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1.  In vitro enhancement of dendritic cell-mediated anti-glioma immune response by graphene oxide 
Nanoscale Research Letters  2014;9(1):311.
Malignant glioma has extremely poor prognosis despite combination treatments with surgery, radiation, and chemotherapy. Dendritic cell (DC)-based immunotherapy may potentially serve as an adjuvant treatment of glioma, but its efficacy generally needs further improvement. Here we explored whether graphene oxide (GO) nanosheets could modulate the DC-mediated anti-glioma immune response in vitro, using the T98G human glioma cell line as the study model. Pulsing DCs with a glioma peptide antigen (Ag) generated a limited anti-glioma response compared to un-pulsed DCs. Pulsing DCs with GO alone failed to produce obvious immune modulation effects. However, stimulating DCs with a mixture of GO and Ag (GO-Ag) significantly enhanced the anti-glioma immune reaction (p < 0.05). The secretion of interferon gamma (IFN-γ) by the lymphocytes was also markedly boosted by GO-Ag. Additionally, the anti-glioma immune response induced by GO-Ag appeared to be target-specific. Furthermore, at the concentration used in this study, GO exhibited a negligible effect on the viability of the DCs. These results suggested that GO might have potential utility for boosting a DC-mediated anti-glioma immune response.
PMCID: PMC4082417  PMID: 25024678
Glioma; Dendritic cell; DC; Graphene oxide; GO; Immunotherapy
2.  Signal Waveform Detection with Statistical Automaton for Internet and Web Service Streaming 
The Scientific World Journal  2014;2014:647216.
In recent years, many approaches have been suggested for Internet and web streaming detection. In this paper, we propose an approach to signal waveform detection for Internet and web streaming, with novel statistical automatons. The system records network connections over a period of time to form a signal waveform and compute suspicious characteristics of the waveform. Network streaming according to these selected waveform features by our newly designed Aho-Corasick (AC) automatons can be classified. We developed two versions, that is, basic AC and advanced AC-histogram waveform automata, and conducted comprehensive experimentation. The results confirm that our approach is feasible and suitable for deployment.
PMCID: PMC4083271  PMID: 25032231
3.  Chronic Infections of West Nile Virus Detected in California Dead Birds 
During 2010 and 2011, 933 recently deceased birds, submitted as part of the dead bird surveillance program, tested positive for West Nile virus RNA at necropsy. The relative amount of RNA measured by qRT-PCR cycles ranged from 8.2 to 37.0 cycle threshold (Ct) and formed a bimodal frequency distribution, with maxima at 20 and 36 Ct and minima at 28–30 Ct. On the basis of frequency distributions among different avian species with different responses to infection following experimental inoculation, field serological data indicating survival of infection, and the discovery of persistent RNA in experimentally infected birds, dead birds collected in nature were scored as “recent” or “chronic” infections on the basis of Ct scores. The percentage of birds scored as having chronic infections was highest during late winter/spring, when all birds were after hatching year, and lowest during late summer, when enzootic transmission was typically highest as indicated by mosquito infections. Our data indicated that intervention efforts should not be based on dead birds with chronic infections unless supported by additional surveillance metrics.
PMCID: PMC3669600  PMID: 23488452
Surveillance; West Nile virus; Dead birds; Chronic infections; Overwintering
4.  Equine Arteritis Virus Does Not Induce Interferon Production in Equine Endothelial Cells: Identification of Nonstructural Protein 1 as a Main Interferon Antagonist 
BioMed Research International  2014;2014:420658.
The objective of this study was to investigate the effect of equine arteritis virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN-β was measured at mRNA and protein levels by quantitative real-time RT-PCR and IFN bioassay using vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP), respectively. Quantitative RT-PCR results showed that IFN-β mRNA levels in EECs infected with EAV VBS were not increased compared to those in mock-infected cells. Consistent with quantitative RT-PCR, Sendai virus- (SeV-) induced type I IFN production was inhibited by EAV infection. Using an IFN-β promoter-luciferase reporter assay, we subsequently demonstrated that EAV nsps 1, 2, and 11 had the capability to inhibit type I IFN activation. Of these three nsps, nsp1 exhibited the strongest inhibitory effect. Taken together, these data demonstrate that EAV has the ability to suppress the type I IFN production in EECs and nsp1 may play a critical role to subvert the equine innate immune response.
