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Extranodal natural killer/T-cell lymphoma, nasal-type (ENKTL) is a distinct subtype of non-Hodgkin lymphoma and most of the patients presented localized disease. Combined modality therapy (CMT), namely chemotherapy combined with radiotherapy, has been recommended for patients with early-stage ENKTL. However, the optimal CMT has not been fully clarified. This study reports the efficacy and toxicity of sequential P-GEMOX (pegaspargase, gemcitabine and oxaliplatin) and radiotherapy in a large Chinese cohort comprising of 202 patients diagnosed with early-stage ENKTL from six medical centers. The observed best overall response rate was 96.0% and 168 (83.2%) patients achieved complete remission. With a median follow-up of 44.1 months, the 3-year progression-free survival (PFS) and overall survival (OS) were 74.6% and 85.2%, respectively. Multivariate analysis suggested that extensive primary tumor (PFS, hazard ratio [HR] 3.660, 95%CI 1.820-7.359, P

Background Postoperative lung cancer patients belong to the high-risk group for venous thromboembolism (VTE). The standardized preventive measures for perioperative VTE in lung cancer are not perfect, especially for the prevention and treatment of catheter-related thrombosis (CRT) caused by carried central venous catheters (CVCs) in lung cancer surgery. Patients and methods This study included 460 patients with lung cancer undergoing video-assisted thoracic surgery (VATS) in our center from July 2020 to June 2021. Patients were randomized into two groups, and intraoperatively-placed CVCs would be carried to discharge. During hospitalization, the control group was treated with low-molecular-weight heparin (LMWH), and the experimental group with LMWH + intermittent pneumatic compression (IPC). Vascular ultrasound was performed at three time points which included before surgery, before discharge, and one month after discharge. The incidence of VTE between the two groups was studied by the Log-binomial regression model. Results CRT occurred in 71.7% of the experimental group and 79.7% of the control group. The multivariate regression showed that the risk of developing CRT in the experimental group was lower than in the control group (Adjusted RR = 0.889 [95%CI0.799–0.989], p = 0.031), with no heterogeneity in subgroups (P for Interaction > 0.05). Moreover, the fibrinogen of patients in the experimental group was lower than control group at follow-up (P = 0.019). Conclusion IPC reduced the incidence of CRT during hospitalization in lung cancer patients after surgery. Trial registration No. ChiCTR2000034511.

The unfolded protein response (UPR) plays a crucial role in Mycoplasma hyopneumoniae (M. hyopneumoniae) pathogenesis. We previously demonstrated that M. hyopneumoniae interferes with the host UPR to foster bacterial adhesion and infection. However, the underlying molecular mechanism of this UPR modulation is unclear. Here, we report that M. hyopneumoniae membrane protein Mhp271 interacts with host GRP78, a master regulator of UPR localized to the porcine tracheal epithelial cells (PTECs) surface. The interaction of Mhp271 with GRP78 reduces the porcine beta-defensin 2 (PBD-2) production, thereby facilitating M. hyopneumoniae adherence and infection. Furthermore, the R1-2 repeat region of Mhp271 is crucial for GRP78 binding and the regulation of PBD-2 expression. Intriguingly, a coimmunoprecipitation (Co-IP) assay and molecular docking prediction indicated that the ATP, rather than the substrate-binding domain of GRP78, is targeted by Mhp271 R1-2. Overall, our findings identify host GRP78 as a target for M. hyopneumoniae Mhp271 modulating the host UPR to facilitate M. hyopneumoniae adherence and infection.

Britannin is a natural pseudoguaiacane sesquiterpene lactone, which is reported to possess a significant anticancer function. However, its anticancer effects in T-cell lymphoblastic lymphoma (T-LBL) have not been studied. We investigated the molecular mechanisms of britannin's effective anticancer activity in T-LBL cells. We detected the proliferation, apoptosis, glucose consumption, and lactate production in T-LBL cells treated with or without britannin. We applied a mouse xenograft for in vivo study. The results showed that the IC50 for britannin in SUP-T1 and MOLT4 cells were 5.661 and 6.043 μM, respectively. Britannin inhibited the growth of T-LBL cells in vitro and in vivo. Besides this, britannin enhanced LC3 puncta formation, as well as LC3II and beclin1 expression in SUP-T1 and MOLT4 cells, while decreased p62 expression, indicating that britannin promoted the autophagy of T-LBL cells in vitro. Moreover, britannin promoted apoptosis and reduced glycolysis of T-LBL cells, which was reversed by the typical autophagic inhibitor chloroquine. Britannin increased the phosphorylation of AMPK, while decreasing the phosphorylation of mTOR and S6K1 in T-LBL cells. Moreover, the induction of autophagy in T-LBL cells by britannin was restrained by Compound C, the inhibitor of AMPK. Taken together, britannin mediated apoptosis and glycolysis of T-LBL cells in an autophagy-dependent manner, which was achieved by regulating AMPK/mTOR/S6K1 signaling, demonstrating its therapeutic potential against T-LBL.

