Construction of a camelid VHH yeast two-hybrid library and the selection of VHH against haemagglutinin-neuraminidase protein of the Newcastle disease virus

Humoral immune response after immunization. Sera from IIama was collected, two-fold diluted and tested by HI using LaSota as antigen. Figure S1 Amplification of VHH through a nested PCR. (A) First round PCR to separate VH from VHH. The upper 900 bp bands represent the VH-CH1-Hinge-CH2 of conventional Abs (lane 1–8). The lower 600 bp bands represent the VHH-Hinge-CH2 of HCAbs (lane 1–8). (B) VHH amplified through nested PCR using 600 bp fragment recovered from first round PCR as template (lane 1–4). M in A and B was the DL2000 DNA marker. C in A and B represent the negative control. Figure S2 PCR identification of inserted VHH. 47 clones were randomly picked to determine the library functional diversity by PCR using universal primers T7 and 3’AD (Table 1). Meanwhile, Sterile water was used as negative controls. 45 clones have amplified the 500 bp VHH fragments (lane 1–47), while negative templates control haven’t amplified any bands (lane C). M indicated the DL2000 DNA marker. Figure S3 Detection of library capacity and library titer. (A) 10-3 dilution plating of the transformed cells calculated a library capacity of 1.25 × 107 independent clones. (B) 10-5 dilution plating of the cultured library indicated a library titer of 3.45 × 108 cfu/mL. Figure S4 Deduced amino acid aligment of 10 random picked VHH. Deduced amino acid sequences were analyzed according to the Kabat numbering. Differences in the sequences are pinked, and the dash represent the missing sequences. Two hallmark Cys residues are labeled by the thick-line boxes. The four conservative hallmark residues of VHH in FR2 are labeled by the dotted line boxes. Figure S5 pGBKT7-HN bait plasmid construction. (A) PCR was carried out to amplify a truncate HN gene (without transmembrane region) from La Sota strain. M, 5000 DNA marker. 1, Truncate HN. C, Negative control. (B) A truncate HN was cloned into pGBKT7 through BamH I and Sal I. M, 5000 DNA marker. 1, Double restriction enzyme digestion of pGBKT7-HN. Figure S6 pHSIE-VHH plasmid construction. (A) 7 positive VHH fragment were amplified from recovered positive clones containing pGADT7-VHH by PCR. M, 5000 DNA marker. 1–7, VHH 1–7. C, Negative control. (B) Double restriction enzyme digestion of pHSIE-VHHs. M, 5000 DNA marker. 1–7, pHSIE-VHH 1–7. Figure S7 Western blot analysis of bait protein expression. 2 mL of Y2HGold(pGBKT7-HN) culture liquid was extracted using yeast protein extraction reagent (Takara). c-Myc tag monoclonal antibody (1:4000 dilution) was used as first antibody and HRP-labeled goat anti-mouse antibody (1:5000) was used as second antibody. The immunoreactive was visualized with cECL Plus Western blotting detection reagent (CWBIO). (DOC 1129 kb

Spatiotemporal Receding Horizon Control with Proactive Interaction Towards Autonomous Driving in Dense Traffic

In dense traffic scenarios, ensuring safety while keeping high task performance for autonomous driving is a critical challenge. To address this problem, this paper proposes a computationally-efficient spatiotemporal receding horizon control (ST-RHC) scheme to generate a safe, dynamically feasible, energy-efficient trajectory in control space, where different driving tasks in dense traffic can be achieved with high accuracy and safety in real time. In particular, an embodied spatiotemporal safety barrier module considering proactive interactions is devised to mitigate the effects of inaccuracies resulting from the trajectory prediction of other vehicles. Subsequently, the motion planning and control problem is formulated as a constrained nonlinear optimization problem, which favorably facilitates the effective use of off-the-shelf optimization solvers in conjunction with multiple shooting. The effectiveness of the proposed ST-RHC scheme is demonstrated through comprehensive comparisons with state-of-the-art algorithms on synthetic and real-world traffic datasets under dense traffic, and the attendant outcome of superior performance in terms of accuracy, efficiency and safety is achieved.Comment: 16 pages, 13 figures, accepted for publication in IEEE Transactions on Intelligent Vehicle

Enhancing laying performance and immunity via probiotic and vitamin additives during induced molting

IntroductionMolting is induced in commercial laying hens to rejuvenate the reproductive system and increase egg production. However, this process causes stress and reduces bird health and performance.MethodsThe experiment was conducted to study the effect of multi probiotics and vitamin additives on induced molting in 240 ISA Brown hens. Hens were randomly divided into four groups receiving probiotic and vitamin additives (I–IV) during different period of molting. During the whole molting process, the laying performance indexes such as egg laying rate, egg quality, ovary weight and oviduct lengths were measured, and the spleen index, serum immunoglobulin, immune response of NDV and AIV vaccine were monitored.ResultsMolted hens resumed 50% egg production in just 37 days, with 1.62% mortality. Egg quality such as egg weight, yolk color, Haugh unit, eggshell strength and protein height were significantly improved. After the second production peak, the reproductive organs and immune organs returned to normal, and the immune antibody titer of NDV vaccine increased significantly.DiscussionMolting with probiotic and vitamin additives improve the laying performance and egg quality, reduce mortality, significantly improve immune function and vaccine titer, and help to enhance disease resistance and maintain production performance of aged laying hens

