Damage Detection of Unwashed Eggs through Video and Deep Learning.

Broken eggs can be harmful to human health but are also unfavorable for transportation and production. This study proposes a video-based detection model for the real-time detection of broken eggs regarding unwashed eggs in dynamic scenes. A system capable of the continuous rotation and translation of eggs was designed to display the entire surface of an egg. We added CA into the backbone network, fusing BiFPN and GSConv with the neck to improve YOLOv5. The improved YOLOV5 model uses intact and broken eggs for training. In order to accurately judge the category of eggs in the process of movement, ByteTrack was used to track the eggs and assign an ID to each egg. The detection results of the different frames of YOLOv5 in the video were associated by ID, and we used the method of five consecutive frames to determine the egg category. The experimental results show that, when compared to the original YOLOv5, the improved YOLOv5 model improves the precision of detecting broken eggs by 2.2%, recall by 4.4%, and mAP:0.5 by 4.1%. The experimental field results showed an accuracy of 96.4% when the improved YOLOv5 (combined with ByteTrack) was used for the video detection of broken eggs. The video-based model can detect eggs that are always in motion, which is more suitable for actual detection than a single image-based detection model. In addition, this study provides a reference for the research of video-based non-destructive testing.

Effects of Stocking Density on Fatty Acid Metabolism by Skeletal Muscle in Mice.

Specific pathogen-free (SPF) grade laboratory animals are kept in specific cages for life. The limited space could affect the characterization of colonization and dynamic changes related to gut microorganisms, and affect adipokines, even further affecting the fat synthesis and muscle quality of animals. The objective of this study was to analyze the stocking density on the dynamic distribution of gut microbiota, fat synthesis and muscle quality of SPF grade Kunming mice. Three housing densities were accomplished by raising different mice per cage with the same floor size. Kunming mice were reared at low stocking density (LSD, three mice a group), medium stocking density (MSD, 5 mice a group), and high stocking density (HSD, 10 mice a group) for 12 weeks. The results demonstrated that the stocking density affected intestinal microbial flora composition. We found that compared with the MSD group, the abundance of Lactobacillus in the LSD group and the HSD group decreased, but the abundance of unclassified_Porphyromonadaceae increased. Moreover, fat synthesis and muscle quality were linked to the intestinal microbial flora and stocking density. Compared with the LSD group and the HSD group, the MSD group had a more balanced gut flora, higher fat synthesis and higher muscle quality. Overall, this study demonstrated that stocking density could affect gut microbiota composition, and reasonable stocking density could improve fat synthesis and muscle quality. Our study will provide theoretical support for the suitable stocking density of laboratory animals.

Optimization of iturin A production from Bacillus subtilis ZK-H2 in submerge fermentation by response surface methodology.

In this research, single-factor and response surface experiments were conducted in the fed-batch fermentation process to improve the yield of iturin A. The effect of adding various concentrations of precursor amino acids l-asparagine (Asn), l-aspartic acid (Asp), l-glutamic acid (Glu), l-glutamine (Gln), l-Serine (Ser) and l-proline (Pro) at different adding times (3 and 12 h) on iturin A production and cell growth was studied. The respective addition of amino acids (Asp 0.28 g/L; Asn 0.36 g/L; Glu 0.20, 0.28 and 0.360 g/L; Gln 0.20, 0.28 and 0.36 g/L; Pro 0.12, 0.20, 0.28 and 0.36 g/L) at 3 h was shown to improve cell growth but did not affect the yield of iturin A. Meanwhile, the individual addition of the same amino acids at 12 h improved cell growth and increased the yield of iturin A. Excellent correlation was obtained between the predicted and measured values, suggesting that the regression model was accurate and reliable; highly significant (P < 0.0001), and the determination coefficient (R2 = 0.975). When 0.0752 g/L Asn; 0.1992 g/L Gln and 0.1464 g/L Pro were added at 12 h, the yield of iturin A reached 0.85 g/L, which is 32.81%-fold higher than that of the initial process. Therefore, this study obtained optimal parameters for iturin A production by the experimental method, and process validation gave high iturin A yields (0.85 g/L) during a 60 h fermentation. These findings could guide an up-scaling of the fermentation process. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-02540-7) contains supplementary material, which is available to authorized users.

