Differentiation of Foot-and-Mouth Disease-Infected pigs from Vaccinated Pigs Using Antibody-Detecting Sandwich ELISA

The presence of serum antibodies for nonstructural proteins of the foot-and-mouth disease virus (FMDV) can differentiate FMDV-infected animals from vaccinated animals. In this study, a sandwich ELISA was developed for rapid detection of the foot-and-mouth disease (FMD) antibodies; it was based on an Escherichia coli-expressed, highly conserved region of the 3ABC nonstructural protein of the FMDV O/TW/99 strain and a monoclonal antibody derived from the expressed protein. The diagnostic sensitivity of the assay was 98.4%, and the diagnostic specificity was 100% for naïve and vaccinated pigs; the detection ability of the assay was comparable those of the PrioCHECK and UBI kits. There was 97.5, 93.4 and 66.6% agreement between the results obtained from our ELISA and those obtained from the PrioCHECK, UBI and CHEKIT kits, respectively. The kappa statistics were 0.95, 0.87 and 0.37, respectively. Moreover, antibodies for nonstructural proteins of the serotypes A, C, Asia 1, SAT 1, SAT 2 and SAT 3 were also detected in bovine sera. Furthermore, the absence of cross-reactions generated by different antibody titers against the swine vesicular disease virus and vesicular stomatitis virus (VSV) was also highlighted in this assay's specificit

PmoB subunit of particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath): The Cu^I sponge and its function

In this study, we describe efforts to clarify the role of the copper cofactors associated with subunit B (PmoB) of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) (M. capsulatus). This subunit exhibits strong affinity toward Cu^I ions. To elucidate the high copper affinity of the subunit, the full-length PmoB, and the N-terminal truncated mutants PmoB_(33–414) and PmoB_(55–414), each fused to the maltose-binding protein (MBP), are cloned and over-expressed into Escherichia coli (E. coli) K12 TB1 cells. The Y374F, Y374S and M300L mutants of these protein constructs are also studied. When this E. coli is grown with the pmoB gene in 1.0 mM Cu^(II), it behaves like M. capsulatus (Bath) cultured under high copper stresswith abundant membrane accumulation and high CuI content. The recombinantPmoB proteins are verified by Western blotting of antibodies directed against the MBP sub-domain in each of the copper-enriched PmoB proteins. Cu K-edge X-rayabsorption near edge spectroscopy (XANES) of the copper ions confirms that all the PmoB recombinants are Cu^I proteins. All the PmoB proteins show evidence of a “dicopper site” according to analysis of the Cu extended X-ray absorption edge fine structure (EXAFS) of the membranes. No specific activities toward methane and propene oxidation are observed with the recombinant membrane-bound PmoB proteins. However, significant production of hydrogen peroxide is observed in the case of the PmoB_(33–414) mutant. Reaction of the dicopper site with dioxygenproduces hydrogen peroxide and leads to oxidation of the CuI ions residing in the C-terminal sub-domain of the PmoB subunit

PmoB subunit of particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath): The Cu^I sponge and its function

In this study, we describe efforts to clarify the role of the copper cofactors associated with subunit B (PmoB) of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) (M. capsulatus). This subunit exhibits strong affinity toward Cu^I ions. To elucidate the high copper affinity of the subunit, the full-length PmoB, and the N-terminal truncated mutants PmoB_(33–414) and PmoB_(55–414), each fused to the maltose-binding protein (MBP), are cloned and over-expressed into Escherichia coli (E. coli) K12 TB1 cells. The Y374F, Y374S and M300L mutants of these protein constructs are also studied. When this E. coli is grown with the pmoB gene in 1.0 mM Cu^(II), it behaves like M. capsulatus (Bath) cultured under high copper stresswith abundant membrane accumulation and high CuI content. The recombinantPmoB proteins are verified by Western blotting of antibodies directed against the MBP sub-domain in each of the copper-enriched PmoB proteins. Cu K-edge X-rayabsorption near edge spectroscopy (XANES) of the copper ions confirms that all the PmoB recombinants are Cu^I proteins. All the PmoB proteins show evidence of a “dicopper site” according to analysis of the Cu extended X-ray absorption edge fine structure (EXAFS) of the membranes. No specific activities toward methane and propene oxidation are observed with the recombinant membrane-bound PmoB proteins. However, significant production of hydrogen peroxide is observed in the case of the PmoB_(33–414) mutant. Reaction of the dicopper site with dioxygenproduces hydrogen peroxide and leads to oxidation of the CuI ions residing in the C-terminal sub-domain of the PmoB subunit

