Evaluation of humoral and cellular immune responses induced by a cocktail of recombinant African swine fever virus antigens fused with OprI in domestic pigs.

BACKGROUND: African swine fever (ASF) is a highly fatal disease in domestic pigs caused by ASF virus (ASFV), for which there is currently no commercial vaccine available. The genome of ASFV encodes more than 150 proteins, some of which have been included in subunit vaccines but only induce limited protection against ASFV challenge. METHODS: To enhance immune responses induced by ASFV proteins, we expressed and purified three fusion proteins with each consisting of bacterial lipoprotein OprI, 2 different ASFV proteins/epitopes and a universal CD4+ T cell epitope, namely OprI-p30-modified p54-TT, OprI-p72 epitopes-truncated pE248R-TT, and OprI-truncated CD2v-truncated pEP153R-TT. The immunostimulatory activity of these recombinant proteins was first assessed on dendritic cells. Then, humoral and cellular immunity induced by these three OprI-fused proteins cocktail formulated with ISA206 adjuvant (O-Ags-T formulation) were assessed in pigs. RESULTS: The OprI-fused proteins activated dendritic cells with elevated secretion of proinflammatory cytokines. Furthermore, the O-Ags-T formulation elicited a high level of antigen-specific IgG responses and interferon-γ-secreting CD4+ and CD8+ T cells after stimulation in vitro. Importantly, the sera and peripheral blood mononuclear cells from pigs vaccinated with the O-Ags-T formulation respectively reduced ASFV infection in vitro by 82.8% and 92.6%. CONCLUSIONS: Our results suggest that the OprI-fused proteins cocktail formulated with ISA206 adjuvant induces robust ASFV-specific humoral and cellular immune responses in pigs. Our study provides valuable information for the further development of subunit vaccines against ASF.

Two-stage deep learning method for sparse-view fluorescence molecular tomography reconstruction.

Fluorescence molecular tomography (FMT) is a preclinical optical tomographic imaging technique that can trace various physiological and pathological processes at the cellular or even molecular level. Reducing the number of FMT projection views can improve the data acquisition speed, which is significant in applications such as dynamic problems. However, a reduction in the number of projection views will dramatically aggravate the ill-posedness of the FMT inverse problem and lead to significant degradation of the reconstructed images. To deal with this problem, we have proposed a deep-learning-based reconstruction method for sparse-view FMT that only uses four perpendicular projection views and divides the image reconstruction into two stages: image restoration and inverse Radon transform. In the first stage, the projection views of the surface fluorescence are restored to eliminate the blur derived from photon diffusion through a fully convolutional neural network. In the second stage, another convolutional neural network is used to implement the inverse Radon transform between the restored projections from the first stage and the reconstructed transverse slices. Numerical simulation and phantom and mouse experiments are carried out. The results show that the proposed method can effectively deal with the image reconstruction problem of sparse-view FMT.

Image restoration for blurry optical images caused by photon diffusion with deep learning.

Optical macroscopic imaging techniques have shown great significance in the investigations of biomedical issues by revealing structural or functional information of living bodies through the detection of visible or near-infrared light derived from different mechanisms. However, optical macroscopic imaging techniques suffer from poor spatial resolution due to photon diffusion in biological tissues. This dramatically restricts the application of optical imaging techniques in numerous situations. In this paper, an image restoration method based on deep learning is proposed to eliminate the blur caused by photon diffusion in optical macroscopic imaging. Two blurry images captured at orthogonal angles are used as the additional information to ensure the uniqueness of the solution and restore the small targets at deep locations. Then a fully convolutional neural network is proposed to accomplish the image restoration, which consists of three sectors: V-shaped network for central view, V-shaped network for side views, and synthetical path. The two V-shaped networks are concatenated to the synthetical path with skip connections to generate the output image. Simulations as well as phantom and mouse experiments are implemented. Results indicate the effectiveness of the proposed method.

Antigenic and immunogenic properties of recombinant proteins consisting of two immunodominant African swine fever virus proteins fused with bacterial lipoprotein OprI.