This study conducted a comprehensive analysis of the clinical significance of N6-methyladenosine (m6A) regulators and their relationship with immune microenvironment characteristics in diffuse large cell lymphoma (DLBCL). Consensus clustering was performed to molecularly discriminate DLBCL subtypesbased on m6A regulators’ expression. Using the Cox and Lasso regression algorithm, survival-associated m6A regulators were identified, and a m6A-based prognostic signature was established. The influence of m6A risk on immune cell infiltration, immune checkpoint genes, cancer immunity cycle, and immunotherapeutic response was evaluated. Potential molecular pathways related to m6A risk were investigated using gene set enrichment analysis. The m6A regulators showed satisfactory performance in distinguishing DLBCL subgroups with distinct clinical traits and outcomes. A six m6A regulator-based prognostic signature was established and validated as an independent predictor, which separated patients into low- and high-risk groups. High-risk m6A indicated worse survival. The B cells naïve, T cells gamma delta, and NK cells resting were the three most affected immune cells by m6A risk. Up-regulated (PDCD1 and KIR3DL1) and down-regulated (TIGIT, IDO1, and BTLA) immune checkpoint genes in the high-risk group were identified. The m6A risk was found to influence several steps in the cancer immunity cycle. Patients with high-risk m6A were more likely to benefit from immunotherapy. Biological function enrichment analysis revealed that high-risk m6A to be tended related to malignant tumor characteristics, while low-risk m6A showed trend to be related to defensive response processes. Collectively, the m6A-based prognostic signature could be a practical prognostic predictor for DLBCL and immune microenvironment characteristics affected by m6A may be part of the mechanism.

Temperature is an important load for ballastless track. However, there is little research on the system dynamic responses when a train travels on a ballastless track under the temperature gradient of ballastless track. Considering the moving train, temperature gradient of slab track, gravity of slab track, and the contact nonlinearity between interfaces of slab track, a dynamic model for a high-speed train runs along the CRTS III slab track on subgrade is developed by a nonlinear coupled way in ANSYS. The system dynamic responses under the temperature gradient of slab track with different amplitudes are theoretically investigated with the model. The results show that: (1) The proportions of the initial force and stress caused by the temperature gradient of slab track are different for different calculation items. The initial fastener tension force and positive slab bending stress have large proportions exceeding 50%. (2) The maximum dynamic responses for slab track are not uniform along the track. The maximum slab bending stress, slab acceleration, concrete base acceleration appear in the slab middle, at the slab end, and at the concrete base end, respectively. (3) The maximum accelerations of track components appear when the fifth or sixth wheel passes the measuring point, and at least two cars should be used. (4) The temperature gradient of slab track has a small influence on the car body acceleration. However, the influences on the slab acceleration, concrete base acceleration, fastener tension force are large, and the influence on the slab bending stress is huge.

With the aim of optimizing the traditional construction process of a fiber-reinforced lattice structure, the present study modified a previously proposed technique called “truss stacking and node gluing.” To explicitly investigate the structural compressive properties (compressive strength and compressive modulus under the flat pressure), the geometrical parameters, material properties, and topological configuration were examined in detail. Additionally, the present study conducted the relevant theoretical analyses to predict the possible destruction modes and compressive properties. All the samples were tested with a universal testing machine at a rate of 2 mm/min using the ASTM-C365 standard. The results showed that compressive properties are positively related to the relative density and negatively related to the aspect ratio. It was also found that the compressive performance for different materials was in the following order (from good to bad): cotton-fiber reinforced epoxy composite (CREC), jute-fiber reinforced epoxy composite (JREC), and nylon-fiber reinforced epoxy composite (NREC). Furthermore, the mixed topological structure performed as well as the square structure, and they both overmatched the diamond structure. Lastly, the accuracy of the theoretical analysis was evaluated by comparing the theoretical values and the experimental values.