Shot Noise in Mesoscopic Conductors

Theoretical and experimental work concerned with dynamic fluctuations has developed into a very active and fascinating subfield of mesoscopic physics. We present a review of this development focusing on shot noise in small electric conductors. Shot noise is a consequence of the quantization of charge. It can be used to obtain information on a system which is not available through conductance measurements. In particular, shot noise experiments can determine the charge and statistics of the quasiparticles relevant for transport, and reveal information on the potential profile and internal energy scales of mesoscopic systems. Shot noise is generally more sensitive to the effects of electron-electron interactions than the average conductance. We present a discussion based on the conceptually transparent scattering approach and on the classical Langevin and Boltzmann-Langevin methods; in addition a discussion of results which cannot be obtained by these methods is provided. We conclude the review by pointing out a number of unsolved problems and an outlook on the likely future development of the field.Comment: 99 two-column pages; 38 .eps figures included. Submitted to Physics Reports. Many minor improvements; typos corrected; references added and update

Development of diagnostic reagents for detecting antibodies to structural and non-structural proteins of foot-and-mouth disease virus

口蹄疫 (FMD)、豬水疱病 (SVD)、水疱性口炎 (VS) 及水疱疹 (VE) 皆屬高度傳染性之動物水疱性疾病，這些疾病於臨床上常被疑惑而不易區別診斷。近年來陸續研發建立能檢測口蹄疫抗體檢測方法且能有效區別其他水疱性疾病，包括sandwich ELISA、sigleplex Luminex及multiplex Luminex (xMAP) 等三種方法。使用的試驗重組蛋白產製於原核大腸桿菌 (E.coli.) 表現系統，經純化而高度保留口蹄疫病毒 (FMDV) O/TW/1997 病毒株的VP1結構蛋白及O/TW/1999病毒株的3ABC非結構蛋白等抗原決定位區域，藉此利用特異性的重組蛋白量產製作單株抗體，以間接結合方式分別建立盤式及微珠等複合型界面檢測平台，應用在血清樣品中的FMDV標的抗體如結構蛋白 (SP-VP1) 及非結構蛋白 (NSP-3ABC) 等評估與分析。在第一項研究中，sandwich ELISA經試驗檢測結果於診斷敏感性 (Dsn) 大致可達98.4%，及於健康和免疫豬隻所測試之診斷特異性 (Dsp) 可達100%，從NSP抗體檢測能力顯示相當於prioCHECK和UBI等商品化試劑，以sandwich ELISA進一步分別與prioCHECK、UBI及CHEKIT等商品化試劑比較分析後，發現一致性分別為97.5%、93.4%及66.6%，其中Kappa統計分析值各為0.95、0.87及0.37。且sandwich ELISA除了可檢測台灣O型口蹄疫病毒株之外，也可檢測其他六種血清型如A、C、Asia 1、SAT 1、SAT 2、SAT 3等牛源血清中的NSP抗體。第二項研究以sigleplex Luminex分析檢測64支感染、307支免疫及280支健康等豬隻血清中的FMDV-NSP抗體，於感染樣品之Dsn可高達100%， Dsp之分析結果顯示免疫樣品可達98.7%，及於健康樣品可達97.5-100%。比較sigleplex Luminex 及商品化3ABC polypeptide blocking ELISA二者之一致性為96.3%，且Kappa統計值為0.92。經試驗顯示sigleplex Luminex與sandwich ELISA二者於感染後的豬隻最早可於第八天檢測到NSP-3ABC抗體。然而，以sigleplex Luminex方法檢測15頭經攻毒而仍出現典型水泡病變的免疫豬皆呈現NSP抗體陽轉反應，sandwich ELISA則只有11頭呈現陽轉反應。第三項研究以xMAP Luminex評估單一血清樣品可同時檢測到FMDV SP-VP1及NSP-3ABC等抗體。經試驗結果顯示，於661支來自感染、健康及免疫等豬隻血清樣品，評估FMDV-SP抗體之Dsn約為90.0-98.7%，及Dsp為93.0-96.5%。然而評估FMDV-NSP抗體之Dsn為90.0%，及Dsp為93.3-99.1%。以xMAP檢測SP及NSP抗體之最早出現時期分別在感染後之第4天及第8天，且可自免疫一次的15頭攻毒豬同時檢測出SP及NSP陽性抗體。以xMAP分別與病毒中和試驗 (VNT) 及一商品化3ABC polypeptide blocking ELISA等試驗檢測感染豬血清樣品，經比較後發現於定性檢測SP及NSP抗體有存在顯著的正相關，然於xMAP及VNT之定量比較後發現二者之相關性頗低，若以xMAP與blocking ELISA 檢測FMDV-NSP抗體之特異性分析則介於93.3 -94.9%。這些研究顯示各種方法之敏感性及特異性皆高於 90 % 以上，可作為區別診斷及免疫狀況等評估之參考，且不會與口蹄疫病毒以外的水疱性疾病如豬水疱病病毒及水疱性口炎病毒等所誘發的抗體產生交叉反應，因此即具有高度的試驗特異性。