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1.  Purification and characterization of a novel defensin from the salivary glands of the black fly, Simulium bannaense 
Parasites & Vectors  2015;8:71.
Background
Black flies (Diptera: Simuliidae) are haematophagous insects that can cause allergic reactions and act as vectors of pathogens. Although their saliva has been thought to contain a diverse array of physiologically active molecules, little information is available on antimicrobial factors in black fly salivary glands, especially no defensins have been reported so far.
Methods
A novel cationic defensin designated SibaDef was purified using reverse phase high-performance liquid chromatography (RP-HPLC) from the salivary glands of the black fly Simulium bannaense. The amino acid sequence of SibaDef was determined by a combination method of automated Edman degradation and cDNA sequencing. The morphologic changes of Gram-positive bacteria Staphylococcus aureus or Bacillus subtilis treated with SibaDef were assessed by scanning electron microscopy (SEM). Quantitative PCR (qPCR) was performed to analyze the expression of SibaDef mRNA in whole bodies of insects after oral infection with the bacteria S. aureus or B. subtilis.
Results
Surprisingly, the phylogenetic analysis of defensin-related amino acid sequences demonstrated that SibaDef is most closely related to defensins from the human body louse Pediculus humanus corporis (Anoplura: Pediculidae), rather than to other dipteran defensins. SibaDef showed potent antimicrobial activities against Gram-positive bacteria with minimal inhibitory concentrations (MICs) ranging from 0.83 μM to 2.29 μM. SEM analysis indicated that SibaDef killed microorganisms through the disruption of cell membrane integrity. The transcript levels of SibaDef in the bacteria-immunized flies increased with the time course, reaching maximum at 36 h and then slowly decreased from that time point.
Conclusions
Our results indicate that SibaDef is involved in the innate humoral response of the black fly S. bannaense, and it might play a significant role in the defence against microorganisms in both sugar and blood meals.
doi:10.1186/s13071-015-0669-9
PMCID: PMC4324660  PMID: 25649358
Insect; Antimicrobial peptide; Defensin; Salivary gland; Black fly; Simulium bannaense
2.  Effects of Number of Animals Monitored on Representations of Cattle Group Movement Characteristics and Spatial Occupancy 
PLoS ONE  2015;10(2):e0113117.
The number of animals required to represent the collective characteristics of a group remains a concern in animal movement monitoring with GPS. Monitoring a subset of animals from a group instead of all animals can reduce costs and labor; however, incomplete data may cause information losses and inaccuracy in subsequent data analyses. In cattle studies, little work has been conducted to determine the number of cattle within a group needed to be instrumented considering subsequent analyses. Two different groups of cattle (a mixed group of 24 beef cows and heifers, and another group of 8 beef cows) were monitored with GPS collars at 4 min intervals on intensively managed pastures and corn residue fields in 2011. The effects of subset group size on cattle movement characterization and spatial occupancy analysis were evaluated by comparing the results between subset groups and the entire group for a variety of summarization parameters. As expected, more animals yield better results for all parameters. Results show the average group travel speed and daily travel distances are overestimated as subset group size decreases, while the average group radius is underestimated. Accuracy of group centroid locations and group radii are improved linearly as subset group size increases. A kernel density estimation was performed to quantify the spatial occupancy by cattle via GPS location data. Results show animals among the group had high similarity of spatial occupancy. Decisions regarding choosing an appropriate subset group size for monitoring depend on the specific use of data for subsequent analysis: a small subset group may be adequate for identifying areas visited by cattle; larger subset group size (e.g. subset group containing more than 75% of animals) is recommended to achieve better accuracy of group movement characteristics and spatial occupancy for the use of correlating cattle locations with other environmental factors.
doi:10.1371/journal.pone.0113117
PMCID: PMC4315582  PMID: 25647571
3.  A Dynamic Integrated Fault Diagnosis Method for Power Transformers 
The Scientific World Journal  2015;2015:459268.
In order to diagnose transformer fault efficiently and accurately, a dynamic integrated fault diagnosis method based on Bayesian network is proposed in this paper. First, an integrated fault diagnosis model is established based on the causal relationship among abnormal working conditions, failure modes, and failure symptoms of transformers, aimed at obtaining the most possible failure mode. And then considering the evidence input into the diagnosis model is gradually acquired and the fault diagnosis process in reality is multistep, a dynamic fault diagnosis mechanism is proposed based on the integrated fault diagnosis model. Different from the existing one-step diagnosis mechanism, it includes a multistep evidence-selection process, which gives the most effective diagnostic test to be performed in next step. Therefore, it can reduce unnecessary diagnostic tests and improve the accuracy and efficiency of diagnosis. Finally, the dynamic integrated fault diagnosis method is applied to actual cases, and the validity of this method is verified.