A simple and efficient method for the extraction and separation of menaquinone homologs from wet biomass of Flavobacterium.

Menaquinone homologs (MK-n), that is, MK-4, MK-5, and MK-6, can be produced by the fermentation of Flavobacterium. In this study, we proposed a simple and efficient method for the extraction of menaquinones from wet cells without the process of drying the biomass. Meanwhile, a rapid and effective solution for the separation of menaquinone homologs was developed using a single organic solvent, which was conducive to the recovery of the solvent. The results showed that the highest yield was obtained with pretreatment using absolute ethanol at a ratio of 6:1 (v/m) for 30 min and then two extractions of 30 min each using methanol at a ratio of 6:1 (v/m). The recovery efficiency of the menaquinones reached to 102.8% compared to the positive control. The menaquinone homologs were effectively separated using methanol as eluent at a flow rate of 0.52 mL/min by a glass reverse-phase C18 silica gel column with a height-to-diameter ratio of 5.5:1. The recovery of menaquinones achieved was 99.6%. In conclusion, the methods for extraction and separation of menaquinone homologs from wet Flavobacterium cells were simple and efficient, which makes them suitable not only on a laboratory scale but also for application on a large scale.

Replacing the decoy epitope of PCV2b capsid protein with a protective epitope enhances efficacy of PCV2b vaccine.

Microbial pathogens may evolve decoy epitopes to evade host immune responses. In recent years, a decoy epitope has been identified in the capsid protein (CP) of porcine circovirus type 2b (PCV2b) to divert the immune response away from protective epitopes in pigs. To avoid the decoy effect, we designed and produced a recombinant PCV2b CP (ΔCP) by replacing the decoy epitope with a neutralizing B cell epitope derived from CP and tested the ability of ΔCP to induce protective antibody responses in mice and pigs. As expected, the ΔCP, unlike inactivated PCV2b vaccines, recombinant PCV2b CP, and natural PCV2b infection, did not induce anti-decoy epitope antibodies. Although unable to form typical virus-like particles (VLPs), the ΔCP could increase the production of the anti-PCV2b antibodies among which no antibody against the decoy epitopes, and therefore induces improved protective immune responses in pigs challenged with PCV2b. These results provide an alternative strategy for development of recombinant subunit vaccines against PCV2b, and possibly other viruses, by replacing the decoy epitope with a protective epitope.

Single immunization with a recombinant multiple-epitope protein induced protection against FMDV type Asia 1 in cattle.

To develop recombinant epitope vaccines against the foot-and-mouth disease virus (FMDV) serotype Asia 1, genes encoding six recombinant proteins (A1-A6) consisting of different combinations of B-cell and T-cell epitopes from VP1 capsid protein (VP1) of FMDV were constructed. These proteins were expressed in Escherichia coli and used to immunize animals. Our results showed that A6 elicited higher titers of neutralizing antibodies after single inoculation in guinea pigs than did the other five recombinant proteins, as determined by micro-neutralization tests. In addition, a strong lymphocyte proliferation response and Th1 type immunity were observed in splenocytes from the mice immunized with A6. Further tests carried out in cattle demonstrated that a single inoculation with A6 generated comparable levels of neutralizing antibodies as inactivated vaccine and protected 4 of 5 cattle against challenge with FMDV type Asia 1. Our results suggest that A6 might be a promising recombinant vaccine against FMDV type Asia 1 in cattle.

Detection of the Assembly and Disassembly of PCV2b Virus-Like Particles Using Fluorescence Spectroscopy Analysis.