Foot-and-mouth disease (FMD), swine vesicular disease (SVD), vesicular stomatitis (VS), and vesicular exanthema (VE) are highly contagious vesicular diseases of animal but are not able to be differentiated clinically. For the purpose of instant detecting FMD and differentiating it from the other vesicular diseases, many methods have been developed and evaluated in recent years. The structural protein VP1 gene of FMDV O/TW/1997 and the non-structural protein 3ABC gene of FMDV O/TW/1999 were constructed, respectively, into expression vectors, which were based on an Escherichia coli expression system. Subsequently, monoclonal antibodies were generated by immunizing mice with the recombinant proteins, and they were employed to develop the complex interface measurement platforms for FMDV tests. Plate and microsphere formats were developed and evaluated for their abilities in antibody detection from serum samples against structural protein (SP-VP1) and non-structural protein (NSP-3ABC) of the FMDV. In those studies, the sandwich enzyme-linked immunosorbent assay (ELISA), singleplex Luminex and multipex Luminex (xMAP) were developed for rapid detection of the FMD antibodies. In the first study, the developed sandwich ELISA demonstrated a diagnostic sensitivity (Dsn) of 98.4 % and a diagnostic specificity (Dsp) of 100 % for naive and vaccinated pigs; the detection ability of the assay was comparable with those of PrioCHECK and UBI kits. There were 97.5, 93.4 and 66.6 % agreement between the results obtained from our sandwich ELISA and those obtained from the PrioCHECK, UBI and CHEKIT kits, respectively. The kappa statistics between our ELISA and the kits were 0.95, 0.87 and 0.37, respectively. Moreover, antibodies to nonstructural proteins of the serotypes A, C, Asia 1, SAT 1, SAT 2 and SAT 3 were also detected in sera of infected cattle. In the secondary study, sera from 64 infected, 307 vaccinated, and 280 naive pigs were tested for the FMDV-NSP antibody by the sigleplex Luminex assay. The Dsn of the assay was 100%. The Dsp of the assay was 98.7% in vaccinated pigs and 97.5% to 100% in naive pigs. Agreement between the results from the singleplex Luminex and those from a 3ABC polypeptide blocking ELISA was 96.3%, and kappa statistics gave a value of 0.92. The singleplex Luminex can detect the immune response to NSP-3ABC in swine as early as eight days post-infection as same as sandwich ELISA. Moreover, the NSP-3ABC antibody in all of the 15 vaccinated but unprotected pigs which presented vesicular lesions were detected by the singleplex Luminex assay, and the antibodies in 11 of the 15 pigs were detected this antibody by the sandwich ELISA. In the third study, an xMAP was optimized to detected antibodies to SP-VP1 and NSP-3ABC of the FMD virus simultaneously in a single serum sample. To detect SP antibodies in 661 sera from infected, naive pigs and vaccinated pigs, the DSn and DSp of the xMAP were 90.0-98.7% and 93.0-96.5%, respectively. To detect NSP antibodies, the DSn was 90% and the DSp ranged from 93.3% to 99.1%. The xMAP can detect the immune response to