Enhanced Hybrid Vision Transformer with Multi-Scale Feature Integration and Patch Dropping for Facial Expression Recognition.

Convolutional neural networks (CNNs) have made significant progress in the field of facial expression recognition (FER). However, due to challenges such as occlusion, lighting variations, and changes in head pose, facial expression recognition in real-world environments remains highly challenging. At the same time, methods solely based on CNN heavily rely on local spatial features, lack global information, and struggle to balance the relationship between computational complexity and recognition accuracy. Consequently, the CNN-based models still fall short in their ability to address FER adequately. To address these issues, we propose a lightweight facial expression recognition method based on a hybrid vision transformer. This method captures multi-scale facial features through an improved attention module, achieving richer feature integration, enhancing the network's perception of key facial expression regions, and improving feature extraction capabilities. Additionally, to further enhance the model's performance, we have designed the patch dropping (PD) module. This module aims to emulate the attention allocation mechanism of the human visual system for local features, guiding the network to focus on the most discriminative features, reducing the influence of irrelevant features, and intuitively lowering computational costs. Extensive experiments demonstrate that our approach significantly outperforms other methods, achieving an accuracy of 86.51% on RAF-DB and nearly 70% on FER2013, with a model size of only 3.64 MB. These results demonstrate that our method provides a new perspective for the field of facial expression recognition.

Quantitative proteome analysis of bovine mammary gland reveals protein dynamic changes involved in peak and late lactation stages.

Mammary gland is an important organ for milk synthesis and secretion. It undergoes dramatic physiological changes to adapt the shift from peak to late lactation stage. Protein plays a final very vital role in many life functions, and the protein changes during different lactation stages potentially reflect the biology of lactation and the functions of mammary gland in cows. In current study, we adopted tandem mass tags label-based quantitative analysis technique and to investigate proteome changes occurring in bovine mammary gland from peak to late lactation stages. A total of 3753 proteins from mammary tissues taken at two lactation points from four individual cows by biopsy were quantified, out of which 179 proteins were expressed differentially between two stages. We observed five new DEPs (AACS, DHCR7, GSTM3, SFRP1 and SFRP4) and nine functional well-studies known proteins (PLIN2, LPIN1, PLIN3, GSN, CD74, MMP2, SOD1, SOD3 and GPX3) related to milk performance and mammary morphology. Bioinformatics analyses of the DEPs showed a majority of the up-regulated proteins during late lactation stage were related to apoptosis and immune process, while the downregulated proteins were mainly involved in localization, lipid metabolic and transport process. This suggests that the mammary gland can adapt to different molecular functions according to the biological need of the animal. From the integrated analysis of the differentially expressed proteins with known quantitative trait loci and genome-wide association study data, we identified 95 proteins may potentially affect milking performance. We expect findings in this study could be a valuable resource for future studies investigating the bovine proteome and functional studies.

Resequencing diverse Chinese indigenous breeds to enrich the map of genomic variations in swine.

To enrich the map of genomic variations in swine, we randomly sequenced 13 domestic and wild individuals from China and Europe. We detected approximately 28.1 million single nucleotide variants (SNVs) and 3.6 million short insertions and deletions (INDELs), of which 2,530,248 SNVs and 3,456,626 INDELs were firstly identified compared with dbSNP 143. Moreover, 208,687 SNVs and 24,161 INDELs were uniquely observed in Chinese pigs, potentially accounting for phenotypic differences between Chinese and European pigs. Furthermore, significantly high correlation between SNV and INDEL was witnessed, which indicated that these two distinct variants may share similar etiologies. We also predicted loss of function genes and found that they were under weaker evolutionary constraints. This study gives interesting insights into the genomic features of the Chinese pig breeds. These data would be useful in the establishment of high-density SNP map and would lay a foundation for facilitating pig functional genomics study.

Identification of a conserved B-cell epitope on the GapC protein of Streptococcus dysgalactiae.

Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched (30)TRINDLT(36) of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of (30)TRINDLT(36), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope (30)TRINDLT(36) showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines.

Development of an indirect ELISA for detection of E. Coli antibodies in cow serum using a recombinant OmpT as antigen.