PMCID: PMC4055586  PMID: 24967365
5.  Bronchial asthma is associated with increased risk of chronic kidney disease 
Bronchial asthma influences some chronic diseases such as coronary heart disease, diabetes mellitus, and hypertension, but the impact of asthma on vital diseases such as chronic kidney disease is not yet verified. This study aims to clarify the association between bronchial asthma and the risk of developing chronic kidney disease.
The National Health Research Institute provided a database of one million random subjects for the study. A random sample of 141 064 patients aged ≥18 years without a history of kidney disease was obtained from the database. Among them, there were 35 086 with bronchial asthma and 105 258 without asthma matched for sex and age for a ration of 1:3. After adjusting for confounding risk factors, a Cox proportional hazards model was used to compare the risk of developing chronic kidney disease during a three-year follow-up period.
Of the subjects with asthma, 2 196 (6.26%) developed chronic kidney disease compared to 4 120 (3.91%) of the control subjects. Cox proportional hazards regression analysis revealed that subjects with asthma were more likely to develop chronic kidney disease (hazard ratio [HR]: 1.56; 95% CI: 1.48-1.64; p < 0.001). After adjusting for sex, age, monthly income, urbanization level, geographic region, diabetes mellitus, hypertension, hyperlipidemia, and steroid use, the HR for asthma patients was 1.40 (95% CI: 1.33-1.48; p = 0.040). There was decreased HRs in steroid use (HR: 0.56; 95% CI: 0.62-0.61; p < 0.001) in the development of chronic kidney disease. Expectorants, bronchodilators, anti-muscarinic agents, airway smooth muscle relaxants, and leukotriene receptor antagonists may also be beneficial in attenuating the risk of chronic kidney disease.
Patients with bronchial asthma may have increased risk of developing chronic kidney disease. The use of steroids or non-steroidal drugs in the treatment of asthma may attenuate this risk.
PMCID: PMC4022436  PMID: 24885269
Bronchial asthma; Chronic kidney diseases; National Health Insurance Research Dataset
6.  Gastric foreign body granuloma caused by an embedded fishbone: A case report 
Fishbones are the most commonly ingested foreign bodies that cause gastrointestinal tract penetration. However, fishbones embedded in the gastrointestinal tract that lead to foreign body granulomas that mimic submucosal tumors are rare. Herein, we describe a 56-year-old woman who presented with a 20-day-history of upper abdominal pain. Endoscopy revealed an elevated lesion in the gastric antrum. An abdominal computed tomography scan showed a mass in the gastric antrum and a linear calcified lesion in the mass. An endoscopic ultrasonography examination revealed a 3.9 cm × 2.2 cm, irregular, hypoechoic mass with indistinct margins in the muscularis propria layer. The patient was initially diagnosed as having a submucosal tumor, and subsequent surgical resection showed that the lesion was a foreign body granuloma caused by an embedded fishbone. Our case indicated that the differential diagnosis of a foreign body granuloma should be considered in cases of elevated lesions in the gastrointestinal tract.
PMCID: PMC3964412  PMID: 24696619
Gastric; Foreign body granuloma; Fishbone; Endoscopic ultrasonography; Computed tomography
7.  The effect of high-top and low-top shoes on ankle inversion kinematics and muscle activation in landing on a tilted surface 
There is still uncertainty concerning the beneficial effects of shoe collar height for ankle sprain prevention and very few data are available in the literature regarding the effect of high-top and low-top shoes on muscle responses during landing. The purpose of this study was to quantify the effect of high-top and low-top shoes on ankle inversion kinematics and pre-landing EMG activation of ankle evertor muscles during landing on a tilted surface.
Thirteen physical education students landed on four types of surfaces wearing either high-top shoes (HS) or low-top shoes (LS). The four conditions were 15° inversion, 30° inversion, combined 25° inversion + 10° plantar flexion, and combined 25° inversion + 20° plantar flexion. Ankle inversion kinematics and EMG data of the tibialis anterior (TA), peroneus longus (PL), and peroneus brevis (PB) muscles were measured simultaneously. A 2 × 4 (shoe × surface) repeated measures ANOVA was performed to examine the effect of shoe and landing surfaces on ankle inversion and EMG responses.