SP and NSP as early as 4 and 8 dpi, respectively, in the experimentally infected pigs Moreover, the SP and NSP antibodies in 15 vaccinated but unprotected pigs were detected by the xMAP. Comparing the abilities in detecting the SP and NSP antibodies in the sera of infected samples, the results from the xMAP had high positive correlation with those from the virus neutralization test (VNT) and commercially available 3ABC polypeptide blocking ELISA. However, the results of xMAP had no quantitative relationship with those of the VNT. Furthermore, the specificity was 93.3-94.9% with the blocking ELISA for detecting the FMDV-NSP antibody. These studies showed that the sensitivity and specificity of the methods developed are higher than 90%, which can be as references for diagnosis and assessment of the immune status. Furthermore, the specificities of these assays were also highlighted by the absence of cross-reactions generated by antibodies against the SVDV and VSV at different titers.Certificate Acknowledgements Contents I Abstract in Chinese VII Abstract in English IX Chapter 1. General Introduction 1 Chapter 2. Literature review 7 2.1 Foot-mouth-disease (FMD) 7 2.1.1 History 7 2.1.2 Susceptible species 7 2.1.3 Pathogenesis 7 2.1.4 Infection routes 8 2.1.5 Transmission 9 2.1.6 Clinical signs 10 2.1.7 Subclinical and persistent infections 11 2.2 Foot-mouth-disease virus (FMDV) 12 2.2.1 Genome 12 2.2.2 Structure of viral particle 13 2.3 Characteristics and functions of viral proteins 14 2.3.1 Structural protein VP1 (1D) 14 2.3.2 Structural protein VP2 (1B) 14 2.3.3 Structural protein VP3 (1C) 15 2.3.4 Structural protein VP4 (1A) 15 2.3.5 Non-structural protein L 16 2.3.6 Non-structural protein 2A 17 2.3.7 Non-structural protein 2B 17 2.3.8 Non-structural protein 2C 17 2.3.9 Non-structural protein 3A 18 2.3.10 Non-structural protein 3B 18 2.3.11 Non-structural protein 3C 19 2.3.12 Non-structural protein 3D 20 2.4 Replication of FMDV in host cell 20 2.4.1 Virus entry 20 2.4.2 Proliferation of viral RNA and assembly of progeny virus 21 2.4.3 Virus release 21 2.5 Serotype-specific of epidemiological characteristics 21 2.5.1 Serotype O 22 2.5.2 Serotype A 22 2.5.3 Serotype C 22 2.5.4 Serotype Asia 1 23 2.5.5 Serotype SAT 1, 2, 3 23 2.6 Laboratory diagnosis 23 2.6.1 Virus neutralization test (VNT) 23 2.6.2 Virus isolation 24 2.6.3 Reverse transcription-polymerase chain reaction (RT-PCR) 25 2.6.4 Reverse transcription loop-mediated isothermal amplification (RT-LAMP) 25 2.6.5 Enzyme linked immunosorbent assay (ELISA) 26 2.6.6 Differentiation between infection and vaccinated animals (DIVA) 26 2.6.7 Chromatographic strip test 27 2.6.8 Luminex assay 28 Chapter 3. Differentiation of foot-and-mouth disease-infected pigs from vaccinated pigs using antibody-detecting sandwich ELISA 30 Introduction 31 Materials and Methods 32 Sera 32 Virus neutralization test (VNT) 32 Expression, purification and identification of the FMDV 3ABC polypeptide 32 Mouse immunization and production of a mAb against 3ABC 33 Sandwich ELISA 33 Commercially available