The aim of this article was to develop an indirect enzyme-linked immunosorbent assay (ELISA) for efficient detection of the infection of E. coli in cattle. OmpT, a highly conserved protease in all E. coli strains, was successfully expressed in E. coli XL-1-Blue strain with PET32a vector. Molecular weight of recombinant protein was identified by analyzing SDS-PAGE and the immunogenicity of OmpT was confirmed by Western Blotting. The recombinant OmpT was then employed as capture antigen in the ELISA. The antigen concentration and serum dilution were determined using a checkerboard titration. Results showed that the optimal concentration of coated antigen was 1 µg/ml at a serum dilution of 1:640 and the cut-off value of the assay was 0.335. In addition, the cross-reactivity assay showed that the OmpT was E. coli specific and the reproducibility experiments displayed good repeatability of the assay. Three hundred and forty cattle serum samples were tested by rOmpT-ELISA and sera coagulation tests. The ELISA has showed relative sensitivity of 100% and specificity of 96.47%. Results of these experiments indicated that the rOmpT-ELISA is a simple, rapid, and convenient method for detection the infection of E. coli with different serotype strains.

Asexual reproduction for improved livestock breeding.

As natural environments deteriorate, genetic improvements to agricultural animals will be required to ensure global food security. Improving livestock production by introducing asexual reproduction (AR) into mainstream animal husbandry can help meet the challenge, but its advantages must be accompanied by social, commercial, and governmental acceptance.

Safety evaluation of transgenic and genome-edited food animals.

There is an urgent need to reform the regulation of transgenic and genome-edited food animals. Now is the time to simplify regulatory safety guidelines based on science before it is too late to have these animals in place to meet societal needs in coming decades.

A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection.

Gene-edited pigs for agricultural and biomedical applications are typically generated using somatic cell nuclear transfer (SCNT). However, SCNT requires the use of monoclonal cells as donors, and the time-consuming and laborious monoclonal selection process limits the production of large populations of gene-edited animals. Here, we developed a rapid and efficient method named RE-DSRNP (reporter RNA enriched dual-sgRNA/CRISPR-Cas9 ribonucleoproteins) for generating gene-edited donor cells. RE-DSRNP takes advantage of the precise and efficient editing features of dual-sgRNA and the high editing efficiency, low off-target effects, transgene-free nature, and low cytotoxic characteristics of reporter RNA enriched RNPs (CRISPR-Cas9 ribonucleoproteins), thus eliminating the need for the selection of monoclonal cells and thereby greatly reducing the generation time of donor cells from 3-4 weeks to 1 week, while also reducing the extent of apoptosis and chromosomal aneuploidy of donor cells. We applied RE-DSRNP to produce cloned pigs bearing a deletion edit of the wild-type p53-induced phosphatase 1 (WIP1) gene: among 32 weaned cloned pigs, 31 (97%) carried WIP1 edits, and 15 (47%) were homozygous for the designed fragment deletion, and no off-target event was detected. The WIP1 knockout (KO) pigs exhibited male reproductive disorders, illustrating the utility of RE-DSRNP for rapidly generating precisely edited animals for functional genomics and disease research. RE-DSRNP's strong editing performance in a large animal and its marked reduction in the required time for producing SCNT donor cells support its application prospects for rapidly generating populations of transgene-free cloned animals.

Long-term, multidomain analyses to identify the breed and allelic effects in MSTN-edited pigs to overcome lameness and sustainably improve nutritional meat production.

Beef and mutton production has been aided by breeding to integrate allelic diversity for myostatin (MSTN), but a lack of diversity in the MSTN germplasm has limited similar advances in pig farming. Moreover, insurmountable challenges with congenital lameness and a dearth of data about the impacts of feed conversion, reproduction, and meat quality in MSTN-edited pigs have also currently blocked progress. Here, in a largest-to-date evaluation of multiple MSTN-edited pig populations, we demonstrated a practical alternative edit-site-based solution that overcomes the major production obstacle of hindlimb weakness. We also provide long-term and multidomain datasets for multiple breeds that illustrate how MSTN-editing can sustainably increase the yields of breed-specific lean meat and the levels of desirable lipids without deleteriously affecting feed-conversion rates or litter size. Apart from establishing a new benchmark for the data scale and quality of genome-edited animal production, our study specifically illustrates how gene-editing site selection profoundly impacts the phenotypic outcomes in diverse genetic backgrounds.