BACKGROUND: African swine fever (ASF) is a highly fatal swine disease, which threatens the global pig industry. There is no commercially available vaccine against ASF and effective subunit vaccines would represent a real breakthrough. METHODS: In this study, we expressed and purified two recombinant fusion proteins, OPM (OprI-p30-modified p54) and OPMT (OprI-p30-modified p54-T cell epitope), which combine the bacterial lipoprotein OprI with ASF virus proteins p30 and p54. Purified recombinant p30 and modified p54 expressed alone or fused served as controls. The activation of dendritic cells (DCs) by these proteins was first assessed. Then, humoral and cellular immunity induced by the proteins were evaluated in mice. RESULTS: Both OPM and OPMT activated DCs with elevated expression of relevant surface molecules and proinflammatory cytokines. Furthermore, OPMT elicited the highest levels of antigen-specific IgG responses, cytokines including interleukin-2, interferon-γ, and tumor necrosis factor-α, and proliferation of lymphocytes. Importantly, the sera from mice vaccinated with OPM or OPMT neutralized more than 86% of ASF virus in vitro. CONCLUSIONS: Our results suggest that OPMT has good immunostimulatory activities and immunogenicity in mice, and might be an appropriate candidate to elicit immune responses in swine. Our study provides valuable information on further development of a subunit vaccine against ASF.

Accuracy improvement of demodulating the stress field with StressUnet in photoelasticity.

Evaluating the stress field based on photoelasticity is of vital significance in engineering fields. To achieve the goal of efficiently demodulating stress distribution and to overcome the limitations of conventional methods, it is essential to develop a deep learning method to simplify and accelerate the process of image acquisition and processing. A framework is proposed to enhance prediction accuracy. By adopting Resnet as the backbone, applying U-Net architecture, and adding a physical constraint module, our model recovers the stress field with higher structural similarity. Under different conditions, our model performs robustly despite complicated geometry and a large stress range. The results prove the universality and effectiveness of our model and offer an opportunity for instant stress detection.

Development of an Indirect Chemiluminescence Immunoassay Using a Multiepitope Recombinant Protein To Specifically Detect Antibodies against Foot-and-Mouth Disease Virus Serotype O in Swine.

Foot-and-mouth disease virus (FMDV) has led to serious losses in animal husbandry worldwide. Seromonitoring of FMDV postvaccination is important for the control and eradication of foot-and-mouth disease (FMD) in regions and countries where vaccination is widespread. However, many commercial kits present high false-positive rates. In this study, a multiepitope-based indirect chemiluminescence immunoassay (ME-CLIA) was developed for specifically detecting antibodies against FMDV serotype O in swine sera. The developed method presented high diagnostic sensitivity and excellent diagnostic specificity, and it could detect a broad spectrum of antibodies against FMDV serotype O. The diagnostic performance, accuracy rate, and analytical sensitivity of ME-CLIA were compared with those of three commercial kits. The immune protection value of multiple-epitope recombinant vaccine detected using ME-CLIA was preliminarily determined by observation of clinical symptoms postimmunization challenge, the results of which indicated that the ME-CLIA can be employed as a matching detection method for evaluating multiple-epitope recombinant vaccine. The percent positive values of ME-CLIA determined using swine vaccinated with inactivated vaccine were significantly positively correlated with the titers of liquid-phase-blocking enzyme-linked immunosorbent assay (ELISA) (LBPE) (r = 0.8361; P < 0.0001). These results indicated that ME-CLIA is suitable for detection of antibodies against FMDV serotype O in swine and for potency evaluation of multiple-epitope and inactivated vaccines.

Development of a competitive chemiluminescence immunoassay using a monoclonal antibody recognizing 3B of foot-and-mouth disease virus for the rapid detection of antibodies induced by FMDV infection.

BACKGROUND: Foot-and-mouth disease (FMD) is a devastating animal disease. Anti-non-structural protein (NSP) antibody detection is very important for confirming suspected cases, evaluating the prevalence of infection, certifying animals for trade and controlling the disease. METHODS: In this study, a competitive chemiluminescence immunoassay (3B-cCLIA) was developed for the rapid detection of antibodies against NSPs in different species of livestock animals using the monoclonal antibody (mAb) 9E2 as a competitive antibody that recognizes NSP 3B. RESULTS: The cut-off value (50%), diagnostic sensitivity (Dsn) (97.20%, 95.71%, and 96.15%) and diagnostic specificity (Dsp) (99.51%, 99.43%, and 98.36) of the assay were estimated by testing a panel of known-background sera from swine, cattle and sheep, respectively. The accuracy rate of the 3B-cCLIA was further validated and subsequently compared with that of two commercial diagnostic kits. The early diagnostic results showed that antibodies recognizing NSPs developed later (approximately 1-2 days) than antibodies recognizing structural proteins. Furthermore, anti-NSP antibody presence in animals vaccinated multiple times (false positives), especially cattle and sheep, was confirmed, and the false-positive rate increased with the number of vaccinations. CONCLUSIONS: These results indicate that the 3B-cCLIA is suitable for the rapid detection of antibodies against FMDV NSP 3B in a wide range of species.