ELISA kits for the detection of antibodies to FMDV NSPs 33 Results 33 Isotype of the mAb 33 Stability of the control sera in the sandwich ELISA 33 Determination of the cut-off value in sandwich ELISA 33 Kinetics of the antibody response to 3ABC 34 Comparison of the sandwich ELISA and the commercially available ELISA kits 34 Identification of recombinant protein to other FMDV serotypes using bovine antiserum 35 Specificity evaluated using antisera against SVDV and VSV 35 Discussion 35 Tables 34-35 Figs 33-35 References 37 Chapter 4. Development of a Luminex assay for the detection of swine antibodies to non-structural proteins of foot-and-mouth disease virus 39 Introduction 40 Materials and Methods 41 Serum sample 41 Virus neutralization test (VNT) 41 Preparation of 3ABC-coupled microspheres 41 Single-signature Luminex assays 42 Tests for comparison 42 Detection of vaccinated but infected pigs by Luminex assay 43 Results 43 Titration of neutralizing antibodies in experimentally infected Pigs 43 Assessment of microsphere coupling efficiency 43 Determination of cutoff value for the Luminex assay 43 Inter-assay repeatability 44 Comparison of the Luminex assay with the commercially available ELISA and in-house methods in detecting serially sampled sera 44 Diagnostic specificity of the Luminex assay in vaccinated Pigs 44 Diagnostic sensitivity and specificity of the Luminex assay 44 Detection of 3ABC NSP antibody in bovine antisera 45 Detection of 3ABC NSP antibody in sera against FMDV O/TW/1997 And FMDV O/TW/1999 strains 45 Analytical specificity of the Luminex assay 45 Detection of FMDV infection in vaccinated animals 45 Discussion 45 Tables 43-46 Figs 43-45 References 47 Chapter 5. Development of a multiplex Luminex assay for detecting swine antibodies to structural and non-structural proteins of foot-and-mouth disease virus in Taiwan 49 Introduction 51 Materials and Methods 51 Serum samples 51 Virus neutralization test (VNT) 52 Expression of recombinant FMD SP and NSP in E. coli 52 SDS-PAGE and Western blotting 53 Preparation of recombinant protein coupled microspheres 53 xMAP assays 54 Comparison of the xMAP to other assays 54 Results 54 Titration of neutralizing antibodies in experimentally infected pigs 54 Expression of SP and NSP polypeptides in E. coli 54 Assessment of microsphere coupling efficiency 55 Determination of the cutoff value for the xMAP assay 55 Comparison of the xMAP with the commercially available ELISA and VNT in detecting sequentially sampled sera 55 Diagnostic sensitivity and specificity to vaccinated pigs of the xMAP 55 Diagnostic sensitivity and specificity of the xMAP in experimental pigs 56 Detection of SP and NSP antibodies in sera against FMDV O/TW/1997 and O/TW/1999 strains 56 Analytical specificity of the xMAP against SVDV antibody 57 Detection of VP1 SP and 3ABC NSP antibodies in bovine antisera 57 Detection of FMDV infection in vaccinated animals 57 Discussion 58 Tables 52-59 Figs 54-58 Reference 60 Chapter 6. General discussion, Conclusion and Perspectives 62 References 67 Appendix 8