doi:10.1155/2015/459268
PMCID: PMC4320846
4.  Combination therapy of ursodeoxycholic acid and budesonide for PBC–AIH overlap syndrome: a meta-analysis 
In this study, a meta-analysis of randomized controlled trials comparing ursodeoxycholic acid (UDCA) monotherapy with combination therapies utilizing UDCA and budesonide was performed. We found that combination therapy with UDCA and budesonide was more effective than UDCA monotherapy for primary biliary cirrhosis–autoimmune hepatitis overlap syndrome. Moreover, compared to prednisone, budesonide has fewer side effects.
doi:10.2147/DDDT.S74515
PMCID: PMC4304592  PMID: 25632224
meta-analysis; UDCA; budesonide; combination therapy; PBC–AIH overlap syndrome
5.  Bacteria can mobilize nematode-trapping fungi to kill nematodes 
Nature Communications  2014;5:5776.
In their natural habitat, bacteria are consumed by bacterivorous nematodes; however, they are not simply passive preys. Here we report a defensive mechanism used by certain bacteria to mobilize nematode-trapping fungi to kill nematodes. These bacteria release urea, which triggers a lifestyle switch in the fungus Arthrobotrys oligospora from saprophytic to nematode–predatory form; this predacious form is characterized by formation of specialized cellular structures or ‘traps’. The bacteria significantly promote the elimination of nematodes by A. oligospora. Disruption of genes involved in urea transport and metabolism in A. oligospora abolishes the urea-induced trap formation. Furthermore, the urea metabolite ammonia functions as a signal molecule in the fungus to initiate the lifestyle switch to form trap structures. Our findings highlight the importance of multiple predator–prey interactions in prey defense mechanisms.
Certain soil fungi form specialized cellular structures or 'traps' to feed on nematodes, which in turn eat bacteria. Here, the authors show that urea released from bacteria induces trap formation in the fungi and this promotes nematode elimination.
doi:10.1038/ncomms6776
PMCID: PMC4275587  PMID: 25514608
6.  New insights into the mechanisms of itch: are pain and itch controlled by distinct mechanisms? 
Pflugers Archiv : European journal of physiology  2013;465(12):10.1007/s00424-013-1284-2.
Itch and pain are closely related but distinct sensations. They share largely overlapping mediators and receptors, and itch-responding neurons are also sensitive to pain stimuli. Itch-mediating primary sensory neurons are equipped with distinct receptors and ion channels for itch transduction, including Mas-related G protein-coupled receptors (Mrgprs), protease-activated receptors (PARs), histamine receptors, bile acid receptor (TGR5), toll-like receptors (TLRs), and transient receptor potential subfamily V1/A1 (TRPV1/A1). Recent progress has indicated the existence of an itch-specific neuronal circuitry. The MrgprA3-expressing primary sensory neurons exclusively innervate the epidermis of skin and their central axons connect with gastrin-releasing peptide receptor (GRPR)-expressing neurons in the superficial spinal cord. Notably, ablation of MrgprA3-expressing primary sensory neurons or GRPR-expressing spinal cord neurons results in selective reduction in itch but not pain. Chronic itch results from dysfunction of the immune and nervous system and can manifest as neural plasticity, despite the fact that chronic itch is often treated by dermatologists. While differences between acute pain and acute itch are striking, chronic itch and chronic pain share many similar mechanisms, including peripheral sensitization (increased responses of primary sensory neurons to itch and pain mediators), central sensitization (hyperactivity of spinal projection neurons and excitatory interneurons), loss of inhibitory control in the spinal cord, and neuro-immune and neuro-glial interactions. Notably, painful stimuli can elicit itch in some chronic conditions (e.g., atopic dermatitis) and some drugs for treating chronic pain are also effective in chronic itch. Thus, itch and pain have more similarities in pathological and chronic conditions.
doi:10.1007/s00424-013-1284-2
PMCID: PMC3796138  PMID: 23636773
Central sensitization; neuro-immune interaction; nociceptor; peripheral sensitization; pruritus; pruriceptor
7.  A Novel Contrast-Induced Acute Kidney Injury Model Based on the 5/6-Nephrectomy Rat and Nephrotoxicological Evaluation of Iohexol and Iodixanol In Vivo 
Contrast-induced acute kidney injury (CI-AKI) is a serious complication in patients after administration of iodinated contrast media. Proper animal models of CI-AKI can help understand the mechanisms involved and prevent the disorder. We used the 5/6-nephrectomized (NE) rat to develop a CI-AKI model and to evaluate differences in the toxic effects on the kidney between iohexol and iodixanol. We found that six weeks after ablative surgery was the preferred time to induce CI-AKI. We compared multiple pretreatment plans and found that dehydration for 48 hours before iodixanol (320, 10 mL/kg) administration was optimal to induce CI-AKI in the 5/6 NE rats. Compared with iodixanol, iohexol induced a significantly greater reduction in renal function, severe renal tissue damage, intrarenal hypoxia, and apoptotic tubular cells. Iohexol and iodixanol resulted in similarly marked increases in levels of inflammation and oxidative stress. In summary, the 5/6 NE rat combined with dehydration for 48 hours is a useful pretreatment to establish a novel and reliable CI-AKI model. Iohexol induced more severe CI-AKI than iodixanol in this model.