No significant differences were observed between the various types of shoes in the maximum ankle inversion angle, the ankle inversion range of motion, and the maximum ankle inversion angular velocity after foot contact for all conditions. However, the onset time of TA and PB muscles was significantly later wearing HS compared to LS for the 15° inversion condition. Meanwhile, the mean amplitude of the integrated EMG from the 50 ms prior to contact (aEMGpre) of TA was significantly lower with HS compared to LS for the 15° inversion condition and the combined 25° inversion + 20° plantarflexion condition. Similarly, the aEMGpre when wearing HS compared to LS also showed a 37.2% decrease in PL and a 31.0% decrease in PB for the combined 25° inversion + 20° plantarflexion condition and the 15° inversion condition, respectively.
These findings provide preliminary evidence suggesting that wearing high-top shoes can, in certain conditions, induce a delayed pre-activation timing and decreased amplitude of evertor muscle activity, and may therefore have a detrimental effect on establishing and maintaining functional ankle joint stability.
PMCID: PMC3943374  PMID: 24548559
High-top/low-top shoe; Ankle inversion; Muscle pre-activity; Tilted surface; Landing
8.  Madelung disease 
PMCID: PMC3537816  PMID: 22733673
9.  Survivin – biology and potential as a therapeutic target in oncology 
OncoTargets and therapy  2013;6:1453-1462.
Survivin is a member of the inhibitor-of-apoptosis proteins (IAPs) family; its overexpression has been widely demonstrated to occur in various types of cancer. Overexpression of survivin also correlates with tumor progression and induces anticancer drug resistance. Interestingly, recent studies reveal that survivin exhibits multiple pro-mitotic and anti-apoptotic functions; the differential functions of survivin seem to be caused by differential subcellular localization, phosphorylation, and acetylation of this molecule. In this review, the complex expression regulations and post-translational modifications of survivin are discussed. This review also discusses how recent discoveries improve our understanding of survivin biology and also create opportunities for developing differential-functioned survivin-targeted therapy. Databases such as PubMed, Scopus® (Elsevier, New York, NY, USA), and SciFinder® (CAS, Columbus, OH, USA) were used to search for literature in the preparation of this review.
PMCID: PMC3804542  PMID: 24204160
survivin; BIRC5; IAP; XIAP; caspase-9; Samc; DIABLO
10.  The Antitumor Effects of Triterpenoid Saponins from the Anemone flaccida and the Underlying Mechanism 
Anemone flaccida Fr. Schmidt, a family of ancient hopanoids, have been used as traditional Asian herbs for the treatments of inflammation and convulsant diseases. Previous study on HeLa cells suggested that triterpenoid saponins from Anemone flaccida Fr. Schmidt may have potential antitumor effect due to their apoptotic activities. Here, we confirmed the apoptotic activities of the following five triterpenoid saponins: glycoside St-I4a (1), glycoside St-J (2), anhuienoside E (3), hedera saponin B (4), and flaccidoside II (5) on human BEL-7402 and HepG2 hepatoma cell lines, as well as the model of HeLa cells treated with lipopolysaccharide (LPS). We found that COX-2/PGE2 signaling pathway, which plays key roles in the development of cancer, is involved in the antitumor activities of these saponins. These data provide the evidence that triterpenoid saponins can induce apoptosis via COX-2/PGE2 pathway, implying a preventive role of saponins from Anemone flaccida in tumor.
PMCID: PMC3804048  PMID: 24191167
11.  Characterization of a porcine intestinal epithelial cell line for influenza virus production 
The Journal of General Virology  2012;93(Pt 9):2008-2016.
We have developed a porcine intestine epithelial cell line, designated SD-PJEC for the propagation of influenza viruses. The SD-PJEC cell line is a subclone of the IPEC-J2 cell line, which was originally derived from newborn piglet jejunum. Our results demonstrate that SD-PJEC is a cell line of epithelial origin that preferentially expresses receptors of oligosaccharides with Sia2-6Gal modification. This cell line is permissive to infection with human and swine influenza A viruses and some avian influenza viruses, but poorly support the growth of human-origin influenza B viruses. Propagation of swine-origin influenza viruses in these cells results in a rapid growth rate within the first 24 h post-infection and the titres ranged from 4 to 8 log10 TCID50 ml−1. The SD-PJEC cell line was further tested as a potential alternative cell line to Madin–Darby canine kidney (MDCK) cells in conjunction with 293T cells for rescue of swine-origin influenza viruses using the reverse genetics system. The recombinant viruses A/swine/North Carolina/18161/02 (H1N1) and A/swine/Texas/4199-2/98 (H3N2) were rescued with virus titres of 7 and 8.25 log10 TCID50 ml−1, respectively. The availability of this swine-specific cell line represents a more relevant substrate for studies and growth of swine-origin influenza viruses.