Viscera Characteristics of MSTN-Edited Heterozygous Pigs.

Myostatin (MSTN) is a protein that negatively regulates growth of skeletal muscle, and inactivation of MSTN improves the mass of skeletal muscle. Our previous work found that MSTN +/- pigs have higher muscle depth and lower fat depth compared to wild type without any developmental problems. Therefore, MSTN-edited pigs are most likely to appear as heterozygotes in the potential future market, but the characteristics of organs in digestive and reproductive system of pigs with MSTN gene editing remains unclear. Here, we investigated the histological of the organs in the digestive system and reproductive system in MSTN gene heterozygotes at adult stages. The length of intestine was further compared between adult heterozygous and wild type pigs. We found no significant differences in histomorphology of organs, including heart, duodenum, jejunum, ileum, cecum, colon, testis, epididymis, ovaries, oviducts and uterus, between individuals from two genotypes. Moreover, there was no significant difference in the average length of intestine in adult pigs. Our data provide a reference for further clarifying the applications of MSTN gene edited pigs.

Multi-Omic Analysis in a Metabolic Syndrome Porcine Model Implicates Arachidonic Acid Metabolism Disorder as a Risk Factor for Atherosclerosis.

Background: The diet-induced gut microbiota dysbiosis has been suggested as a major risk factor for atherothrombosis, however, the detailed mechanism linking these conditions is yet to be fully understood. Methods: We established a long-term excessive-energy diet-induced metabolic syndrome (MetS) inbred Wuzhishan minipig model, which is characterized by its genetic stability, small size, and human-like physiology. The metabolic parameters, atherosclerotic lesions, gut microbiome, and host transcriptome were analyzed. Metabolomics profiling revealed a linkage between gut microbiota and atherothrombosis. Results: We showed that white atheromatous plaque was clearly visible on abdominal aorta in the MetS model. Furthermore, using metagenome and metatranscriptome sequencing, we discovered that the long-term excessive energy intake altered the local intestinal microbiota composition and transcriptional profile, which was most dramatically illustrated by the reduced abundance of SCFAs-producing bacteria including Bacteroides, Lachnospiraceae, and Ruminococcaceae in the MetS model. Liver and abdominal aorta transcriptomes in the MetS model indicate that the diet-induced gut microbiota dysbiosis activated host chronic inflammatory responses and significantly upregulated the expression of genes related to arachidonic acid-dependent signaling pathways. Notably, metabolomics profiling further revealed an intimate linkage between arachidonic acid metabolism and atherothrombosis in the host-gut microbial metabolism axis. Conclusions: These findings provide new insights into the relationship between atherothrombosis and regulation of gut microbiota via host metabolomes and will be of potential value for the treatment of cardiovascular diseases in MetS.

Autophagy in Staphylococcus aureus Infection.

Staphylococcus aureus is an invasive, facultative intracellular pathogen that can colonize niches in various host organisms, making it difficult for the host immune system to completely eliminate. Host autophagy is an intracellular clearance pathway involved in degrading S. aureus. Whereas the accessory gene regulatory system of S. aureus that controls virulence factors could resist the host immune defenses by evading and even utilizing autophagy. This article reviews the interaction between autophagy and S. aureus, providing insights on how to use these mechanisms to improve S. aureus infection control.

Effect of Abrupt Changes in the Cross-Sectional Area of a Pipe on Flame Propagation Characteristics of CH4/Air Mixtures.

To study the flame propagation characteristics of methane/air premixed gas in the pipeline with a sudden change of the pipe cross-sectional area, six kinds of customized pipes are used to study the methane/air premixed gas with a concentration of 9.5%. The results show that when the initial smooth flame front encounters an abrupt change in the cross-sectional area, the flame front becomes disordered and a turbulent flame is formed. A greater change in the cross-sectional area results in more severe flame turbulence. Compared with larger cross-section pipes set at the ignition end and downstream end, when the large cross-sectional area pipe is set in the middle of the pipe, the flame propagation process receives the secondary mutation induction effect of the abrupt cross section and the turbulence effect is stronger. The maximum propagation velocity and pressure are observed in configuration with the larger pipe in the middle of the pipe network. Moreover, when the cross-sectional area of this larger pipe increases, the flame is more substantially influenced by longitudinal expansion, the maximum propagation velocity and maximum overpressure increase accordingly, and the pressure oscillations are more obvious.