The unicost set covering problem and the attribute reduction problem are NP-complete problems. In this paper, the relationship between these two problems are discussed. Based on the transformability between attribute reductions and minimal solutions in unicost set covering models, two methods are provided. One is to induce an information table from a given unicost set covering model. With no doubt, it shows that the unicost set covering problem can be investigated by rough set theory. The other is to induce a unicost set covering model from a given information table. Similarly, it shows that the attribute reduction problem can be studied by set covering theory. As an application of the proposed theoretical results, a rough set heuristic algorithm is presented for the unicost set covering problem.

Foam-filled two-dimensional lattice structures were designed, and their compression performance was studied relative to corresponding structures without the foam. The experimental results showed that the compressive load of foam-filled lattice structures improved greatly compared with foam-unfilled specimens. The specific energy absorption (SEA) of foam-unfilled specimens exceeded that of the corresponding foam-filled lattice structure. The maximum energy absorption efficiency of the foam-unfilled lattice structure exceeded 1.5, while that of the foam-filled lattice structure was less than 1. The theoretically predicted compression performance was close to the experimental results. The wood-based lattice structure exhibited excellent specific strength and stiffness compared with other structures.

To investigate the effects of the core configuration on the out-of-plane compressive behavior of a wood-based 2D straight column lattice truss sandwich structure, structures with different types of core configurations made of oriented particleboard (OSB) and birch (Betula) dowels were manufactured by using a simple slotting and adhesive bonding approach. An out-of-plane compression experiment was conducted to measure the structural load capacity, equivalent compressive strength, and modulus of elasticity (MOE). Numerical and theoretical analyses were conducted to verify the experimental results. The results show that the ultimate strength in the out-of-plane compression of eight different configurations followed a linear relationship with the relative density of the core (the ratio of the section area of the core to the bottom surface). Type IV (four columns with a diameter of 6 mm) and type V (one column with a diameter of 12 mm) with identical density (both 3.14%) showed similar compressive strength. The compressive MOE of type IV and type V differed greatly. The theoretical displacement is less than the actual displacement because the panel is weak. Therefore, 5 mm birch veneers were used to enhance the OSB panel. The compressive MOE of the reinforced structure could be increased by up to 78%.

Background: N6-methyladenosine (m6A) and immune microenvironment infiltration have been widely reported to play important roles in various cancers. However, in diffuse large cell lymphoma (DLBCL), the clinical significance of m6A regulators and their relationship with immune microenvironment infiltration have not been illuminated.Methods: The expression of m6A regulators in DLBCL was investigated using The Cancer Genome Atlas and Genotype-Tissue Expression databases. The capacity of m6A regulators in dividing molecular clusters of DLBCL was determined using Consensus Clustering algorithm and validated via principal component analysis. The clinical traits and prognosis difference in m6A-sorted clusters were revealed. The m6A prognostic signature was established and validated based on Gene Expression Omnibus dataset using univariate Cox and LASSO regression analysis. The immune cell infiltration and the expression of immune checkpoint genes in m6A low/high-risk DLBCL were studied. Gene set enrichment analysis (GSEA) was adopted to unveil the underlying molecular mechanism in m6A low/high-risk DLBCL.Results: Differentially expressed m6A regulators were able to molecularly discriminate DLBCL as two clusters based on consensus clustering and principal component analysis. A six m6A regulators-based risk prediction signature was established and validated as an independent predictor, which separated patients into m6A low- and high-risk groups. High-risk m6A indicates worse survival, for which the predictive AUC at 1-, 2-, and 5-year achieved 0.605, 0.640, and 0.652, respectively. Immune cell infiltration analysis revealed the B cells naïve and T cells gamma delta were the top increased and decreased immune cells in high-risk m6A patients. Up-regulated (PDCD1 and KIR3DL1) and down-regulated (TIGIT, DO1, and BTLA) immune checkpoint genes in high-risk m6A patients were identified. GSEA analysis unveiled high-risk m6A related to tumor proliferation associated process, while low-risk m6A related to defense response associated process. Conclusions: This study provided a comprehensive analysis for the clinical significance of m6A regulators and their association with immune microenvironment infiltration. An m6A regulators-based risk signature may be applied for the risk stratification of DLBCL patients, thus may facilitate the clinical management of DLBCL. Immune microenvironment was found to be closely related to m6A risk, which may be part of the mechanism of m6A regulators in DLBCL.