PMCID: PMC3542131  PMID: 22739061
12.  Use of Scented Sugar Bait Stations to Track Mosquito-Borne Arbovirus Transmission in California 
Journal of medical entomology  2012;49(6):1466-1472.
Laboratory and field research was conducted to determine if Culex tarsalis Coquillett expectorated West Nile virus (WNV) during sugar feeding and if a lure or bait station could be developed to exploit this behavior for WNV surveillance. Experimentally infected Cx. tarsalis repeatedly expectorated WNV onto filter paper strips and into vials with wicks containing sucrose that was readily detectable by a quantitative reverse transcriptase-polymerase chain reaction assay. Few females (33%, n = 27) became infected by imbibing sugar solutions spiked with high concentrations (107 plaque forming units/ml) of WNV, indicating sugar feeding stations probably would not be a source of WNV infection. In nature, sugar bait stations scented with the floral attractant phenyl acetaldehyde tracked WNV transmission activity in desert but not urban or agricultural landscapes in California. When deployed in areas of the Coachella Valley with WNV activity during the summer of 2011, 27 of 400 weekly sugar samples (6.8%) tested positive for WNV RNA by reverse transcriptase-polymerase chain reaction. Prevalence of positives varied spatially, but positive sugar stations were detected before concurrent surveillance measures of infection (mosquito pools) or transmission (sentinel chicken seroconversions). In contrast, sugar bait stations deployed in urban settings in Los Angeles or agricultural habits near Bakersfield in Kern County supporting WNV activity produced 1 of 90 and 0 of 60 positive weekly sugar samples, respectively. These results with sugar bait stations will require additional research to enhance bait attractancy and to understand the relationship between positive sugar stations and standard metrics of arbovirus surveillance.
PMCID: PMC3544359  PMID: 23270177
surveillance; West Nile virus; sugar feeding; bait station; Culex tarsalis
13.  Cyclophilin Inhibitors Block Arterivirus Replication by Interfering with Viral RNA Synthesis 
Journal of Virology  2013;87(3):1454-1464.
Virus replication strongly depends on cellular factors, in particular, on host proteins. Here we report that the replication of the arteriviruses equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) is strongly affected by low-micromolar concentrations of cyclosporine A (CsA), an inhibitor of members of the cyclophilin (Cyp) family. In infected cells, the expression of a green fluorescent protein (GFP) reporter gene inserted into the PRRSV genome was inhibited with a half-maximal inhibitory concentration (IC50) of 5.2 μM, whereas the GFP expression of an EAV-GFP reporter virus was inhibited with an IC50 of 0.95 μM. Debio-064, a CsA analog that lacks its undesirable immunosuppressive properties, inhibited EAV replication with an IC50 that was 3-fold lower than that of CsA, whereas PRRSV-GFP replication was inhibited with an IC50 similar to that of CsA. The addition of 4 μM CsA after infection prevented viral RNA and protein synthesis in EAV-infected cells, and CsA treatment resulted in a 2.5- to 4-log-unit reduction of PRRSV or EAV infectious progeny. A complete block of EAV RNA synthesis was also observed in an in vitro assay using isolated viral replication structures. The small interfering RNA-mediated knockdown of Cyp family members revealed that EAV replication strongly depends on the expression of CypA but not CypB. Furthermore, upon fractionation of intracellular membranes in density gradients, CypA was found to cosediment with membranous EAV replication structures, which could be prevented by CsA treatment. This suggests that CypA is an essential component of the viral RNA-synthesizing machinery.
PMCID: PMC3554155  PMID: 23152531
14.  Double-balloon enteroscopy in small bowel tumors: A Chinese single-center study 
AIM: To analyze the clinical characteristics of small bowel tumors detected by double-balloon enteroscopy (DBE) and to evaluate the diagnostic value of DBE in tumors.