Monitoring the assembly and disassembly of virus-like particles (VLPs) is important in developing effective VLP-based vaccines. We tried to establish a simple and rapid method to evaluate the status of VLP assembly using fluorescence spectroscopic analysis (FSA) while developing a VLP-based vaccine against porcine circovirus type 2b (PCV2b). We synthesized the gene coding for PCV2b capsid protein (CP). The CP was expressed in Escherichia coli in a soluble form, dialyzed into three different buffers, and assembled into VLPs. The immunogenicity of the VLPs was evaluated by an enzyme-linked immunosorbent assay using the sera of mice immunized with inactivated PCV2b. The VLP assembly was detected using transmission electron microscopy and FSA. The assembled VLPs showed a distinct FSA curve with a peak at 320 nm. We found that the assembly status was related to the immunogenicity, fluorescence intensity, and morphology of the VLP. The FSA assay was able to monitor the various denatured statuses of PCV2b VLPs treated with ß-mercaptoethanol or ß-mercaptoethanol plus urea. We have demonstrated that FSA can be used to detect the assembly of PCV2b VLPs produced in E. coli. This provides a simple solution for monitoring VLP assembly during the production of VLP-based vaccines.

Influence of hydrophilic amino acids and GC-content on expression of recombinant proteins used in vaccines against foot-and-mouth disease virus in Escherichia coli.

Epitope-based protein expression in Escherichia coli can be improved by adjusting its amino acid composition and encoding genes. To that end, we analyzed 24 recombinant epitope proteins (rEPs) that carry multiple epitopes derived from VP1 protein of foot-and-mouth disease virus. High level expression of the rEPs was attributed to a high content of Arg, Asn, Asp and Thr, a low content of Gln, Pro and Lys, a high content of hydrophilic amino acids and a higher isoelectric point value resulting from abundant Arg. It is also attributed to the appropriate guanine and cytosine content in the encoding genes. The data provide a reference for adjusting the amino acid composition in designing epitope-based proteins used in vaccines and for adjusting the synonymous codons to improve their expressions in E. coli.

Immunization with a HSP65-HER2 fusion peptide selectively eliminates HER2(+) B16 melanoma cells in a xenograft tumor mouse model.

HER2/neu peptide-based vaccines can eliminate human tumors overexpressing the human epidermal growth factor receptor 2 (HER2/neu), but the efficacy of this therapeutic strategy is suboptimal. Heat shock proteins (HSPs) are capable of eliciting efficient cytotoxic T lymphocyte (CTL) responses by cross-presentation. To evaluate whether immunization with a HSP65-HER2 fusion peptide could selectively eliminate HER2(+) B16 melanoma cells in a xenograft tumor mouse model, a HSP65-HER2 fusion peptide was incubated with immature dendritic cells (iDCs) in vitro to determine whether loading of iDCs with HSP65-HER2 could induce the expression of the immunomodulatory cell surface molecule, CD86. In vivo mouse immunizations with HSP65-HER2 or PBS (control) were performed to determine the antitumor effects by longitudinally monitoring changes in tumor volume, weight, and incidence. The effects on percentages of HER2(+) B16 cells in tumors were assessed by confocal microscopy and flow cytometry. The results indicated that loading of iDCs with HSP65-HER2 induced the expression of CD86 in vitro, suggesting that the hybrid antigen was able to stimulate an immune response. Immunization with HSP65-HER2 had no significant influence on tumor weight or volume but significantly reduced tumor incidence (62.5 % in mice injected with 25 µg of HSP65-HER2 vs. 100 % in PBS-injected controls; P < 0.05). Confocal microscopy and flow cytometry analyses revealed that HSP65-HER2 immunization significantly reduced the percentages of HER2(+) B16 cells in xenografted tumors (1.86 % vs. 30.56 % in PBS-injected controls; P = 0.01). Our findings suggest that immunization with the HSP65-HER2 fusion peptide selectively eliminates HER2(+) B16 melanoma cells in a xenograft tumor mouse model and may represent a novel and efficacious targeted therapy of HER2/neu(+) tumors.

MF59 formulated with CpG ODN as a potent adjuvant of recombinant HSP65-MUC1 for inducing anti-MUC1+ tumor immunity in mice.

