub-therapeutic dose (1/10 and 1/100 of therapeutic doses) of enrofloxacin could effectively select for mutation in GI tract E. coli at the later course of enrofloxacin treatment and during the cessation periods. Surprisingly, very low dose of enrofloxacin (1/1000 therapeutic dose) could also select for mutation in GI tract E. coli at the later course of enrofloxacin treatment, only with slightly lower efficiency. No enrofloxacin-resistant E. coli could be selected at all test levels of enrofloxacin during long term treatment and the strength of antibiotic treatment does not alter the overall level of E. coli in rat GI tract. This study demonstrated that long term antibiotic treatment seems to be the major trigger for the development of target mutations in GI tract E. coli, which provided insight into the rational use of antibiotics in animal husbandry.
doi:10.3389/fmicb.2014.00468
PMCID: PMC4154546  PMID: 25237308
target mutation; rat GI tract E. coli; enrofloxacin; resistance development
25.  Crystal Structure of a Conserved Hypothetical Protein MJ0927 from Methanocaldococcus jannaschii Reveals a Novel Quaternary Assembly in the Nif3 Family 
BioMed Research International  2014;2014:171263.
A Nif3 family protein of Methanocaldococcus jannaschii, MJ0927, is highly conserved from bacteria to humans. Although several structures of bacterial Nif3 proteins are known, no structure representing archaeal Nif3 has yet been reported. The crystal structure of Methanocaldococcus jannaschii MJ0927 was determined at 2.47 Å resolution to understand the structural differences between the bacterial and archaeal Nif3 proteins. Intriguingly, MJ0927 is found to adopt an unusual assembly comprising a trimer of dimers that forms a cage-like architecture. Electrophoretic mobility-shift assays indicate that MJ0927 binds to both single-stranded and double-stranded DNA. Structural analysis of MJ0927 reveals a positively charged region that can potentially explain its DNA-binding capability. Taken together, these data suggest that MJ0927 adopts a novel quartenary architecture that could play various DNA-binding roles in Methanocaldococcus jannaschii.
doi:10.1155/2014/171263
PMCID: PMC4163360  PMID: 25243119

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