Social acceptance for commercialization of genetically modified food animals.

Genetically modified food animals (GMFAs) are needed to address early the cumulative effects of livestock production on the environment, and to accommodate future food demands. In 2020 China and the USA, the world's two largest economies, embarked on regulatory reforms to boost the commercialization of such animals. However, gaining social acceptance of GMFAs for commercialization remains a global challenge. We propose a framework that focuses on social license for commercialization of GMFAs by defining four classes of improvement using precision genetics: (1) animals equivalent to natural variation to obtain the improved effect of cross-breeding (ENV); (2) animals with an inactivated gene that could occur via natural mutation (ENC-); (3) animals harboring a natural genetic sequence isolated from another species (ENC+); and (4) animals with synthetic sequences encoding novel genes (BNE). Our approach can guide regulators and the public to support orderly commercialization of GMFAs.

CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance.

Porcine reproductive and respiratory syndrome virus (PRRSV) and transmissible gastroenteritis virus (TGEV) are two highly infectious and lethal viruses causing major economic losses to pig production. Here, we report generation of double-gene-knockout (DKO) pigs harboring edited knockout alleles for known receptor proteins CD163 and pAPN and show that DKO pigs are completely resistant to genotype 2 PRRSV and TGEV. We found no differences in meat-production or reproductive-performance traits between wild-type and DKO pigs, but detected increased iron in DKO muscle. Additional infection challenge experiments showed that DKO pigs exhibited decreased susceptibility to porcine deltacoronavirus (PDCoV), thus offering unprecedented in vivo evidence of pAPN as one of PDCoV receptors. Beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit pAPN or CD163 for entry.

Pig epidemics are the biggest threat to the pork industry. In 2019 alone, hundreds of billions of dollars worldwide were lost due to various pig diseases, many of them caused by viruses. The porcine reproductive and respiratory virus (PRRS virus for short), for instance, leads to reproductive disorders such as stillbirths and premature labor. Two coronaviruses ­ the transmissible gastroenteritis virus (or TGEV) and the porcine delta coronavirus ­ cause deadly diarrhea and could potentially cross over into humans. Unfortunately, there are still no safe and effective methods to prevent or control these pig illnesses, but growing disease-resistant pigs could reduce both financial and animal losses. Traditionally, breeding pigs to have a particular trait is a slow process that can take many years. But with gene editing technology, it is possible to change or remove specific genes in a single generation of animals. When viruses infect a host, they use certain proteins on the surface of the host's cells to find their inside: the PRRS virus relies a protein called CD163, and TGEV uses pAPN. Xu, Zhou, Mu et al. used gene editing technology to delete the genes that encode the CD163 and pAPN proteins in pigs. When the animals were infected with PRRS virus or TGEV, the non-edited pigs got sick but the gene-edited animals remained healthy. Unexpectedly, pigs without CD163 and pAPN also coped better with porcine delta coronavirus infections, suggesting that CD163 and pAPN may also help this coronavirus infect cells. Finally, the gene-edited pigs reproduced and produced meat as well as the control pigs. These experiments show that gene editing can be a powerful technology for producing animals with desirable traits. The gene-edited pigs also provide new knowledge about how porcine viruses infect pigs, and may offer a starting point to breed disease-resistant animals on a larger scale.

Structural Variant Detection by Large-scale Sequencing Reveals New Evolutionary Evidence on Breed Divergence between Chinese and European Pigs.