MF59 is an oil-in-water emulsion adjuvant approved for influenza vaccines for human use in Europe. Due to its Th2 inducing properties, MF59 is seldom tested for cancer vaccines. In this study, MF59 formulated with a C-type CpG oligodeoxynucleotide (YW002) was tested for its Th1 adjuvant activity to induce immune responses to HSP65-MUC1, a recombinant fusion protein incorporating a mycobacterial heat shock protein (HSP65) and mucin 1, cell surface associated (MUC1) derived peptide. Combination of YW002 with MF59 (MF59-YW002) could confer a potent Th1 biasing property to the adjuvant, which enhanced the immunogenicity of HSP65-MUC1 to induce significantly higher levels of specific IgG2c, increased IFN-γ mRNA expression in splenocytes and the generation of antigen-specific cytotoxic T lymphocytes in mice. When prophylactically applied, MF59-YW002 adjuvant containing HSP65-MUC1 inhibited the growth of MUC1+ B16 melanoma and prolonged the survival of tumor-bearing mice. In contrast, adjuvant containing MF59 with HSP65-MUC1 in the absence of YW002, promoted the growth of MUC1+ B16 melanoma in mice. These results suggest that MF59 plus CpG oligodeoxynucleotide might be developed as an efficient adjuvant for tumor vaccines against melanoma, and possibly other tumors.

Recombinant hepatitis B virus surface antigen formulated with B-type CpG oligodeoxynucleotide induces therapeutic immunity against hepatitis B virus surface antigen-expressing liver cancer cells in mice.

To develop a therapeutic vaccine against hepatitis B virus surface antigen (HBsAg)-expressing liver cancer, we tried to prepare a vaccine by formulating recombinant HBsAg with BW006, a B type CpG oligodeoxynucleotide (ODN) with Th1-biasing activity, and examined its potency of inducing therapeutic immunity against HBsAg-expressing liver cancer cells in mice. When applied therapeutically, BW006 could assist HBsAg to induce vigorous immune responses capable of inhibiting the growth of HBsAg-expressing liver cancer cells and prolonging the survival of mice bearing HBsAg-expressing liver cancer cells. In vivo and in vitro experiments showed that the BW006-adjuvanted HBsAg enhanced the production of IgG2a antibodies, interferon-γ, and interleukin-12 and facilitated the generation of specific cytotoxic T lymphocyte that killed the HBsAg-expressing liver cancer cells. These results suggest that the BW006-adjuvanted HBsAg might be developed into a candidate tumor vaccine for the treatment of HBsAg-expressing liver cancer.

Correlation between efficacy and structure of recombinant epitope vaccines against bovine type O foot and mouth disease virus.

To develop recombinant epitope vaccines against foot-and-mouth disease virus (FMDV), genes coding for six recombinant proteins (rP1­rP6) consisting of different combinations of B cell and T cell epitope from VP1 capsid protein (VP1) of type O FMDV were constructed and the 3D structure of these proteins analyzed. This revealed a surface-exposed RGD sequence of B cell epitopes in all six recombinant proteins as that in VP1 of FMDV and rP1, rP2 and rP4 globally mimicked the backbone conformation of the VP1. rP1, rP2 and rP4 stimulated guinea pigs to produce higher level of neutralizing antibodies capable of protecting suckling mice against FMDV challenge. rP1 stimulated cattle to produce FMDV-neutralizing antibody. The data suggest that an efficient recombinant epitope vaccine against FMDV should share local similarities with the natural VP1 of FMDV.

A CpG oligodeoxynucleotide potentiates the anti-tumor effect of HSP65-Her2 fusion protein against Her2 positive B16 melanoma in mice.

Although being promising tumor vaccine candidates in animal models, heat shock protein (HSP)-based tumor vaccines have not yet succeeded in the clinical trials, implying the necessity to be formulated with appropriate adjutants to enhance their immunogenicity. In this study, we investigated whether a B-class CpG ODN (BW006), a TLR9 agonist, could facilitate HSP65-Her2, a recombinant protein between mycobacterial HSP65 and Her2-derived peptide, to induce vigorous anti-tumor activity against Her2 positive tumors in mice both prophylactically and therapeutically. It was found that BW006 could enhance prophylactic and therapeutic effect of HSP65-Her2 with improved survival of the mice bearing Her2(+) B16 melanoma and HSP65-Her2 specific Th1 response.