doi:10.1155/2014/427560
PMCID: PMC4244948  PMID: 25478060
8.  Cryptococcus inositol utilization modulates the host protective immune response during brain infection 
Background
Cryptococcus neoformans is the most common cause of fungal meningitis among individuals with HIV/AIDS, which is uniformly fatal without proper treatment. The underlying mechanism of disease development in the brain that leads to cryptococcal meningoencephalitis remains incompletely understood. We have previously demonstrated that inositol transporters (ITR) are required for Cryptococcus virulence. The itr1aΔ itr3cΔ double mutant of C. neoformans was attenuated for virulence in a murine model of intra-cerebral infection; demonstrating that Itr1a and Itr3c are required for full virulence during brain infection, despite a similar growth rate between the mutant and wild type strains in the infected brain.
Results
To understand the immune pathology associated with infection by the itr1aΔ itr3cΔ double mutant, we investigated the molecular correlates of host immune response during mouse brain infection. We used genome-wide transcriptome shotgun sequencing (RNA-Seq) and quantitative real-time PCR (qRT-PCR) methods to examine the host gene expression profile in the infected brain. Our results show that compared to the wild type, infection of mouse brains by the mutant leads to significant activation of cellular networks/pathways associated with host protective immunity. Most of the significantly differentially expressed genes (SDEG) are part of immune cell networks such as tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) regulon, indicating that infection by the mutant mounts a stronger host immune response compared to the wild type. Interestingly, a significant reduction in glucuronoxylomannan (GXM) secretion was observed in the itr1aΔ itr3cΔ mutant cells, indicating that inositol utilization pathways play a role in capsule production.
Conclusions
Since capsule has been shown to impact the host response during Cryptococcus-host interactions, our results suggest that the reduced GXM production may contribute to the increased immune activation in the mutant-infected animals.
Electronic supplementary material
The online version of this article (doi:10.1186/s12964-014-0051-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12964-014-0051-0
PMCID: PMC4172957  PMID: 25201772
Cryptococcus neoformans; Cryptococcal meningoencephalitis; Host immune response; Glucuronoxylomannan; Capsule production; Inositol transporters; Genome-wide transcriptome; Quantitative real-time PCR; Cellular networks; Immune pathways
9.  Tripartite Interactions of Barley Yellow Dwarf Virus, Sitobion avenae and Wheat Varieties 
PLoS ONE  2014;9(9):e106639.
The tripartite interactions in a pathosystem involving wheat (Triticum aestivum L.), the Barley yellow dwarf virus (BYDV), and the BYDV vector aphid Sitobion avenae were studied under field conditions to determine the impact of these interactions on aphid populations, virus pathology and grain yield. Wheat varietal resistance to BYDV and aphids varied among the three wheat varieties studied over two consecutive years. The results demonstrated that (1) aphid peak number (APN) in the aphid + BYDV (viruliferous aphid) treatment was greater and occurred earlier than that in the non-viruliferous aphid treatment. The APN and the area under the curve of population dynamics (AUC) on a S. avenae-resistant variety 98-10-30 was significantly lower than on two aphid-susceptible varieties Tam200(13)G and Xiaoyan6. (2) The production of alatae (PA) was greater on the variety 98-10-30 than on the other varieties, and PA was greater in the aphid + BYDV treatment on 98-10-30 than in the non-viruliferous aphid treatment, but this trend was reversed on Tam200(13)G and Xiaoyan6. (3) The BYDV disease incidence (DIC) on the variety 98-10-30 was greater than that on the other two varieties in 2012, and the disease index (DID) on Tam200(13)G was lower than on the other varieties in the aphid + BYDV and BYDV treatments in 2012, but not in 2011 when aphid vector numbers were generally lower. (4) Yield loss in the aphid + BYDV treatment tended to be greater than that in the aphid or BYDV alone treatments across varieties and years. We suggested that aphid population development and BYDV transmission tend to promote each other under field conditions. The aphids + BYDV treatment caused greater yield reductions than non-viruliferous aphids or virus treatment. Wheat varietal resistance in 98-10-30 affects the aphid dispersal, virus transmission and wheat yield loss though inhibits aphid populations from increasing.
doi:10.1371/journal.pone.0106639
PMCID: PMC4153664  PMID: 25184214
10.  Survivin promotion of melanoma metastasis requires upregulation of α5 integrin 
Carcinogenesis  2013;34(9):2137-2144.