In this study, we performed a genome-wide SV detection among the genomes of thirteen pigs from diverse Chinese and European originated breeds by next genetation sequencing, and constrcuted a single-nucleotide resolution map involving 56,930 putative SVs. We firstly identified a SV hotspot spanning 35 Mb region on the X chromosome specifically in the genomes of Chinese originated individuals. Further scrutinizing this region by large-scale sequencing data of extra 111 individuals, we obtained the confirmatory evidence on our initial finding. Moreover, thirty five SV-related genes within the hotspot region, being of importance for reproduction ability, rendered significant different evolution rates between Chinese and European originated breeds. The SV hotspot identified herein offers a novel evidence for assessing phylogenetic relationships, as well as likely explains the genetic difference of corresponding phenotypes and features, among Chinese and European pig breeds. Furthermore, we employed various SVs to infer genetic structure of individuls surveyed. We found SVs can clearly detect the difference of genetic background among individuals. This clues us that genome-wide SVs can capture majority of geneic variation and be applied into cladistic analyses. Characterizing whole genome SVs demonstrated that SVs are significantly enriched/depleted with various genomic features.

Production of mouse monoclonal antibody against Streptococcus dysgalactiae GapC protein and mapping its conserved B-cell epitope.

Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in animals. To identify the conserved B-cell epitope of S. dysgalactiae GapC, a mouse monoclonal antibody 1E11 (mAb1E11) against GapC was generated and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12). Eleven positive clones recognized by mAb1E11 were identified, most of which matched the consensus motif TGFFAKK. Sequence of the motif exactly matched amino acids 97-103 of the S. dysgalactiae GapC. In addition, the epitope (97)TGFFAKK(103) showed high homology among different streptococcus species. Site-directed mutagenic analysis further confirmed that residues G98, F99, F100 and K103 formed the core of (97)TGFFAKK(103), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1E11. Collectively, the identification of conserved B-cell epitope within S. dysgalactiae GapC highlights the possibility of developing the epitope-based vaccine.

Identification of a Conserved Linear B-Cell Epitope of Streptococcus dysgalactiae GapC Protein by Screening Phage-Displayed Random Peptide Library.

The GapC of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce protective humoral immune response in animals. However, B-cell epitopes on the S. dysgalactiae GapC have not been well identified. In this study, a monoclonal antibody (mAb5B7) against the GapC1-150 protein was prepared. After passive transfer, mAb5B7 could partially protect mice against S. dysgalactiae infection. Eleven positive phage clones recognized by mAb5B7 were identified by screening phage-displayed random 12-peptide library, most of which matched the consensus motif DTTQGRFD. The motif sequence exactly matches amino acids 48-55 of the S. dysgalactiae GapC protein. In addition, the motif 48DTTQGRFD55 shows high homology among various streptococcus species. Site-directed mutagenic analysis further confirmed that residues D48, T50, Q51, G52 and F54 formed the core motif of 48DTTQGRFD55. This motif was the minimal determinant of the B-cell epitope recognized by the mAb5B7. As expected, epitope-peptide evoked protective immune response against S. dysgalactiae infection in immunized mice. Taken together, this identified conserved B-cell epitope within S. dysgalactiae GapC could provide very valuable insights for vaccine design against S. dysgalactiae infection.

Cross-protective effect of a novel multi-antigen-chimeric vaccine against Streptococcus and Staphylococcus aureus infection in mice.

Staphylococcal and streptococcal species are the most common pathogens that cause bovine mastitis. Induction of a broad-spectrum protective immunity against staphylococci and streptococci by combining multiple antigens into a single vaccine is highlighted. To develop a universal vaccine candidate, a GapC1-tIsdB-TRAP (GIT) construct was generated. The GIT contained the truncated GapC from Streptococcus dysgalactiae, and truncated IsdB and full-length TRAP from Staphylococcus aureus. The humoral and cellular immune responses elicited by GIT were evaluated in mice. Antibody levels against GIT displayed a consistent tendency with antibody levels against GapC, IsdB and TRAP. The level of IFN-γ was higher in the GIT group than in the IsdB group (P<0.05), and the level of IL-4 was higher in the GIT group than in the GapC or TRAP groups (P<0.05). The GIT group showed an improved protection against Streptococcus in comparison with GapC group. A significant difference in S. aureus challenge test was detected between the GIT group and the IsdB or TRAP groups (P<0.05) in per cent survival of mice, and a synergistic immunoprotection against S. aureus or S. dysgalactiae was produced in the GIT group. These results suggested that the GIT would be a promising common vaccine candidate against S. aureus and Streptococcus.