METHODS: Four hundred and forty consecutive DBE examinations were performed in 400 patients (250 males and 150 females, mean age 46.9 ± 16.3 years, range 14-86 years) between January 2007 and April 2012. Of these, 252 patients underwent the antegrade approach, and 188 patients underwent the retrograde approach. All the patients enrolled in our study were suspected of having small bowel diseases with a negative etiological diagnosis following other routine examinations, such as upper and lower gastrointestinal endoscopy and radiography tests. Data on tumors, such as clinical information, endoscopic findings and operation results, were retrospectively collected.
RESULTS: Small bowel tumors were diagnosed in 78 patients, of whom 67 were diagnosed using DBE, resulting in a diagnostic yield of 16.8% (67/400); the other 11 patients had negative DBE findings and were diagnosed through surgery or capsule endoscopy. Adenocarcinoma (29.5%, 23/78), gastrointestinal stromal tumor (24.4%, 19/78) and lymphoma (15.4%, 12/78) were the most common tumors. Among the 78 tumors, 60.3% (47/78) were located in the jejunum, and the overall number of malignant tumors was 74.4% (58/78). DBE examinations were frequently performed in patients with obscure gastrointestinal bleeding (47.4%) and abdominal pain (24.4%). The positive detection rate for DBE in the 78 patients with small bowel tumors was 85.9% (67/78), which was higher than that of a computed tomography scan (72.9%, 51/70). Based on the operation results, the accuracy rates of DBE for locating small bowel neoplasms, such as adenocarcinoma, gastrointestinal stromal tumor and lymphoma, were 94.4%, 100% and 100%, respectively. The positive biopsy rates for adenocarcinoma and lymphoma were 71.4% and 60%, respectively.
CONCLUSION: DBE is a useful diagnostic tool with high clinical practice value and should be considered the gold standard for the investigation of small bowel tumors.
PMCID: PMC3691029  PMID: 23801870
Double-balloon enteroscopy; Small bowel tumors; Diagnosis; Capsule endoscopy; Endoscopic findings
15.  Correction: Changes in Innate and Permissive Immune Responses after HBV Transgenic Mouse Vaccination and Long-Term-siRNA Treatment 
PLoS ONE  2013;8(6):10.1371/annotation/d0e70062-ac3b-47a6-ab1e-402ed08affa4.
PMCID: PMC3731338
16.  Improvements in the Quantitative Assessment of Cerebral Blood Volume and Flow with the Removal of Vessel Voxels from MR Perfusion Images 
BioMed Research International  2013;2013:382027.
Objective. To improve the quantitative assessment of cerebral blood volume (CBV) and flow (CBF) in the brain voxels from MR perfusion images. Materials and Methods. Normal brain parenchyma was automatically segmented with the time-to-peak criteria after cerebrospinal fluid removal and preliminary vessel voxel removal. Two scaling factors were calculated by comparing the relative CBV and CBF of the segmented normal brain parenchyma with the absolute values in the literature. Using the scaling factors, the relative values were converted to the absolute CBV and CBF. Voxels with either CBV > 8 mL/100 g or CBF > 100 mL/100 g/min were characterized as vessel voxels and were excluded from the quantitative measurements. Results. The segmented brain parenchyma with normal perfusion was consistent with the angiographic findings for each patient. We confirmed the necessity of dual thresholds including CBF and CBV for proper removal of vessel voxels. The scaling factors were 0.208 ± 0.041 for CBV, and 0.168 ± 0.037, 0.172 ± 0.037 for CBF calculated using standard and circulant singular value decomposition techniques, respectively. Conclusion. The automatic scaling and vessel removal techniques provide an alternative method for obtaining improved quantitative assessment of CBV and CBF in patients with thromboembolic cerebral arterial disease.
PMCID: PMC3613063  PMID: 23586033
17.  Changes in Innate and Permissive Immune Responses after HBV Transgenic Mouse Vaccination and lLong-Term-siRNA Treatment 
PLoS ONE  2013;8(3):e57525.