Survivin is an apoptotic and mitotic regulator that is overexpressed in melanoma and a poor prognostic marker in patients with metastatic disease. We recently showed that Survivin enhances melanoma cell motility through Akt-dependent upregulation of α5 integrin. However, the functional role of Survivin in melanoma metastasis is not clearly understood. We found that overexpression of Survivin in LOX and YUSAC2 human melanoma cells increased colony formation in soft agar, and this effect was abrogated by knockdown of α5 integrin by RNA interference. We employed melanoma cell xenografts to determine the in vivo effect of Survivin overexpression on melanoma metastasis. Although Survivin overexpression did not affect primary tumor growth of YUSAC2 or LOX subcutaneous tumors, or indices of proliferation or apoptosis, it significantly increased expression of α5 integrin in the primary tumors and formation of metastatic colonies in the lungs. Additionally, Survivin overexpression resulted in enhanced lung colony formation following intravenous (i.v.) injection of tumor cells in vivo and increased adherence to fibronectin-coated plastic in vitro. Importantly, in vivo inhibition of α5 integrin via intraperitoneal injection of an α5β1 integrin-blocking antibody significantly slowed tumor growth and reduced Survivin-enhanced pulmonary metastasis. Knockdown of α5 integrin in cells prior to i.v. injection also blocked Survivin-enhanced lung colony formation. These findings support a direct role for Survivin in melanoma metastasis, which requires α5 integrin and suggest that inhibitors of α5 integrin may be useful in combating this process.
doi:10.1093/carcin/bgt155
PMCID: PMC3765044  PMID: 23640047
11.  The Role of Interleukin-28b Gene Polymorphisms in Chinese Patients With Chronic Hepatitis C Treated With Pegylated Interferon and Ribavirin 
Hepatitis Monthly  2014;14(8):e18793.
Background:
Interleukin-28B (IL28B) single nucleotide polymorphism (SNP) rs8099917 has been described to be associated with response to treatment with pegylated interferon and ribavirin (PEG-IFN/RBV) in patients with chronic hepatitis C from the North America, Europe, Asia countries like Japan and Taiwan. Whether this holds true for Chinese patients remains unknown.
Objectives:
We aimed to study the effects of IL28B rs8099917 on antiviral therapy responses in Chinese patients with hepatitis C.
Patients and Methods:
IL28B rs8099917 was genotyped in 263 patients with hepatitis C virus (HCV) infection and 244 healthy controls in Tianjin, China using TaqMan SNP genotyping method. The roles of rs8099917 and clinical characteristics in antiviral treatment were analyzed by logistic regression.
Results:
Among 263 patients with chronic HCV infection, 223 had a TT genotype (84.8%). Frequencies of TG/GG genotypes in patients with hepatitis C were significantly different from those of healthy controls (15.2% vs. 9.0%; P = 0.033). Patients with HCV infection had a higher G allele frequency than healthy controls (7.8% vs. 4.7%; P = 0.044). Univariate analysis revealed no significant association between rs8099917 and sustained virological response (SVR) (P = 0.612). However, it was found that HCV genotypes 2a/3a, age, prothrombin time (PT), albumin (ALB) and cholesterol (CHO) were associated with SVR. In multivariate analysis, only ALB was significantly an independent predictor of SVR (OR = 1.223; 95%CI: 1.046−1.430; P = 0.011).
Conclusions:
In contrast with T, rs8099917 G is a susceptible allele to HCV in China. ALB can independently predict SVR. Rs8099917 may play a quiet role to predict treatment response of patients with hepatitis C who received PEG−IFN/RBV therapy in China.
doi:10.5812/hepatmon.18793
PMCID: PMC4199147  PMID: 25337144
China; Polymorphism; Hepatitis C; Interleukin-28B; Therapy
12.  Fbp1-Mediated Ubiquitin-Proteasome Pathway Controls Cryptococcus neoformans Virulence by Regulating Fungal Intracellular Growth in Macrophages 
Infection and Immunity  2014;82(2):557-568.
Cryptococcus neoformans is a human fungal pathogen that often causes lung and brain infections in immunocompromised patients, with a high fatality rate. Our previous results showed that an F-box protein, Fbp1, is essential for Cryptococcus virulence independent of the classical virulence factors, suggesting a novel virulence control mechanism. In this study, we show that Fbp1 is part of the ubiquitin-proteasome system, and we further investigated the mechanism of Fbp1 function during infection. Time course studies revealed that the fbp1Δ mutant causes little damage in the infected lung and that the fungal burden in the lung remains at a low but persistent level throughout infection. The fbp1Δ mutant cannot disseminate to other organs following pulmonary infection in the murine inhalation model of cryptococcosis but still causes brain infection in a murine intravenous injection model, suggesting that the block of dissemination of the fbp1Δ mutant is due to its inability to leave the lung. The fbp1Δ mutant showed a defect in intracellular proliferation after phagocytosis in a Cryptococcus-macrophage interaction assay, which likely contributes to its virulence attenuation. To elucidate the molecular basis of the SCF(Fbp1) E3 ligase function, we analyzed potential Fbp1 substrates based on proteomic approaches combined with phenotypic analysis. One substrate, the inositol phosphosphingolipid-phospholipase C1 (Isc1), is required for fungal survival inside macrophage cells, which is consistent with the role of Fbp1 in regulating Cryptococcus-macrophage interaction and fungal virulence. Our results thus reveal a new determinant of fungal virulence that involves the posttranslational regulation of inositol sphingolipid biosynthesis.