Recombinant heat shock protein 65 carrying PADRE and HBV epitopes activates dendritic cells and elicits HBV-specific CTL responses.

BCG Hsp65 and PADRE have been shown to be potent to enhance antigen specific immunity. In order to explore the possibility to utilize them for the development of HBV therapeutic vaccine, a chimeric protein, Hsp65-HBV, was created by fusing PADRE and epitopes from HBV to the carboxyl-terminus of BCG Hsp65 and expressed in E. coli. We evaluated its effects on human dendritic cell maturation and specific CTL induction in vitro. Results showed that Hsp65-HBV could activate human dendritic cells by up-regulating the expressions of HLA-A2, HLA-DR and CD86, companioning with high level of IL-12 secretion. Furthermore, Hsp65-HBV matured DCs could significantly stimulate human autologous CD8(+) T cell proliferation and induce HBV-specific CTLs. Hsp65-HBV was also shown to generate HBsAg-specific CTLs in vivo in mice. These results indicated that Hsp65-HBV might be a candidate for the treatment of chronic HBV infection.

Detection of circulating anti-mucin 1 (MUC1) antibodies in breast tumor patients by indirect enzyme-linked immunosorbent assay using a recombinant MUC1 protein containing six tandem repeats and expressed in Escherichia coli.

Mucin 1 (MUC1), a tumor-associated antigen, is a transmembrane glycoprotein expressed by normal epithelial cells and overexpressed by carcinomas of epithelial origin. Autoantibodies against MUC1 are often found in circulation, either free or bound to immune complexes, which might contribute to limit tumor outgrowth and dissemination by antibody-dependent cell-mediated cytotoxicity, and were found favorably predictive of survival in early breast cancer patients. There is no commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting the anti-MUC1 antibodies in human serum thus far. To detect circulating anti-MUC1 antibodies, we established an indirect ELISA (I-ELISA) using a recombinant MUC1 protein containing six tandem repeat sequences of MUC1 after the antigenicity and specificity of the protein were confirmed. The I-ELISA had a sensitivity of 91.3% and a specificity of 94.1% when a competitive I-ELISA was used as a reference test. The results showed that more patients with benign breast tumors (P = 0.001) and breast cancer patients before primary treatment (P = 0.010) were found to have anti-MUC1 IgG than healthy women; anti-MUC1 IgG before primary treatment was found more than after primary treatment (P = 0.016) in breast cancer patients. Interestingly, the anti-MUC1 IgG serum level was reversely correlated to that of CA15-3 antigen in advanced-stage patients (r = -0.4294, P = 0.046). Our study has demonstrated the suitability of the established I-ELISA for detecting circulating anti-MUC1 antibodies in human serum. Furthermore, we found that circulating anti-MUC1 antibodies may still bind MUC1 shed into blood in stage IV breast cancer, which can support the use of MUC1-target immune therapy strategies.

Purification of clinical-grade recombinant HSP65-MUCI fusion protein.

HSP65-MUCI is a fusion protein between BCG (Bacille Calmette-Guerin)-derived HSP65 (heat-shock protein 65) and human MUCI (mucin I) VNTR (variable number of tandem repeats)-domain peptides that has shown antitumour efficacy. China's Food and Drug Administration has recently approved a Phase I clinical trial using HSP65-MUCI for the treatment of MUCI-positive breast cancer. In order to produce sufficient quantities of clinical-grade HSP65-MUCI, we established a pilot-scale purification scheme comprising two steps of column chromatography: HIC (hydrophobic-interaction chromatography) and IEX (ion-exchange chromatography). The pH values of the buffers used in homogenization and HIC were adjusted to pH 9.0 to maintain protein stability and prevent protein degradation. Using this manufacturing process, we obtained clinical-grade HSP65-MUCI with a yield of 400 mg per 70 g of wet cell pellet and >96% purity.