Currently, no licensed therapy can thoroughly eradicate hepatitis B virus (HBV) from the body, including interferon α and inhibitors of HBV reverse-transcription. Small interfering RNA (siRNA) seem to be a promising tool for treating HBV, but had no effect on the pre-existing HBV covalently closed circular DNA. Because it is very difficult to thoroughly eradicate HBV with unique siRNAs, upgrading the immune response is the best method for fighting HBV infection. Here, we aim to explore the immune response of transgenic mice to HBV vaccination after long-term treatment with siRNAs and develop a therapeutic approach that combines siRNAs with immunopotentiators.
Methodology/Principal Findings
To explore the response of transgenic mice to hepatitis B vaccine, innate and acquired immunity were detected after long-term treatment with siRNAs and vaccination. Antiviral cytokines and level of anti-hepatitis B surface antigen antibody (HBsAg-Ab) were measured after three injections of hepatitis B vaccine.
Functional analyses indicated that toll-like receptor-mediated innate immune responses were reinforced, and antiviral cytokines were significantly increased, especially in the pSilencer4.1/HBV groups. Analysis of CD80+/CD86+ dendritic cells in the mouse liver indicated that dendritic cell antigen presentation was strengthened. Furthermore, the siRNA-treated transgenic mice could produce detectable HBsAg-Ab after vaccination, especially in the CpG oligonucleotide vaccine group.
For the first time, our studies demonstrate that siRNAs with CpG HBV vaccine could strengthen the immune response and break the immune tolerance status of transgenic mice to HBV. Thus, siRNAs and HBV vaccine could provide a sharp double-edged sword against chronic HBV infection.
PMCID: PMC3589400  PMID: 23472088
18.  CVD Growth of Large Area Smooth-edged Graphene Nanomesh by Nanosphere Lithography 
Scientific Reports  2013;3:1238.
Current etching routes to process large graphene sheets into nanoscale graphene so as to open up a bandgap tend to produce structures with rough and disordered edges. This leads to detrimental electron scattering and reduces carrier mobility. In this work, we present a novel yet simple direct-growth strategy to yield graphene nanomesh (GNM) on a patterned Cu foil via nanosphere lithography. Raman spectroscopy and TEM characterizations show that the as-grown GNM has significantly smoother edges than post-growth etched GNM. More importantly, the transistors based on as-grown GNM with neck widths of 65-75 nm have a near 3-fold higher mobility than those derived from etched GNM with the similar neck widths.
PMCID: PMC3566595  PMID: 23393620
19.  A Naturally Occurring Mutation Within the Probe-Binding Region Compromises a Molecular-Based West Nile Virus Surveillance Assay for Mosquito Pools (Diptera: Culicidae) 
Journal of medical entomology  2012;49(4):939-941.
A naturally occurring mutation was detected within the probe binding region targeting the envelope gene sequence of West Nile virus used in real-time polymerase chain reaction assays to test mosquito pools and other samples. A single C→T transition 6nt from the 5′ end of the 16mer in the envelope gene probe-binding region at genomic position 1,194 reduced assay sensitivity. The mutation first was detected in 2009 and persisted at a low prevalence into 2011. The mutation caused a 0.4% false negative error rate during 2011. These data emphasized the importance of confirmational testing and redundancy in surveillance systems relying on highly specific nucleic acid detection platforms.
PMCID: PMC3541937  PMID: 22897055
surveillance; mosquito pool; qRT-PCR; probe binding region; mutation
20.  Structural gene (prME) chimeras of St Louis encephalitis virus and West Nile virus exhibit altered in vitro cytopathic and growth phenotypes 
The Journal of General Virology  2012;93(Pt 1):39-49.
Despite utilizing the same avian hosts and mosquito vectors, St Louis encephalitis virus (SLEV) and West Nile virus (WNV) display dissimilar vector-infectivity and vertebrate-pathogenic phenotypes. SLEV exhibits a low oral infection threshold for Culex mosquito vectors and is avirulent in avian hosts, producing low-magnitude viraemias. In contrast, WNV is less orally infective to mosquitoes and elicits high-magnitude viraemias in a wide range of avian species. In order to identify the genetic determinants of these different phenotypes and to assess the utility of mosquito and vertebrate cell lines for recapitulating in vivo differences observed between these viruses, reciprocal WNV and SLEV pre-membrane and envelope protein (prME) chimeric viruses were generated and growth of these mutant viruses was characterized in mammalian (Vero), avian (duck) and mosquito [Aedes (C6/36) and Culex (CT)] cells. In both vertebrate lines, WNV grew to 100-fold higher titres than SLEV, and growth and cytopathogenicity phenotypes, determined by chimeric phenotypes, were modulated by genetic elements outside the prME gene region. Both chimeras exhibited distinctive growth patterns from those of SLEV in C6/36 cells, indicating the role of both structural and non-structural gene regions for growth in this cell line. In contrast, growth of chimeric viruses was indistinguishable from that of virus containing homologous prME genes in CT cells, indicating that structural genetic elements could specifically dictate growth differences of these viruses in relevant vectors. These data provide genetic insight into divergent enzootic maintenance strategies that could also be useful for the assessment of emergence mechanisms of closely related flaviviruses.