doi:10.1128/IAI.00994-13
PMCID: PMC3911387  PMID: 24478071
13.  Functional Proteomics Approaches for the Identification of Transnitrosylase and Denitrosylase Targets 
Methods (San Diego, Calif.)  2013;62(2):151-160.
Protein S-nitrosylation is a dynamic post-translational modification (PTM) of specific cysteines within a target protein. Both proteins and small molecules are known to regulate the attachment and removal of this PTM, and proteins exhibiting such a function are transnitrosylase or denitrosylase candidates. With the advent of the biotin switch technique coupled to high-throughput proteomics workflows, the identification and quantification of large numbers of S-nitrosylated proteins and peptides is now possible. Proper analysis and interpretation of high throughout and quantitative proteomics data will help identify specific transnitrosylase and denitrosylase target peptide sequences and contribute to an understanding of the function and regulation of specific S-nitrosylation events. Here we describe the application of a quantitative proteomics approach using isotope-coded affinity tags (ICAT) in the biotin switch approach for the identification of transnitrosylation and denitrosylation targets of thioredoxin 1, an enigmatic protein with both reported transnitrosylase and denitrosylase activities.
doi:10.1016/j.ymeth.2013.02.002
PMCID: PMC3726571  PMID: 23428400
Biotin switch; ICAT; proteomics; S-nitrosylation; thioredoxin
14.  Three-dimensional reconstruction method for measuring the knee valgus angle of the femur in northern Chinese adults*  
The purpose of this study was to establish a method for measuring the knee valgus angle from the anatomical and mechanical axes on three-dimensional reconstruction imaging models, and to use this method for estimating an average knee valgus angle value for northern Chinese adults. Computed tomographic angiography data in DICOM format for 128 normal femurs from 64 adult subjects were chosen for analysis. After the femur images were subjected to three-dimensional reconstruction, the deepest point in the intercondylar notch (point A), the midpoint of the medullary cavity 20 cm above the knee-joint line (point B), and the landmark of the femoral head rotation center (point C) were identified on each three-dimensional model. The knee valgus angle was defined as the angle enclosed by the distal femoral anatomical axis (line AB) and the femoral mechanical axis (line AC). The average (mean±SD) of knee valgus angle for the 128 femurs was 6.20°±1.20° (range, 3.05° to 10.64°). Significant positive correlations were found between the knee valgus angles of the right and left sides and between the knee valgus angle and age. During total knee arthroplasty, choosing a valgus cut angle of approximately 6° may achieve a good result in reestablishing the natural mechanical alignment of the lower extremity for patients of northern Chinese ethnicity. Larger valgus cut angles should be chosen for older patients.
doi:10.1631/jzus.B1400019
PMCID: PMC4129092  PMID: 25091990
Knee valgus angle; Three-dimensional reconstruction femoral model; Total knee arthroplasty; Northern Chinese adults; Preoperative design
15.  Holmium Laser Enucleation versus Transurethral Resection in Patients with Benign Prostate Hyperplasia: An Updated Systematic Review with Meta-Analysis and Trial Sequential Analysis 
PLoS ONE  2014;9(7):e101615.
Background
Holmium laser enucleation (HoLEP) in surgical treatment of benign prostate hyperplasia (BPH) potentially offers advantages over transurethral resection of the prostate (TURP).
Methods
Published randomized controlled trials (RCTs) were identified from PubMed, EMBASE, Science Citation Index, and the Cochrane Library up to October 10, 2013 (updated on February 5, 2014). After methodological quality assessment and data extraction, meta-analysis was performed using STATA 12.0 and Trial Sequential Analysis (TSA) 0.9 software.
Results
Fifteen studies including 8 RCTs involving 855 patients met the criteria. The results of meta-analysis showed that: a) efficacy indicators: there was no significant difference in quality of life between the two groups (P>0.05), but compared with the TURP group, Qmax was better at 3 months and 12 months, PVR was less at 6, 12 months, and IPSS was lower at 12 months in the HoLEP, b) safety indicators: compared with the TURP, HoLEP had less blood transfusion (RR 0.17, 95% CI 0.06 to 0.47), but there was no significant difference in early and late postoperative complications (P>0.05), and c) perioperative indicators: HoLEP was associated with longer operation time (WMD 14.19 min, 95% CI 6.30 to 22.08 min), shorter catheterization time (WMD −19.97 h, 95% CI −24.24 to −15.70 h) and hospital stay (WMD −25.25 h, 95% CI −29.81 to −20.68 h).