Development of an indirect ELISA to specifically detect antibodies against African swine fever virus: bioinformatics approaches.

BACKGROUND: African swine fever (ASF), characterized by acute, severe, and fast-spreading, is a highly lethal swine infectious disease caused by the African swine fever virus (ASFV), which has caused substantial economic losses to the pig industry worldwide in the past 100 years. METHODS: This study started with bioinformatics methods and verified the epitope fusion protein method's reliability that does not rely on traditional epitope identification. Meanwhile, it will also express and purify the constructed genes through prokaryotic expression and establish antibody detection methods. RESULTS: The results indicated that the protein had good reactivity and did not cross-react with other swine diseases. The receiver-operating characteristic analysis was performed to verify the determination. The area under the receiver-operating characteristic curve was 0.9991 (95% confidence interval 0.9973 to 1.001). CONCLUSIONS: It was proved that the recombinant protein is feasible as a diagnostic antigen to distinguish ASFV and provides a new idea for ASFV antibody detection.

Robustness and accuracy improvement of data processing with 2D neural networks for transient absorption dynamics.

To achieve the goal of efficiently analyzing transient absorption spectra without arbitrary assumption and to overcome the limitations of conventional methods in fitting ability and highly noised backgrounds, it is essential to develop new tools to achieve more accurate and robust prediction based on the intrinsic properties of a spectrum even under strong noise. In this work, Lasso regression and neural network were combined to achieve an effective fitting. Compared to the conventional global fitting method, our network could automatically determine the exponential form on each wave unit, in which the accuracy was as high as 97%. Thereafter, the lifetime with the corresponding amplitude ratio could be easily predicted by the neural network on each wave unit. This kind of prediction is difficult to achieve by global fitting due to the limitation of computational resources. Furthermore, more accurate fitting even under weak signals could be achieved for the mean square error (MSE) decreasing by more than 100 times on average compared to conventional global fitting methods. Attributed to its improved accuracy and robustness, our developed algorithm could be readily applied to analyze time-resolved transient spectra.

Improving the Subtype Classification of Non-small Cell Lung Cancer by Elastic Deformation Based Machine Learning.

Non-invasive image-based machine learning models have been used to classify subtypes of non-small cell lung cancer (NSCLC). However, the classification performance is limited by the dataset size, because insufficient data cannot fully represent the characteristics of the tumor lesions. In this work, a data augmentation method named elastic deformation is proposed to artificially enlarge the image dataset of NSCLC patients with two subtypes (squamous cell carcinoma and large cell carcinoma) of 3158 images. Elastic deformation effectively expanded the dataset by generating new images, in which tumor lesions go through elastic shape transformation. To evaluate the proposed method, two classification models were trained on the original and augmented dataset, respectively. Using augmented dataset for training significantly increased classification metrics including area under the curve (AUC) values of receiver operating characteristics (ROC) curves, accuracy, sensitivity, specificity, and f1-score, thus improved the NSCLC subtype classification performance. These results suggest that elastic deformation could be an effective data augmentation method for NSCLC tumor lesion images, and building classification models with the help of elastic deformation has the potential to serve for clinical lung cancer diagnosis and treatment design.

Radiomics With Attribute Bagging for Breast Tumor Classification Using Multimodal Ultrasound Images.

OBJECTIVES: We aimed to develop radiomics with attribute bagging, which leverages multimodal ultrasound (US) images to improve the classification accuracy of breast tumors. METHODS: A retrospective study was conducted. B-mode US, shear wave elastographic, and contrast-enhanced US images of 178 patients with 181 tumors (67 malignant and 114 benign) were included. Radiomics with attribute bagging consisted of extraction of 1226 radiomic features and analysis of them with attribute bagging. Histologic examination results acted as the reference standard. Radiomics with several feature selection algorithms were used for comparison. Cross-validation and a holdout test were performed to evaluate their performances. RESULTS: The accuracy, sensitivity, specificity, and area under the receiver operating characteristic curve of radiomics with attribute bagging with the multimodal US images were 84.12%, 92.86%, 78.80%, and 0.919, respectively, exceeding all the comparison methods. CONCLUSIONS: Radiomics with attribute bagging combined with multimodal US images has the potential to be used for accurate diagnosis of breast tumors in the clinic.

3D deep encoder-decoder network for fluorescence molecular tomography.