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.

A mixed two- and three-dimensional model is developed to simulate the dynamic track-tunnel-soil interaction in underground railway with ballastless track taking into account the moving train comprising a number of carriages, the track irregularities, and interaction among various components of the system. The effects of different track irregularities on the environmental vibration generated by underground railway with direct fixation and floating slab tracks are investigated. The results show that random track irregularities have large effects on the environmental vibration compared with perfectly smooth track condition. For direct fixation tracks, the environmental vibration induced by the irregularity of short wavelength is more significant than that induced by the irregularity of medium wavelength. However, for floating slab tracks, the environmental vibration induced by the irregularity of short wavelength is less significant than that induced by the irregularity of medium wavelength. The track irregularity samples measured by track geometry measuring car should therefore be used together with the short-wavelength random track irregularity for accurate prediction of the environmental vibration induced by underground railway with direct fixation track.

Bluetongue virus (BTV) has been found to trigger autophagy to favor its replication, but the underlying mechanisms have not been clarified. Here, we show that cellular energy metabolism is involved in BTV-induced autophagy. Cellular ATP synthesis was impaired by BTV1 infection, causing metabolic stress, which was responsible for activation of autophagy, since the conversion of LC3 and aggregation of GFP-LC3 (autophagy markers) were suppressed when infection-caused energy depletion was reversed via MP (metabolic substrate) treatment. The reduced virus yields with MP further supported this view. Overall, our findings suggest that BTV1-induced disruption of cellular energy metabolism contributes to autophagy, and this provides new insights into BTV-host interactions.

Bluetongue (BT) is an arbovirus-borne disease of ruminants caused by bluetongue virus (BTV) that has the potential to have a serious economic impact. Currently available commercial vaccines include attenuated vaccines and inactivated vaccines, both of which have achieved great success in the prevention and control of BTV. However, these vaccines cannot distinguish between infected animals and immunized animals. To control outbreaks of BTV, the development of labeled vaccines is urgently needed. In this study, we used the plasmid-based reverse genetics system (RGS) of BTV to rescue four recombinant viruses in which HA (influenza hemagglutinin) tags were inserted at different sites of VP2. In vitro, the recombinant tagged viruses exhibited morphologies, plaque, and growth kinetics similar to the parental BTV-16, and expressed both VP2 and HA tag. Subsequently, the selected recombinant tagged viruses were prepared as inactivated vaccines to immunize IFNAR(-/-) mice and sheep, and serological detection results of anti-HA antibody provided discriminative detection. In summary, we used plasmid-based RGS to rescue BTV recombinant viruses with HA tags inserted into VP2, and detected several sites on VP2 that can accommodate HA tags. Some of the recombinant tagged viruses have potential to be developed into distinctive inactivated vaccines.

Bluetongue virus (BTV) is an important pathogen of wild and domestic ruminants. We have previously reported that BTV1 infection induced autophagy for its own benefit, but how this occurs remains unclear. Here, the classical autophagy features including autophagsomes formation, GFP-LC3 dots and LC3-II conversation were shown in BTV1-infected cells, we also found the endoplasmic reticulum (ER) stress was triggered by BTV1 infection, which was demonstrated by the increased transcription level of the ER stress marker GRP78 and the expanded morphology of ER. During ER stress, PERK and eIF2α phosphorylation increased along with BTV1 infection, consistent with the elevated LC3 level, indicating that the PERK pathway of the unfolded protein response (UPR) was activated. In addition, both the blockage of PERK by GSK2656157 or knockdown of eIF2α by siRNA reduced the level of LC3, which suggested that the PERK-eIF2α pathway contributed to autophagy induced by BTV1. Furthermore, inactivation of PERK or silencing of eIF2α both significantly reduced the expression of VP2 protein and the viral yields in the supernatants. In sum, these data suggest that ER stress mediates autophagy via the PERK-eIF2α pathway and contributes to BTV1 replication, thus offering new insight into the molecular mechanisms of the BTV-host interaction.