Vaccination with B16 tumor cell lysate plus recombinant Mycobacterium tuberculosis Hsp70 induces antimelanoma effect in mice.

Tumor cell lysate (TCL) has an advantage of containing an extensive repertoire of tumor antigens but requires proper adjuvants to enhance its immunogenicity when used as an efficient tumor vaccine. Mycobacterium tuberculosis-derived heat shock protein 70 (TBHsp70) has been shown to assist crosspresentation of exogenously applied tumor antigens and activate innate immunity against tumor cells. In this study, TBHsp70-B16TCL, a preparation generated by mixing recombinant TBHsp70 and TCL of B16 melanoma cells directly, was tested for its immunogenicity as a tumor vaccine. The TBHsp70-B16TCL induced a significant inhibition of the growth and metastasis of B16 melanoma in mice and prolonged the survival of B16 melanoma-bearing mice. The inhibition was correlated with the specific immune responses induced by TBHsp70-B16TCL. The data suggest that recombinant TBHsp70-adjuvanted TCL might be developed into effective tumor vaccines for melanomas and possibly for other tumors.

A recombinant truncated FMDV 3AB protein used to better distinguish between infected and vaccinated cattle.

To distinguish the antibodies induced by Foot-and-mouth disease virus (FMDV) infection from those induced by vaccination, a recombinant N-terminal truncated FMDV non-structural protein (NSP) of 3AB, designated as r3aB, was constructed by deleting 80 amino acids displayed about 30% homology to transposase IS4 family protein of Escherichia coli, expressed in E. coli BL21 (DE3) and then purified. The r3aB was majorly expressed in soluble fraction and presented as homogeneous monomers after purification. Using r3aB as coating antigen, an indirect ELISA was established to specifically identify antibodies induced by FMDV infection but not those induced by vaccination. Compared with 3AB, r3aB was more specific to catch antibodies against NSP. The performance of this assay was validated by two commercial FMDV NSP ELISA kits. The result suggested that the r3aB coated ELISA could be developed into a kit to better distinguish between infected and vaccinated cattle.

Vaccination with TCL plus MHSP65 induces anti-lung cancer immunity in mice.

To develop effective anti-lung cancer vaccines, we directly mixed mycobacterial heat shock protein 65 (MHSP65) and tumor cell lysate (TCL) from Lewis lung cancer cells in vitro and tested its efficacy on stimulating anti-tumor immunity. Our results showed that MHSP65-TCL immunization significantly inhibited the growth of lung cancer in mice and prolonged the survival of lung cancer bearing mice. In vivo and in vitro data suggest that MHSP65-TCL could induce specific CTL responses and non-specific immunity, both of which could contribute to the tumor inhibition. Thus, this report provides an easy approach to prepare an efficient TCL based tumor vaccine.

[Expression, purification and activity analysis of BCG HSP70.].

AIM: To get BCG HSP70 protein with excellent biologic activity through E.coli. expression and purification. METHODS: BCG HSP70 gene was amplified by PCR and inserted into vector pMD18-T. After confirmed by sequencing, the gene was subcloned into expression vector pET28a. Recombinant pET28a/HSP70 was transformed into E.coli. BL21(DE3). Recombinant BCG HSP70 protein was expressed with IPTG induction and the purified protein was then identified by SDS-PAGE and Western blot. And its effect on the proliferation of mouse splenocytes was observed. RESULTS: Gene encoding BCG HSP70 which was identical with that published in GenBank was successfully obtained by PCR. SDS-PAGE analysis showed a protein with relative molecular mass of 70 000 was expressed. When the purified protein was detected by Western blot analysis, a specific protein with a molecular mass of 70 000 could be visualized. The purity of the purified protein was about 96.5%. The purified protein could stimulate the proliferation of mouse splenocytes significantly. CONCLUSION: BCG HSP70 is expressed and purified successfully, which would lay a foundation for further research on BCG HSP70 and BCG.