Conclusions
In conventional meta-analyses, there is no clinically relevant difference in early and late postoperative complications between the two techniques, but HoLEP is preferable due to advantage in the curative effect, less blood transfusion rate, shorter catheterization duration time and hospital stay. However, trial sequential analysis does not allow us to draw any solid conclusion in overall clinical benefit comparison between the two approaches. Further large, well-designed, multicentre/international RCTs with long-term data and the comparison between the two approaches remain open.
doi:10.1371/journal.pone.0101615
PMCID: PMC4086899  PMID: 25003963
16.  Differential Accessibility of a Rotavirus VP6 Epitope in Trimers Comprising Type I, II, or III Channels as Revealed by Binding of a Human Rotavirus VP6-Specific Antibody 
Journal of Virology  2014;88(1):469-476.
Previous human antibody studies have shown that the human VH1–46 antibody variable gene segment encodes much of the naturally occurring human B cell response to rotavirus and is directed to virus protein 6 (VP6). It is currently unknown why some of the VH1-46-encoded human VP6 monoclonal antibodies inhibit viral transcription while others do not. In part, there are affinity differences between antibodies that likely affect inhibitory activity, but we also hypothesize that there are differing modes of binding to VP6 that affect the ability to block the transcriptional pore on double-layered particles. Here, we used a hybrid method approach for antibody epitope mapping, including single-particle cryo-electron microscopy (cryo-EM) and enhanced amide hydrogen-deuterium exchange mass spectrometry (DXMS) to determine the location and mode of binding of a VH1-46-encoded antibody, RV6-25. The structure of the RV6-25 antibody–double-layered particle (DLP) complex indicated a very complex binding pattern that revealed subtle differences in accessibility of the VP6 epitope depending on its position in the type I, II, or III channels. These subtle variations in the presentation or accessibility of the RV VP6 capsid layer led to position-specific differences in occupancy for binding of the RV6-25 antibody. The studies also showed that the location of binding of the noninhibitory antibody RV6-25 on the apical surface of RV VP6 head domain does not obstruct the transcription pore upon antibody binding, in contrast to binding of an inhibitory antibody, RV6-26, deeper in the transcriptional pore.
doi:10.1128/JVI.01665-13
PMCID: PMC3911710  PMID: 24155406
17.  B-Cell Epitopes in GroEL of Francisella tularensis 
PLoS ONE  2014;9(6):e99847.
The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other’s binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange–mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL.
doi:10.1371/journal.pone.0099847
PMCID: PMC4072690  PMID: 24968190
18.  Transcriptional Regulatory Cascades in Runx2-Dependent Bone Development 
The development of the musculoskeletal system is a complex process that involves very precise control of bone formation and growth as well as remodeling during postnatal life. Although the understanding of the transcriptional mechanisms of osteogenesis has increased considerably, the molecular regulatory basis, especially the gene regulatory network of osteogenic differentiation, is still poorly understood. This review provides the reader with an overview of the key transcription factors that govern bone formation, highlighting their function and regulation linked to Runt-related transcription factor 2 (Runx2). Runx2 as the master transcription factor of osteoblast differentiation, Twist, Msh homeobox 2 (Msx2), and promyelocytic leukemia zinc-finger protein (PLZF) acting upstream of Runx2, Osterix (Osx) acting downstream of Runx2, and activating transcription factor 4 (ATF4) and zinc-finger protein 521 (ZFP521) acting as cofactors of Runx2 are discussed, and their relevance for tissue engineering is presented. References are provided for more in-depth personal study.
doi:10.1089/ten.teb.2012.0527
PMCID: PMC3627420  PMID: 23150948
20.  Direct and Indirect Impacts of Infestation of Tomato Plant by Myzus persicae (Hemiptera: Aphididae) on Bemisia tabaci (Hemiptera: Aleyrodidae) 
PLoS ONE  2014;9(4):e94310.