Epizootic haemorrhagic disease is a non-contagious infectious viral disease of wild and domestic ruminants caused by epizootic hemorrhagic disease virus (EHDV). EHDV belongs to the genus Orbivirus within the family Reoviridae and is transmitted by insects of the genus Culicoides. The impact of epizootic haemorrhagic disease is underscored by its designation as a notifiable disease by the Office International des Epizooties. The EHDV genome consists of 10 linear dsRNA segments (Seg1-Seg10). Until now, no reverse genetics system (RGS) has been developed to generate replication-competent EHDV entirely from cloned cDNA, hampering detailed functional analyses of EHDV biology. Here, we report the generation of viable EHDV entirely from cloned cDNAs. A replication-competent EHDV-2 (Ibaraki BK13 strain) virus incorporating a marker mutation was rescued by transfection of BHK-21 cells with expression plasmids and in vitro synthesized RNA transcripts. Using this RGS, two additional modified EHDV-2 viruses were also generated: one that contained a duplex concatemeric Seg9 gene and another that contained a duplex concatemeric Seg10 gene. The modified EHDV-2 with a duplex Seg9 gene was genetically stable during serial passage in BHK-21 cells. In contrast, the modified EHDV-2 with a duplex Seg10 gene was unstable during serial passage, but displayed enhanced replication kinetics in vitro when compared with the WT virus. This RGS provides a new platform for the investigation of EHDV replication, pathogenesis and novel EHDV vaccines.

Bluetongue virus (BTV) is the etiological agent of bluetongue (BT) disease, a noncontagious insect-transmitted disease of international importance. To date, 26 BTV serotypes have been recognized worldwide. Methods to discriminate BTV serotypes in clinical samples are essential to epidemiological surveillance efforts and BTV vaccination programs. The BTV VP2 major outer capsid protein, encoded by genomic segment 2 (Seg-2), is the most highly variable BTV protein and is the primary determinant of the virus serotype. Here, we report the development of rapid and reliable real-time RT-PCR assays to detect and discriminate 22 BTV serotypes on the basis of VP2-encoding genomic sequences. Serotype-specific primers and probes detected only the targeted BTV serotype and displayed no cross-amplification of off-target BTV serotypes or other closely related Reoviridae and Bunyaviridae family members. The real-time RT-PCR assays developed were highly sensitive, and the majority of serotype-specific reactions could detect template when present at ≥10 copies. These BTV serotype-specific real-time RT-PCR assays represent a rapid, sensitive, and reliable method for the identification, differentiation and quantification of 22 BTV serotypes.

In this paper, we aim to provide a rough set method to deal with a class of set covering problem called the unicost set covering problem, which is a well-known problem in binary optimization. Firstly, by constructing a Multi-Relation Granular Computing (GrC) model of a given unicost set covering problem, the problem can be equivalently converted to the knowledge reduction problem in rough set theory. Thus, various kinds of efficient knowledge reduction methods in rough set theory can be used to solve the unicost set covering problem. Secondly, a commonly used reduction algorithm based on information entropy is proposed to compute a local minimum of the unicost set covering problem. Finally, the feasibility and efficiency of the proposed algorithm is examined by an example.

The Bluetongue virus (BTV) VP7 protein represents an important group-specific antigen that can serve as a basis for diagnostic tests. Here, we report the generation of a novel BTV group-specific monoclonal antibody (Mab; herein named 4H7) that recognizes a conformational epitope in the VP7 protein. We used a phage-displayed peptide screen and site-directed mutagenesis to define the VP7 amino acid residues that most strongly contribute to the conformational epitope recognized by Mab 4H7. Amino acid residues at positions 175, 185, 186, and 278 of the BTV VP7 protein strongly contributed to Mab 4H7 binding. These key amino acid residues are conserved among all BTV serotypes, whereas related Orbiviruses possess at least one amino acid substitution at these positions. We developed a competitive enzyme-linked immunosorbent assay (c-ELISA) using Mab 4H7 and recombinant BTV VP7 protein to detect serum antibodies against this BTV group-specific VP7 epitope. The c-ELISA was used to screen 833 clinical samples collected from animals in three provinces of China. BTV seroprevalence in the three provinces ranged from 25.42 to 47.45 %. This work provides the foundation for a new diagnostic c-ELISA that can be further applied to BTV surveillance activities and informs our understanding of the structure of the BTV VP7 protein.