PMCID: PMC3352334  PMID: 21940408
21.  Envelope and pre-membrane protein structural amino acid mutations mediate diminished avian growth and virulence of a Mexican West Nile virus isolate 
The Journal of General Virology  2011;92(Pt 12):2810-2820.
The hallmark attribute of North American West Nile virus (WNV) strains has been high pathogenicity in certain bird species. Surprisingly, this avian virulent WNV phenotype has not been observed during its geographical expansion into the Caribbean, Central America and South America. One WNV variant (TM171-03-pp1) isolated in Mexico has demonstrated an attenuated phenotype in two widely distributed North American bird species, American crows (AMCRs) and house sparrows (HOSPs). In order to identify genetic determinants associated with attenuated avian replication of the TM171-03-pp1 variant, chimeric viruses between the NY99 and Mexican strains were generated, and their replicative capacity was assessed in cell culture and in AMCR, HOSP and house finch avian hosts. The results demonstrated that mutations in both the pre-membrane (prM-I141T) and envelope (E-S156P) genes mediated the attenuation phenotype of the WNV TM171-03-pp1 variant in a chicken macrophage cell line and in all three avian species assayed. Inclusion of the prM-I141T and E-S156P TM171-03-pp1 mutations in the NY99 backbone was necessary to achieve the avian attenuation level of the Mexican virus. Furthermore, reciprocal incorporation of both prM-T141I and E-P156S substitutions into the Mexican virus genome was necessary to generate a virus that exhibited avian virulence equivalent to the NY99 virus. These structural changes may indicate the presence of new evolutionary pressures exerted on WNV populations circulating in Latin America or may signify a genetic bottleneck that has constrained their epiornitic potential in alternative geographical locations.
PMCID: PMC3352571  PMID: 21865445
22.  Long-Term Nitrogen Addition Leads to Loss of Species Richness Due to Litter Accumulation and Soil Acidification in a Temperate Steppe 
PLoS ONE  2012;7(10):e47369.
Although community structure and species richness are known to respond to nitrogen fertilization dramatically, little is known about the mechanisms underlying specific species replacement and richness loss. In an experiment in semiarid temperate steppe of China, manipulative N addition with five treatments was conducted to evaluate the effect of N addition on the community structure and species richness.
Methodology/Principal Findings
Species richness and biomass of community in each plot were investigated in a randomly selected quadrat. Root element, available and total phosphorus (AP, TP) in rhizospheric soil, and soil moisture, pH, AP, TP and inorganic N in the soil were measured. The relationship between species richness and the measured factors was analyzed using bivariate correlations and stepwise multiple linear regressions. The two dominant species, a shrub Artemisia frigida and a grass Stipa krylovii, responded differently to N addition such that the former was gradually replaced by the latter. S. krylovii and A. frigida had highly-branched fibrous and un-branched tap root systems, respectively. S. krylovii had higher height than A. frigida in both control and N added plots. These differences may contribute to the observed species replacement. In addition, the analysis on root element and AP contents in rhizospheric soil suggests that different calcium acquisition strategies, and phosphorus and sodium responses of the two species may account for the replacement. Species richness was significantly reduced along the five N addition levels. Our results revealed a significant relationship between species richness and soil pH, litter amount, soil moisture, AP concentration and inorganic N concentration.
Our results indicate that litter accumulation and soil acidification accounted for 52.3% and 43.3% of the variation in species richness, respectively. These findings would advance our knowledge on the changes in species richness in semiarid temperate steppe of northern China under N deposition scenario.