Fluorescence molecular tomography (FMT) is a promising and noninvasive in vivo functional imaging modality. However, the quality of FMT reconstruction is limited by the simplified linear model of photon propagation. Here, an end-to-end three-dimensional deep encoder-decoder (3D-En-Decoder) network is proposed to improve the quality of FMT reconstruction. It directly establishes the nonlinear mapping relationship between the inside fluorescent source distribution and the boundary fluorescent signal distribution. Thus the reconstruction inaccuracy caused by the simplified linear model can be fundamentally avoided by the proposed network. Both numerical simulations and phantom experiments were carried out, and the results demonstrated that the 3D-En-Decoder network can greatly improve image quality and significantly reduce reconstruction time compared with conventional methods.

Preparation Mechanism and Properties of Orthoboric Acid Nanowires with Honeycomb-Like Structure.

Orthoboric acid (H3BO3) nanowires with honeycomb-like structure are successfully synthesized on boron nitride (BN) substrate using Fe powder as a catalyst. The formation of the nanowires is a two-step process (solid-liquid-solid growth mechanism), including the formation of B2O3 and its reaction with water to form H3BO3. The fundamental properties of H3BO3 nanowires, including their morphology, crystal structure, optical properties, thermal stability, and electrochemical properties, were systematically studied in order to expand their application fields. When innovatively investigated as anode materials for lithium ion batteries, the nanowires show a high discharge specific capacity of 243 mA h g-1 in the first cycle at a current density of 100 mA g-1. Further modification is essential for improving its cycling stability, such as surface coating and doping ions. Orthoboric acid nanowires with high first discharge capacity are believed to have great prospects for the development of novel anode materials for lithium ion batteries.

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos.

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5µM RepSox and 50nM LBH589 (RepSox+LBH589) for 24h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P<0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox+LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox+LBH589 group. Moreover, RepSox+LBH589 improved epigenetic reprogramming. In summary, RepSox+LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the invitro development of porcine SCNT embryos.

The cyclin-dependent kinase inhibitor, JNJ-7706621, improves in vitro developmental competence of porcine parthenogenetic activation and somatic cell nuclear transfer embryos.

In this study we examined the effects of JNJ-7706621, a cyclin-dependent kinase inhibitor, on the in vitro growth of pig embryos that had been produced either by parthenogenetic activation (PA) or somatic cell nuclear transfer (SCNT). A significantly higher percentage of PA embryos reached the blastocyst stage by Day 7 after exposure to 10µM JNJ-7706621 for 4h compared with embryos exposed to 5µgmL-1 cytochalasin B for 4h (P<0.05). Similarly, the rate of Tyr15 phosphorylation of the complex of cyclin and p34cdc2 (CDK1) was significantly elevated in the JNJ-7706621-treated embryos compared with embryos exposed to cytochalasin B or non-treated controls (P<0.05). In contrast, Thr161 phosphorylation of CDK1 was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated as well as the non-treated group (P<0.05). Similarly, the level of M-phase-promoting factor (MPF) in embryos was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated and non-treated groups (P<0.05). In addition, more SCNT embryos reached the blastocyst stage after treatment with JNJ-7706621 than following exposure to cytochalasin B (P<0.05). In conclusion, these results reveal that exposure to 10µM JNJ-7706621 for 4h improves early development of PA and SCNT porcine embryos by suppressing the activity of CDK1 and a concomitant reduction in the level of MPF.

Quisinostat treatment improves histone acetylation and developmental competence of porcine somatic cell nuclear transfer embryos.

Abnormal epigenetic modifications are considered a main contributing factor to low cloning efficiency. In the present study, we explored the effects of quisinostat, a novel histone deacetylase inhibitor, on blastocyst formation rate in porcine somatic-cell nuclear transfer (SCNT) embryos, on acetylation of histone H3 lysine 9 (AcH3K9), and on expression of POU5F1 protein and apoptosis-related genes BAX and BCL2. Our results showed that treatment with 10 nM quisinostat for 24 hr significantly improved the development of reconstructed embryos compared to the untreated group (19.0 ± 1.6% vs. 10.2 ± 0.9%; p < 0.05). Quisinostat-treated SCNT embryos also possessed significantly increased AcH3K9 at the pseudo-pronuclear stage (p < 0.05), as well as improved immunostaining intensity for POU5F1 at the blastocyst stage (p < 0.05). While no statistical difference in BAX expression was observed, BCL2 transcript abundance was significantly different in the quisinostat-treated compared to the untreated control group. Of the 457 quisinostat-treated cloned embryos transferred into three surrogates, six fetuses developed from the one sow that became pregnant. These findings suggested that quisinostat can regulate gene expression and epigenetic modification, facilitating nuclear reprogramming, and subsequently improving the developmental competence of pig SCNT embryos and blastocyst quality.