Real-Time Parallel Trajectory Optimization with Spatiotemporal Safety Constraints for Autonomous Driving in Congested Traffic

Multi-modal behaviors exhibited by surrounding vehicles (SVs) can typically lead to traffic congestion and reduce the travel efficiency of autonomous vehicles (AVs) in dense traffic. This paper proposes a real-time parallel trajectory optimization method for the AV to achieve high travel efficiency in dynamic and congested environments. A spatiotemporal safety module is developed to facilitate the safe interaction between the AV and SVs in the presence of trajectory prediction errors resulting from the multi-modal behaviors of the SVs. By leveraging multiple shooting and constraint transcription, we transform the trajectory optimization problem into a nonlinear programming problem, which allows for the use of optimization solvers and parallel computing techniques to generate multiple feasible trajectories in parallel. Subsequently, these spatiotemporal trajectories are fed into a multi-objective evaluation module considering both safety and efficiency objectives, such that the optimal feasible trajectory corresponding to the optimal target lane can be selected. The proposed framework is validated through simulations in a dense and congested driving scenario with multiple uncertain SVs. The results demonstrate that our method enables the AV to safely navigate through a dense and congested traffic scenario while achieving high travel efficiency and task accuracy in real time.Comment: 8 pages, 7 figures, accepted for publication in the 26th IEEE International Conference on Intelligent Transportation Systems (ITSC 2023

Incremental Bayesian Learning for Fail-Operational Control in Autonomous Driving

Abrupt maneuvers by surrounding vehicles (SVs) can typically lead to safety concerns and affect the task efficiency of the ego vehicle (EV), especially with model uncertainties stemming from environmental disturbances. This paper presents a real-time fail-operational controller that ensures the asymptotic convergence of an uncertain EV to a safe state, while preserving task efficiency in dynamic environments. An incremental Bayesian learning approach is developed to facilitate online learning and inference of changing environmental disturbances. Leveraging disturbance quantification and constraint transformation, we develop a stochastic fail-operational barrier based on the control barrier function (CBF). With this development, the uncertain EV is able to converge asymptotically from an unsafe state to a defined safe state with probabilistic stability. Subsequently, the stochastic fail-operational barrier is integrated into an efficient fail-operational controller based on quadratic programming (QP). This controller is tailored for the EV operating under control constraints in the presence of environmental disturbances, with both safety and efficiency objectives taken into consideration. We validate the proposed framework in connected cruise control (CCC) tasks, where SVs perform aggressive driving maneuvers. The simulation results demonstrate that our method empowers the EV to swiftly return to a safe state while upholding task efficiency in real time, even under time-varying environmental disturbances.Comment: 8 pages, 8 figures, accepted for publication in the 22nd European Control Conference (ECC 2024

A single amino acid substitution alter antigenicity of Glycosylated protein 4 of HP-PRRSV

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is an important pig endemic disease in pork-producing countries worldwide. The etiology, porcine reproductive and respiratory syndrome virus (PRRSV), is characterized by fast antigen variability. Glycosylated protein 4 (GP4) is a minor protein in PRRSV virion, but contributes to induce protective immune responses. However, the antigenic characterization of PRRSV GP4 and the role of the mutations in this protein in PRRSV evolution are not clear. METHODS: Peptides chip scanning and peptide based ELISA was used to analyze the antigenic characterization of HP-PRRSV GP4. A total of 142 peptides printed on a chip were used to reveal the antigen reaction characteristics of the HP-PRRSV. The reactions of these peptides with HP-PRRSV-specific pig serum were scanned and quantified using the software PepSlide® Analyzer by fluorescence intensity. The active reaction regions (AR) were identified based on the scanning results and then the amino acids (aa) sequences of AR(s) is aligned among PRRSV strains for further identify the key aa site(s) impact the antigenicity of the protein. Peptide based ELISA is then reacted with PRRSV positive sera derived from pig inoculated with different PRRSV strains for further analysis the role of specific amino acid in AR. RESULTS: The intensity plot was used to show the reactions of the peptides with PRRSV serum and it showed that enormously different response happened to various parts of GP4. The highest reaction intensity value reached 6401.5 against one peptide with the sequence DIKTNTTAASDFVVL. An AR from S29 to G56 was identified. Sequence alignment revealed various mutations in site 43 and possibly played an important role in this AR. Peptides ELISA reaction with sera from pigs inoculated with different PRRSV strain revealed that the change of aa in site 43 reduced the reaction of the peptide with PRRSV positive sera derived from pigs inoculated with the peptide related PRRSV strains. CONCLUSION: In this study, one AR covering S29 to G56 was identified in GP4. The aa in site 43 play an important role in determining the antigenic character of GP4. The continual mutations (S → G → D → N) occurred in this site alter the antigenicity of PRRSV GP4. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-016-0586-3) contains supplementary material, which is available to authorized users