VON: A Scalable Peer-to-Peer Network for Virtual Environments

[[abstract]]The scalability of large-scale networked virtual environments (NVEs) such as today's massively multiplayer online games (MMOGs) faces inherent limits imposed by client-server architectures. We identify an emerging research direction that applies peer-to-peer (P2P) networks in order to realize more scalable and affordable NVEs. The central issue for P2P-based NVE (P2P-NVE) systems is to correctly and efficiently maintain the topology of all participating peers by solving the neighbor discovery problem. We also propose the Voronoi-based overlay network (VON), a simple and efficient design that maintains the P2P topology in a fully-distributed, low-latency, and message-efficient manner. Simulation results show that by bounding the per-node resource consumption, VON can be fundamentally more scalable than existing methods while achieving high topology consistency and reliability.[[incitationindex]]SCI[[booktype]]紙

Relationships among burnout, job dissatisfaction, psychosocial work conditions and minor mental disorders of precarious employment in Taiwan

Background: Precarious employment is a major determinant of mental health outcomes. The COVID-19 pandemic and development of digital economic platforms have enhanced the ratio of precarious employment relationship. The aim of this study was to explore the relationships among burnout, job dissatisfaction, psychosocial work conditions and minor mental disorders of precarious employment. Methods: A cross-sectional study was conducted, using the questionnaire from a national survey of employees in 2013. Minor mental disorder was measured using the five-item brief symptom rating scale (BSRS-5). 1909 males and 1499 females, with a total of 3408 non-standard employees aged 20 to 65, including short-term and temporary precarious employment, have been analyzed. Also obtained were participants’ sex, age, type of industry, status of shift work, job dissatisfaction, burnout as well as psychosocial work conditions. Results: The prevalence of minor mental disorders among precarious work condition in man and women were 16.08% and 19.35%, respectively. When we adjusted age and status of shift work, associations between minor mental disorders and female, job dissatisfaction, increased scores in burnout, and high psychological demand of work was noticed. When we further categorized by sex, it was found that job dissatisfaction and increased scores in burnout were significantly related with an increased risk for minor mental disorders in both male and female workers. The odds of minor mental disorders was significantly related with an increased scores in psychological demand of work among female precarious workers. Conclusions: This research study provides directions for future researches

Comparison of Fragmentation and Dusting Modality Using Holmium YAG Laser during Ureteroscopy for the Treatment of Ureteral Stone: A Single-Center’s Experience

Laser ureteroscopic lithotripsy (URSL) is an efficacious treatment for ureteral stones. There have been few previous studies comparing the different energy and frequency settings for URSL in a single center. We compared these two laser modalities, which were simultaneously used in our medical center for the treatment of ureteral stones. Patients who underwent fragmentation or dusting laser URSL between September 2018 and June 2020 were retrospectively reviewed. We compared patients who underwent fragmentation and dusting laser and assessed the enhancing factors for stone free rate. There were a total of 421 patients with ureteral stones who met the study criteria. There was no significant difference between the characteristics of both groups. The fragmentation group had a better stone free rate and a lower retropulsion rate compared with the dusting group. Multivariate analysis revealed that stone basket use, no upper ureteral stone or pyuria significantly improved the stone free rate. Both laser modes were effective and safe for ureteral lithotripsy although the fragmentation system showed slightly higher effectiveness and lower complication rate

Impacts of Cross-Linkers on Biological Effects of Mesoporous Silica Nanoparticles

Chemically synthesized cross-linkers play decisive roles in variable cargos attached to nanoparticles (NPs). Previous studies reported that surface properties, such as the size, charge, and surface chemistry, are particularly important determinants influencing the biological fate and actions of NPs and cells. Recent studies also focused on the relationship of serum proteins with the surface properties of NPs (also called the protein corona), which is recognized as a key factor in determining the cytotoxicity and biodistribution. However, there is concern that cross-linkers conjugated onto NPs might induce undesirable biological effects. Cell responses induced by cross-linkers have not yet been precisely elucidated. Herein, using mesoporous silica nanoparticles (MSNs) the surfaces of which were separately conjugated with four popular heterobifunctional cross-linkers, <i>i.e.</i>, <i>N</i>-[α-maleimidoacetoxy]­succinimide ester (AMAS), <i>m</i>-maleimidobenzoyl-<i>N</i>-hydroxysuccinimide ester (MBS), succinimidyl 4-(<i>N</i>-maleimidomethyl)­cyclohexane-1-carboxylate (SMCC), and maleimide poly­(ethylene glycol) succinimidyl carboxymethyl ester (MAL-PEG-SCM), we investigated cross-linker-conjugated MSNs to determine whether they can cause cytotoxicity, or enhance reactive oxygen species (ROS) generation, nuclear factor (NF)-κB activation, and p-p38 or p21 protein expressions in RAW264.7 macrophage cells. Furthermore, we also separately conjugated two biomolecules containing TAT peptides and bovine serum albumin (BSA) as model systems to study their cell responses in detail. Finally, <i>in vivo</i> mice studies evaluated the biodistribution and blood assays (biochemistry and complete blood count) of PEG-derivative NPs, and results suggested that TAT peptides caused significant white blood cell (WBC)-related cell and platelet abnormalities, as well as liver and kidney dysfunction compared to BSA when conjugated onto MSNs. The results showed that attention to cross-linkers should be considered an issue in the surface modification of NPs. We anticipate that our results could be helpful in developing biosafety nanomaterials