Fiber-type distribution and expression of myosin heavy chain isoforms in newborn heterozygous myostatin-knockout pigs.

OBJECTIVES: To explore the effects of heterozygous myostatin-knockout (MSNT+/-) on muscle characteristics, specifically fiber-type distribution and expression of myosin heavy chain isoforms in pigs. RESULTS: The fiber cross-sectional area of the semitendinosus and semimembranosus muscles were much larger in MSTN+/- pigs at birth than in wild-type (WT) pigs. MSTN+/- pigs had a higher proportion of fast-type fibers and lower succinate dehydrogenase activity in muscles than WT pigs. The myosin heavy chain IIB mRNA level in both two muscles was ~ threefold higher in MSTN+/- pigs compared with WT pigs. CONCLUSION: MSTN+/- pigs exhibit a disproportionate increase in muscle mass and can have a higher body weight due to fiber hypertrophy, a change in the fiber-type distribution, and alteration of myosin heavy chain isoforms levels, leading to more fast glycolytic fibers.

CDK inhibitor SU9516 induces tetraploid blastocyst formation from parthenogenetically activated porcine embryos.

OBJECTIVE: To examine the effect of SU9516, a cyclin-dependent kinase inhibitor, on the induction of tetraploid blastocyst formation in porcine embryos by parthenogenetic activation. RESULTS: Karyotype analysis of blastocysts showed that in the SU9516-treatment group 56% were tetraploid, whereas in the cytochalasin B (CB) group 67% were diploid. The level of maturation-promoting factor (MPF) in stimulated embryos treated with 10 µM SU9516 for 4 h was lower than in embryos treated with CB group (103 vs. 131 pg/ml). The mRNA expression levels of Nanog significantly increased in SU9516-treated embryos than CB group. CONCLUSION: SU9516 can induce tetraploid blastocyst formation at high efficiency. SU9516 can significantly influence the in vitro developmental competence of porcine parthenogenetically activated embryos by influencing the level of MPF and the gene related apoptosis and pluripotency.

RepSox improves viability and regulates gene expression in rhesus monkey-pig interspecies cloned embryos.

OBJECTIVE: To investigate the effect of the small molecule, RepSox, on the expression of developmentally important genes and the pre-implantation development of rhesus monkey-pig interspecies somatic cell nuclear transfer (iSCNT) embryos. RESULTS: Rhesus monkey cells expressing the monomeric red fluorescent protein 1 which have a normal (42) chromosome complement, were used as donor cells to generate iSCNT embryos. RepSox increased the expression levels of the pluripotency-related genes, Oct4 and Nanog (p < 0.05), but not of Sox2 compared with untreated embryos at the 2-4-cell stage. Expression of the anti-apoptotic gene, Bcl2, and the pro-apoptotic gene Bax was also affected at the 2-4-cell stage. RepSox treatment also increased the immunostaining intensity of Oct4 at the blastocyst stage (p < 0.05). Although the blastocyst developmental rate was higher in the group treated with 25 µM RepSox for 24 h than in the untreated control group (2.4 vs. 1.2%, p > 0.05), this was not significant. CONCLUSION: RepSox can improve the developmental potential of rhesus monkey-pig iSCNT embryos by regulating the expression of pluripotency-related genes.

The developmental competence of oocytes parthenogenetically activated by an electric pulse and anisomycin treatment.

OBJECTIVE: The aim of this study was to investigate the developmental competence of oocytes parthenogenetically activated by an electric pulse (EP) and treated with anisomycin and to determine whether this method is applicable to somatic cell nuclear transfer (SCNT). RESULTS: Embryos derived from porcine oocytes parthenogenetically activated by an EP and treatment with 0.01 µg/mL anisomycin had a significantly improved in vitro developmental capacity. Furthermore, 66.6% of blastocysts derived from these embryos had a diploid karyotype. The blastocyst formation rate of cloned embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 0.01 µg/mL anisomycin for 4 h. The level of maturation-promoting factor was significantly decreased in oocytes activated by an EP and treated with anisomycin. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR. CONCLUSION: Our results demonstrate that porcine oocyte activation via an EP in combination with anisomycin treatment can lead to a high blastocyst formation rate in parthenogenetic activation and SCNT experiments.