The NS2 protein of Bluetongue virus (BTV) is an important non-structural protein and plays important roles in viral replication and assembly. In this study, one monoclonal antibody (mAb), 4D4, was raised against BTV8 NS2. Phage display technology was used and identified the consensus binding motif SNYD recognized by mAb 4D4. To define the minimal region required for antibody binding, a panel of synthetic peptides encompassing SNYD derived from the BTV8 NS2 was then used to more specifically define the 4D4 epitope as 149RSNYDV154. Furthermore, amino acid sequence alignments of different BTV serotypes and other orbiviruses suggested that this epitope is highly conserved among the BTV serotypes. The mAb reagent generated in this study may be applied to the development of BTV diagnosis and surveillance programs and the epitope defined here can lead to important insights into how BTV might interact with the sheep's immune system.

In the background of huge information integration and connection in information science, understanding of the whole world has become one of the most important requirements. In order to gain a comprehensive understanding of individuals, this paper proposed person-oriented modeling methodology to construct personal virtual image which consists of personal comprehensive information, event, task and interactive rules, imposing great significance on subjective initiative and personal practice. Basic Information Units and Standard Tree Structure are employed together to depict individuals from a comprehensive perspective. This paper demonstrates how to establish an appropriate information category in detail on the basis of sociology and philosophy related theories to guarantee the completeness and rationality of the structure of personal virtual image and takes personal credit scoring as an example to prove the feasibility and effectiveness of personal virtual image model.

The stability of recombinant bovine interferon-γ (rbIFN-γ) produced by a baculovirus expression system was investigated under different storage conditions: freezing-thawing and storage for 30 days at temperatures of −80, 4, 25, and 37°C. Antiviral activity was not significantly decreased by freeze-thawing at least five times. Furthermore, although not statistically different, antiviral activity gradually decreased as temperature increased. These findings suggest that rbIFN-γ possesses high thermal and freeze-thaw stability.

A nationwide investigation into polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs) in market seafood was conducted for the first time in this study. Total PCDD/F concentrations in fatty fish ranged from 0.13 to 8.64 pg g −1 wet weight (mean 2.05 pg g −1 wet weight), total PCB concentrations ranged from 38.9 to 3514 pg g −1 wet weight (mean 1133 pg g −1 wet weight), and total PBDE concentrations ranged from 42.8 to 913 pg g −1 wet weight (mean 322 pg g −1 wet weight). Corresponding mean toxicity equivalent (TEQ) values for total PCDD/F and dioxin-like PCB were 0.25 pg g −1 wet weight (WHO 98-TEQ) and 0.32 pg g −1 wet weight (WHO 98-TEQ), respectively. OCDD, PCB-138 and PBDE-47 were the dominant compounds according to their respective congeners. WHO 98-TEQ PCDD/PCDF/PCB for fatty fish and shell fish were 0.60 and 0.070 pg g −1 wet weight, respectively, lower than the standard set by the European Commission. The contamination levels and profiles were compared with those documented in previous publications.

Bluetongue virus (BTV) is the causative agent of bluetongue (BT), an important sheep disease that caused great economic loss to the sheep industry. There are 26 BTV serotypes based on the outer protein VP2. However, the serotypes BTV-1 and BTV-16 are the two most prevalent serotypes in China. Vaccination is the most effective method of preventing viral infections. Therefore, the need for an effective vaccine against BTV is urgent. In this study, DNA vaccines and recombinant fowlpox virus (rFPV) vaccines expressing VP2 alone or VP2 in combination with VP5 or co-expressing the VP2 and VP5 proteins of BTV-1 were evaluated in both mice and sheep. Several strategies were tested in mice, including DNA vaccine prime and boost, rFPV vaccine prime and boost, and DNA vaccine prime and rFPV vaccine boost. We then determined the best vaccine strategy in sheep. Our results indicated that a strategy combining a DNA vaccine prime (co-expressing VP2 and VP5) followed by an rFPV vaccine boost (co-expressing VP2 and VP5) induced a high titer of neutralizing antibodies in sheep. Therefore, our data suggest that a DNA vaccine consisting of a pCAG-(VP2+VP5) prime and an rFPV-(VP2+VP5) boost is an important candidate for the design of a novel vaccine against BTV-1.