Cyclooxygenase-2 Facilitates Newcastle Disease Virus Proliferation and Is as a Target for Canthin-6-One Antiviral Activity

Cyclooxygenase-2 (COX-2), one of the mediators of inflammation in response to viral infection, plays an important role in host antiviral defense system. But its role in Newcastle disease virus (NDV) proliferation process remains unclear. This study revealed that inhibition of COX-2 could benefit NDV proliferation and overexpression of COX-2 dose-dependently suppressed NDV proliferation. Overexpression of COX-2 also showed inhibitory effect on NDV-induced endoplasmic reticulum (ER)-stress and autophagy, also promoted the expression of antiviral genes. However, prostaglandin E2 (PGE2), the major product of COX-2, had indistinctive effects on NDV proliferation. At variant time point post viral infection, a tight regulation pattern of COX-2 by NDV was observed. Using inhibitors and siRNA against signaling molecules, the nuclear factor-κB (NF-κB) and melanoma differentiation-associated gene 5 (MDA5) were identified as critical factors for NDV induced COX-2 expression. Nonetheless, at late stage of NDV proliferation, substantial suppression of COX-2 protein synthesis could be detected, accompanied by a decrease in mRNA half-life. Furthermore, three C ring-truncated canthin-6-one analogs were used to activate COX-2 expression and showed inhibitory effect on NDV proliferation with the effective concentrations on μM level. Taken together, these results illustrated a novel NDV-regulated cellular mechanism and indicated that COX-2 is an important regulator of NDV proliferation which can serve as a potential target for anti-NDV agents.</p

IncHI1 plasmids mediated the tet(X4) gene spread in Enterobacteriaceae in porcine

The tigecycline resistance gene tet(X4) was widespread in various bacteria. However, limited information about the plasmid harboring the tet(X4) gene spread among the different species is available. Here, we investigated the transmission mechanisms of the tet(X4) gene spread among bacteria in a pig farm. The tet(X4) positive Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Enterobacter hormaeche were identified in the same farm. The whole genome sequencing (WGS) analysis showed that the K. pneumoniae belonged to ST727 (n = 11) and ST3830 (n = 1), E. cloacae and E. hormaeche belonged to ST524 (n = 1) and ST1862 (n = 1). All tet(X4) genes were located on the IncHI1 plasmids that could be conjugatively transferred into the recipient E. coli C600 at 30°C. Moreover, a fusion plasmid was identified that the IncHI1 plasmid recombined with the IncN plasmid mediated by ISCR2 during the conjugation from strains B12L to C600 (pB12L-EC-1). The fusion plasmid also has been discovered in a K. pneumoniae (K1L) that could provide more opportunities to spread antimicrobial resistance genes. The tet(X4) plasmids in these bacteria are derived from the same plasmid with a similar structure. Moreover, all the IncHI1 plasmids harboring the tet(X4) gene in GenBank belonged to the pST17, the newly defined pMLST. The antimicrobial susceptibility testing was performed by broth microdilution method showing the transconjugants acquired the most antimicrobial resistance from the donor strains. Taken together, this report provides evidence that IncHI1/pST17 is an important carrier for the tet(X4) spread in Enterobacteriaceae species, and these transmission mechanisms may perform in the environment

A NADC30-like PRRSV causes serious intestinal infections and tropism in piglets

Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic loss to China's swine industry. Currently, a novel type 2 PRRSV, called the NADC30-like strain, is epidemic in numerous provinces of China. In this study, a NADC30-Like PRRSV strain was isolated in primary alveolar macrophage (PAM) cells from fecal samples collected from a local pig farm, which suffered severe diarrhea. A pathogenicity comparison study was conducted in 6–week‐old piglets by inoculating highly pathogenic HP-PRRSV and NADC30-Like PRRSV isolates. RT-qPCR revealed detection of NADC30-Like PRRSV but not the HP-PRRSV in the intestine. PRRSV infection-related lesions were observed in the intestine were further confirmed by histopathological and immunohistochemically examination (IHC). In addition, severe virus infections were also detected by RT-qPCR. Based on clinical observation and pathogenicity experiments, we confirmed that NADC30-Like PRRSV gained more tissue tropism, especially in the small intestine. This may be the one reason explaining why NADC-Like 30 PRRSV become a major epidemic strain in China since the first outbreak in 2013