The impacts of infestation by the green peach aphid (Myzus persicae) on sweetpotato whitefly (Bemisia tabaci) settling on tomato were determined in seven separate experiments with whole plants and with detached leaves through manipulation of four factors: durations of aphid infestation, density of aphids, intervals between aphid removal after different durations of infestation and the time of whitefly release, and leaf positions on the plants. The results demonstrated that B. tabaci preferred to settle on the plant leaves that had not been infested by aphids when they had a choice. The plant leaves on which aphids were still present (direct effect) had fewer whiteflies than those previously infested by aphids (indirect effect). The whiteflies were able to settle on the plant which aphids had previously infested, and also could settle on leaves with aphids if no uninfested plants were available. Tests of direct factors revealed that duration of aphid infestation had a stronger effect on whitefly landing preference than aphid density; whitefly preference was the least when 20 aphids fed on the leaves for 72 h. Tests of indirect effects revealed that the major factor that affected whitefly preference for a host plant was the interval between the time of aphid removal after infestation and the time of whitefly release. The importance of the four factors that affected the induced plant defense against whiteflies can be arranged in the following order: time intervals between aphid removal and whitefly release > durations of aphid infestation > density of aphids > leaf positions on the plants. In conclusion, the density of aphid infestation and time for which they were feeding influenced the production of induced compounds by tomatoes, the whitefly responses to the plants, and reduced interspecific competition.
doi:10.1371/journal.pone.0094310
PMCID: PMC3978044  PMID: 24710393
21.  Structural insights into RNA encapsidation and helical assembly of the Toscana virus nucleoprotein 
Nucleic Acids Research  2014;42(9):6025-6037.
Toscana virus is an emerging bunyavirus in Mediterranean Europe where it accounts for 80% of pediatric meningitis cases during the summer. The negative-strand ribonucleic acid (RNA) genome of the virus is wrapped around the virally encoded nucleoprotein N to form the ribonucleoprotein complex (RNP). We determined crystal structures of hexameric N alone (apo) and in complex with a nonameric single-stranded RNA. RNA is sequestered in a sequence-independent fashion in a deep groove inside the hexamer. At the junction between two adjacent copies of Ns, RNA binding induced an inter-subunit rotation, which opened the RNA-binding tunnel and created a new assembly interface at the outside of the hexamer. Based on these findings, we suggest a structural model for how binding of RNA to N promotes the formation of helical RNPs, which are a characteristic hallmark of many negative-strand RNA viruses.
doi:10.1093/nar/gku229
PMCID: PMC4027202  PMID: 24688060
22.  Drosophila Pheromone-Sensing Neurons Expressing the ppk25 Ion Channel Subunit Stimulate Male Courtship and Female Receptivity 
PLoS Genetics  2014;10(3):e1004238.
As in many species, gustatory pheromones regulate the mating behavior of Drosophila. Recently, several ppk genes, encoding ion channel subunits of the DEG/ENaC family, have been implicated in this process, leading to the identification of gustatory neurons that detect specific pheromones. In a subset of taste hairs on the legs of Drosophila, there are two ppk23-expressing, pheromone-sensing neurons with complementary response profiles; one neuron detects female pheromones that stimulate male courtship, the other detects male pheromones that inhibit male-male courtship. In contrast to ppk23, ppk25, is only expressed in a single gustatory neuron per taste hair, and males with impaired ppk25 function court females at reduced rates but do not display abnormal courtship of other males. These findings raised the possibility that ppk25 expression defines a subset of pheromone-sensing neurons. Here we show that ppk25 is expressed and functions in neurons that detect female-specific pheromones and mediates their stimulatory effect on male courtship. Furthermore, the role of ppk25 and ppk25-expressing neurons is not restricted to responses to female-specific pheromones. ppk25 is also required in the same subset of neurons for stimulation of male courtship by young males, males of the Tai2 strain, and by synthetic 7-pentacosene (7-P), a hydrocarbon normally found at low levels in both males and females. Finally, we unexpectedly find that, in females, ppk25 and ppk25-expressing cells regulate receptivity to mating. In the absence of the third antennal segment, which has both olfactory and auditory functions, mutations in ppk25 or silencing of ppk25-expressing neurons block female receptivity to males. Together these results indicate that ppk25 identifies a functionally specialized subset of pheromone-sensing neurons. While ppk25 neurons are required for the responses to multiple pheromones, in both males and females these neurons are specifically involved in stimulating courtship and mating.
Author Summary
Drosophila mating behaviors serve as an attractive model to understand how external sensory cues are detected and used to generate appropriate behavioral responses. Pheromones present on the cuticle of Drosophila have important roles in stimulating male courtship toward females and inhibiting male courtship directed at other males. Recently, stimulatory pheromones emitted by females and inhibitory pheromones emitted by males have been shown to stimulate distinct subsets of gustatory neurons on the legs. We have previously shown that a DEG/ENaC ion channel subunit, ppk25, is involved in male courtship toward females but not in inhibition of male-male courtship. Here we show that ppk25 is specifically expressed and functions in a subset of gustatory neurons that mediate physiological and behavioral responses to female-specific stimulatory pheromones. Furthermore, ppk25 is also required for the function of those neurons to activate male courtship in response to other pheromones that are not female-specific. In addition to their roles in males, we find that ppk25, and the related DEG/ENaC subunits ppk23 and ppk29, also stimulate female mating behavior. In conclusion, these results show that, in both sexes, ppk25 functions in a group of neurons with a specialized role in stimulating mating behaviors.
doi:10.1371/journal.pgen.1004238
PMCID: PMC3967927  PMID: 24675786
23.  Deciphering the Mechanism of β-Aminobutyric Acid-Induced Resistance in Wheat to the Grain Aphid, Sitobion avenae 
PLoS ONE  2014;9(3):e91768.