Many approaches for generating large quantities of recombinant protein in Escherichia coli fuse the protein of interest to a protein tag to enhance solubility and improve recovery. However, the fusion tags can confound downstream applications, as the fusion partner can alter the structure and biological activity of the recombinant protein and proteolytic removal of the fusion tags can be expensive. Here we describe a new system for production of native proteins in E. coli that allows for removal of the fusion tag via intracellular self-cleavage by the human rhinovirus 3C (HRV3C) protease. This system allows for parallel cloning of target protein coding sequences into six different expression vectors, each with a different fusion partner tag to enhance solubility during induction. Temperature-regulated expression of the HRV3C protease allows for intracellular removal of the fusion tag following induction, and the liberated recombinant protein can be purified by affinity chromatography by virtue of a short six-histidine tag. This system will be an attractive approach for the expression and purification of recombinant proteins free of solubility-enhancing fusion tags, and should be amenable to high-throughput applications.

The fan-chirp transform is a transform method that matches the characteristics of the speech signal. We use the curve fitting tool to study the probability distribution of speech spectra obtained by the fan-chirp transform in order to apply the results to the statistical model-based speech processing. The experimental results demonstrate that the clean speech spectra are best described with Gamma distribution for the real part, imaginary part, and amplitude. For the white noise, the real part and imaginary part of speech spectra are best described with the Laplacian model while the amplitude is best modeled with the Gamma distribution. In other noisy cases, the real part, imaginary part, and amplitude of spectra are all best described with Gamma distribution. The phase spectrum is a nonuniform distribution for the clean speech while it is uniform for the noisy speech.

VP5, the outer capsid protein of bluetongue virus (BTV), plays an important role in viral penetration and antibody-mediated viral neutralization. Therefore, VP5 represents an important target for development of vaccines and diagnostic tests. In this study, we use bioinformatic tools to predict nine antigenic B cell epitopes in the VP5 protein of a BTV serotype 4 (BTV4) isolate from China. Further, we generate five BTV4 VP5-specific monoclonal antibodies (MAbs) and define their corresponding epitopes using a set of VP5-derived peptides expressed as maltose-binding protein (MBP) fusion proteins. The five identified epitopes map to amino acids 119–134, 257–272, 286–301, 322–337, and 481–496 of the VP5 protein. Importantly, the epitopes identified using VP5-derived peptides do not correlate with our bioinformatic prediction of antibody epitopes. Identification and characterization of BTV4 VP5 protein epitopes may aid the development of diagnostic tools and provide information with which to study the structure of the BTV VP5 protein.

The VP2 protein of bluetongue virus (BTV) is an important structural protein and is the principal antigen responsible for BTV serotype specificity. In this study, we mapped the reactivity of two BTV16-specific monoclonal antibodies (MAbs) and identified two novel serotype-specific linear B cell epitopes on the BTV16 VP2 protein. By screening a series of peptides derived from the BTV16 VP2 protein and expressed as mannose-binding protein fusions, we determined that the linear epitopes recognized by the VP2-specific MAbs 3 G10 and 2B4 were located within the peptides 34EWSGHDVTEIPNRRMF49 and 540KNEDPYVKRTVKPIRA555, respectively. To define the minimal region required for antibody binding within these peptide regions, a series of progressively shorter peptides were synthesized and evaluated for 3 G10 and 2B4 binding. This work defined the motifs 34EWSGHDVTEIPNRRMF49 and 543DPYVKRTVK555 as the minimal linear peptides required for 3 G10 and 2B4 binding, respectively. Alignment of amino acid sequences from a number of BTV16 strains isolated from different regions indicated that these two epitopes are highly conserved among BTV16 strains. Furthermore, these two epitopes are not conserved among other BTV serotypes or prototype members of the genus Orbivirus in the family Reoviridae, as shown by sequence alignments. The MAb reagents and linear epitopes defined here provide the basis for the development of epitope-based serotype-specific differential diagnostic tools and may be useful in the design of epitope-based vaccines.