PMCID: PMC3470592  PMID: 23077603
23.  Reduced Avian Virulence and Viremia of West Nile Virus Isolates from Mexico and Texas 
A West Nile virus (WNV) isolate from Mexico (TM171-03) and BIRD1153, a unique genotype from Texas, have exhibited reduced murine neuroinvasive phenotypes. To determine if murine neuroinvasive capacity equates to avian virulence potential, American crow (Corvus brachyrhynchos) and house sparrows (Passer domesticus) were experimentally inoculated with representative murine neuroinvasive/non-neuroinvasive strains. In both avian species, a plaque variant from Mexico that was E-glycosylation competent produced higher viremias than an E-glycosylation–incompetent variant, indicating the potential importance of E-glycosylation for avian replication. The murine non-neuroinvasive BIRD1153 strain was significantly attenuated in American crows but not house sparrows when compared with the murine neuroinvasive Texas strain. Despite the loss of murine neuroinvasive properties of nonglycosylated variants from Mexico, our data indicate avian replication potential of these strains and that unique WNV virulence characteristics exist between murine and avian models. The implications of reduced avian replication of variants from Mexico for restricted WNV transmission in Latin America is discussed.
PMCID: PMC3183789  PMID: 21976584
24.  Nonstructural Protein 2 of Porcine Reproductive and Respiratory Syndrome Virus Inhibits the Antiviral Function of Interferon-Stimulated Gene 15 
Journal of Virology  2012;86(7):3839-3850.
Type I interferon (alpha/beta interferon [IFN-α/β]) stimulates the expression of interferon-stimulated gene 15 (ISG15), which encodes a ubiquitin-like protein, ISG15. Free ISG15 and ISG15 conjugates function in diverse cellular pathways, particularly regulation of antiviral innate immune responses. In this study, we demonstrate that ISG15 overexpression inhibits porcine reproductive and respiratory syndrome virus (PRRSV) replication in cell culture and that the antiviral activity of interferon is reduced by inhibition of ISG15 conjugation. PRRSV nonstructural protein 2 (nsp2) was previously identified as a potential antagonist of ISG15 production and conjugation. The protein contains a papain-like protease domain (PLP2) that plays a crucial role in the proteolytic cleavage of the PRRSV replicase polyproteins. PLP2 was also proposed to belong to the ovarian tumor domain-containing superfamily of deubiquitinating enzymes (DUBs), which is capable of inhibiting ISG15 production and counteracting ISG15 conjugation to cellular proteins. To determine whether this immune antagonist function could be selectively inactivated, we engineered a panel of mutants with deletions and/or mutations at the N-terminal border of the nsp2 PLP2-DUB domain. A 23-amino-acid deletion (amino acids 402 to 424 of the ORF1a-encoded protein) largely abolished the inhibitory effect of nsp2 on ISG15 production and conjugation, but no viable recombinant virus was recovered. A 19-amino-acid deletion (amino acids 402 to 420), in combination with a downstream point mutation (S465A), partially relieved the ISG15 antagonist function and yielded a viable recombinant virus. Taken together, our data demonstrate that ISG15 and ISGylation play an important role in the response to PRRSV infection and that nsp2 is a key factor in counteracting the antiviral function of ISG15.
PMCID: PMC3302520  PMID: 22258253
25.  Local electrical potential detection of DNA by nanowire-nanopore sensors 
Nature Nanotechnology  2011;7(2):119-125.
Nanopores could potentially be used to perform single molecule DNA sequencing at low cost and with high throughput1–4. Although single-base resolution and differentiation have been demonstrated with nanopores using ionic current measurements5–7, direct sequencing has not been achieved due to difficulties in recording very small (~pA) ionic current at a bandwidth consistent with fast translocation speeds1–3. Here we show that solid-state nanopores can be combined with silicon nanowire field-effect transistors (FETs) to create sensors in which detection is localised and self-aligned at the nanopore. Well-defined FET signals associated with DNA translocation are recorded when an ionic strength gradient is imposed across the nanopores. Measurements and modelling show that FET signals are generated by highly-localized changes in the electrical potential during DNA translocation and that the nanowire-nanopore sensors could enable large-scale integration with a high intrinsic bandwidth.
PMCID: PMC3273648  PMID: 22157724

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