The non-protein amino acid β-aminobutyric acid (BABA) can induce plant resistance to a broad spectrum of biotic and abiotic stresses. However, BABA-induced plant resistance to insects is less well-studied, especially its underlying mechanism. In this research, we applied BABA to wheat seedlings and tested its effects on Sitobion avenae (F.). When applied as a soil drench, BABA significantly reduced weights of S. avenae, whereas foliar spray and seed treatment had no such effects. BABA-mediated suppression of S. avenae growth was dose dependent and lasted at least for 7 days. The aminobutyric acid concentration in phloem sap of BABA-treated plants was higher and increased with BABA concentrations applied. Moreover, after 10 days of treatment, the aminobutyric acid content in BABA-treated plants was still higher than that in control treatment. Sitobion avenae could not discriminate artificial diet containing BABA from standard diet, indicating that BABA itself is not a deterrent to this aphid. Also S. avenae did not show preference for control plants or BABA-treated plants. Consistent with choice test results, S. avenae had similar feeding activities on control and BABA-treated plants, suggesting that BABA did not induce antifeedants in wheat seedlings. In addition, aminobutyric acid concentration in S. avenae feeding on BABA-treated plants was significantly higher than those feeding on control plants. Sitobion avenae growth rate was reduced on the artificial diet containing BABA, indicating that BABA had direct toxic effects on this aphid species. These results suggest that BABA application reduced S. avenae performance on wheat seedlings and the mechanism is possibly due to direct toxicity of high BABA contents in plant phloem.
doi:10.1371/journal.pone.0091768
PMCID: PMC3961263  PMID: 24651046
24.  The Effects of Needling Fenglong (ST40) and Neiguan (PC6) on IL-17 of ApoE-Gene-Knockout Mice's Liver 
The aim of the present paper was to observe the effects of needling ST40 and PC6 on IL-17 of ApoE−/− mice with fatty liver. Forty male ApoE−/− mice were randomized into Needling-Acupoint Group, Simvastatin Intragastric Administration Group, Needling Nonacupoint Group, and Model Group. Each was fed with high fat diet for 8 weeks since 16 weeks of age; after 8 weeks of intervention, mice were sacrificed and tested for various examinations. Result showed that the body weight, TC, and serum IL-17 in Needling-Acupoint Group decreased. Compared with Model Group, the immunohistochemical expressions of IL-17 in liver tissue were significantly decreased among the other three groups. In conclusion, acupuncture was able to lower the expression of IL-17 level both in serum and liver tissue in ApoE−/− mice, which is helpful to reduce the inflammation and defers the progress from fatty liver to cirrhosis.
doi:10.1155/2014/691863
PMCID: PMC3979069  PMID: 24778705
25.  Extracellular caspase-6 drives murine inflammatory pain via microglial TNF-α secretion 
The Journal of Clinical Investigation  2014;124(3):1173-1186.
Increasing evidence indicates that the pathogenesis of neuropathic pain is mediated through spinal cord microglia activation. The intracellular protease caspase-6 (CASP6) is known to regulate neuronal apoptosis and axonal degeneration; however, the contribution of microglia and CASP6 in modulating synaptic transmission and pain is unclear. Here, we found that CASP6 is expressed specifically in C-fiber axonal terminals in the superficial spinal cord dorsal horn. Animals exposed to intraplantar formalin or bradykinin injection exhibited CASP6 activation in the dorsal horn. Casp6-null mice had normal baseline pain, but impaired inflammatory pain responses. Furthermore, formalin-induced second-phase pain was suppressed by spinal injection of CASP6 inhibitor or CASP6-neutralizing antibody, as well as perisciatic nerve injection of CASP6 siRNA. Recombinant CASP6 (rCASP6) induced marked TNF-α release in microglial cultures, and most microglia within the spinal cord expressed Tnfa. Spinal injection of rCASP6 elicited TNF-α production and microglia-dependent pain hypersensitivity. Evaluation of excitatory postsynaptic currents (EPSCs) revealed that rCASP6 rapidly increased synaptic transmission in spinal cord slices via TNF-α release. Interestingly, the microglial inhibitor minocycline suppressed rCASP6 but not TNF-α–induced synaptic potentiation. Finally, rCASP6-activated microglial culture medium increased EPSCs in spinal cord slices via TNF-α. Together, these data suggest that CASP6 released from axonal terminals regulates microglial TNF-α secretion, synaptic plasticity, and inflammatory pain.
doi:10.1172/JCI72230
PMCID: PMC3934175  PMID: 24531553

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