VP7 is a major group-specific protein of the bluetongue virus (BTV), and is therefore a candidate for use as a diagnostic reagent. In this study, BALB/c mice were immunized with BTV16, and the lymphocyte hybridoma technique and indirect ELISA screening method were employed to obtain two strains of hybridoma cells secreting specific monoclonal antibodies (MAbs) to BTV16. Eukaryotic recombinant plasmids coding for 10 segments of BTV16 separately were transfected into BHK-21 cells, respectively, followed by immunofluorescence, showing that two MAbs only reacted with BTV-VP7. Western blot analysis showed the same result. Indirect immunofluorescence results indicated that two of the MAbs present different response spectrums with BTV1~24 serotypes. These results indicate that these MAbs may be good candidates for a specific diagnostic method and functional exploration of the VP7 protein.

Bluetongue virus (BTV) VP5 protein is an important antigenic protein which is centrally involved in serotype determination and the virus entry process. Very little is known about the B-cell epitopes on the BTV VP5 protein recognized by humoral immune responses. In this study, we generated five BTV16 VP5 protein-specific monoclonal antibodies (MAbs), named 3B11, 2B10, 1H7, 4A6 and 3G9, and defined the linear epitopes recognized by MAbs using a series of peptides expressed as maltose-binding protein (MBP)-fusion polypeptides. Three novel linear B-cell epitopes were identified: 3B11 and 3G9 recognized the motif ITANTREIQHIKEE; 2B10 recognized the motif LSGID; and 4A6 recognized the motif STMVKEYRQKIDALKA. Exact sequences corresponding to the three motifs identified were found in the BTV16 VP5 protein ((310)ITANTREIQHIKEE(323), (265)LSGID(269) and (188)STMVKEYRQKIDALKA(203)). These motifs represent the minimal linear peptide sequence required for MAb reactivity, as binding of each MAb was abolished when additional amino acids were removed from the amino and carboxy termini of the peptide. Amino acid sequence alignment indicated that three epitopes were totally conserved among different BTV16 strains. The MAbs generated along with identified epitopes will be useful for examining VP5 protein function and the development of epitope-based marker vaccines against BTV.

The present study identified a linear B-cell epitope in the Eastern equine encephalitis virus (EEEV) E2 glycoprotein by screening a phage-displayed random 12-mer peptide library using an EEEV E2 specific monoclonal antibody (mAb) 7C11 and defined L/F-E/R-Y-T-W-G/R-N-H/W-P as the consensus binding motif. A sequence (321EGLEYTWGNHPP332) encompassing this consensus motif was found in the EEEV E2 glycoprotein and synthesized for further epitope confirmation. Meanwhile, the corresponding epitope peptides in E2 protein of associated alphaviruses were synthesized for specificity identification. Results showed the mAb 7C11 and murine antisera all reacted strongly against the synthesized polypeptide of EEEV antigen complex, but no reaction with Western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) was detected. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against EEEV.

We report here the complete genomic sequence of the Chinese bluetongue virus serotype 1 (BTV1) strain SZ97/1. This work is the first to document the complete genomic sequence of a BTV1 strain from China and represents the second complete sequence of BTV1 in the world. The sequence information provided here will help determine the geographic origin of Chinese BTV1 and provide data to facilitate future analyses of the genetic diversity and phylogenetic relationships of BTV strains.

We report here the complete genomic sequence of the Chinese bluetongue virus serotype 16 (BTV16) strain BN96/16. This work is the first to document the complete genomic sequence (segments 1 to 10) of a BTV16 strain. The sequence information provided herein will help determine the geographic origin of BTV16 and define the phylogenetic relationship of BTV16 to other BTV strains.

The stability of recombinant bovine interferon-γ (rbIFN-γ) produced by a baculovirus expression system was investigated under different storage conditions: freezing-thawing and storage for 30 days at temperatures of -80, 4, 25, and 37°C. Antiviral activity was not significantly decreased by freeze-thawing at least five times. Furthermore, although not statistically different, antiviral activity gradually decreased as temperature increased. These findings suggest that rbIFN-γ possesses high thermal and freeze-thaw stability.

On purpose to develop network-based engineering education, we produce a new ISP (In System Programming) tool that is capable of remotely designing, debugging or upgrading digital systems and products made of PLD by PSTN. The students can do all digital design and research work at home or anywhere as long as they have a telephone at hand. This tool can be used by schools or universities, where the courses of Digital Logic Circuits or Digital System Design are learned.