Isorhamnetin Regulates Programmed Death Ligand-1 Expression by Suppressing the EGFR-STAT3 Signaling Pathway in Canine Mammary Tumors.

Programmed death ligand-1 (PD-L1) is highly expressed in a variety of cancer cells and suggests a poorer prognosis for patients. The natural compound isorhamnetin (ISO) shows promise in treating cancers and causing damage to canine mammary tumor (CMT) cells. We investigated the mechanism of ISO in reducing PD-L1 expression in CMT cells. Clustered, regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) was used to mediate CD274 knockout in U27 cells. Then, monoclonal cells were screened and cultured. Nucleotide sequencing and expression of PD-L1 were detected. Additionally, we examined cell migration, invasion, and damage. Immunofluorescent staining of PD-L1 was examined in U27 cells. The signaling pathways were measured by Western blotting. Murine xenotransplantation models and murine immunocompetent allograft mammary tumor models were established to evaluate the effect of ISO therapy. Expression of Ki-67, caspase3, and PD-L1 were analyzed by immunohistochemistry. A pull-down assay was used to explore which proteins could bind to ISO. Canine EGFR protein was purified and used to detect whether it directly binds to ISO using a surface plasmon resonance assay. ISO inhibited the EGFR-STAT3-PD-L1 signaling pathway and blocked cancer growth, significantly increasing the survival rate of healthy cells. The cell membrane receptor EGFR was identified as a direct target of ISO. ISO could be exploited as an antineoplastic treatment of CMT by targeting EGFR to suppress PD-L1 expression.

Homoharringtonine induces apoptosis of mammary carcinoma cells by inhibiting the AKT/mTOR signaling pathway.

Mammary tumour is the most common type of tumour in dogs, especially in unneutered female dogs. Homoharringtonine (HHT) is a natural alkaloid that can be used to treat various types of human tumour. However, the inhibitory effect and mechanism of HHT on canine mammary carcinomas (CMC) remain unclear. This study aimed to evaluate the inhibitory effect of HHT on CMC in vitro and determine its underlying molecular mechanism. The effects of HHT on the cytotoxicity of CMC U27 cells were evaluated by the cell counting kit-8, wound healing, and Transwell assays. HHT-induced apoptosis of U27 cells was detected by JC-1 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Moreover, the gene expression of B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) were analysed using quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the protein expression of protein kinase B/mammalian target of rapamycin (AKT/mTOR) and mitochondrial apoptosis proteins were determined by western blotting. Furthermore, mammary tumour-bearing mouse models were established using 4T1 cells to evaluate the therapeutic effect of HHT. It was found that HHT could significantly down-regulated the protein expression of p-AKT, p-mTOR, and Bcl-2, and up-regulated the protein expression of P53, Bax, cleaved caspase-3, and cleaved caspase-9. In addition, HHT significantly suppressed both tumour volume and mass in mammary tumour mice. In conclusion, HHT damages CMC cells by inhibiting the AKT/mTOR signalling pathway and inducing mitochondrial apoptosis. Such findings lay a theoretical foundation for the clinical treatment of CMC and provide more options for clinical medication.

Genetic specialization of key bifidobacterial phylotypes in multiple mother-infant dyad cohorts from geographically isolated populations.

Little has been known about symbiotic relationships and host specificity for symbionts in the human gut microbiome so far. Bifidobacteria are a paragon of the symbiotic bacteria biota in the human gut. In this study, we characterized the population genetic structure of three bifidobacteria species from 58 healthy mother-infant pairs of three ethnic groups in China, geographically isolated, by Rep-PCR, multi-locus sequence analysis (MLSA), and in vitro carbohydrate utilization. One hundred strains tested were incorporated into 50 sequence types (STs), of which 29 STs, 17 STs, and 4 STs belong to B. longum subsp. longum, B. breve, and B. animalis subsp. lactis, respectively. The conspecific strains from the same mother-child pair were genetically very similar, supporting the vertical transmission of Bifidobacterium phylotypes from mother to offspring. In particular, results based on allele profiles and phylogeny showed that B. longum subsp. longum and B. breve exhibited considerable intraspecies genetic heterogeneity across three ethnic groups, and strains were clustered into ethnicity-specific lineages. Yet almost all strains of B. animalis subsp. lactis were incorporated into the same phylogenetic clade, regardless of ethnic origin. Our findings support the hypothesis of co-evolution between human gut symbionts and their respective populations, which is closely linked to the lifestyle of specific bacterial lineages. Hence, the natural and evolutionary history of Bifidobacterium species would be an additional consideration when selecting bifidobacterial strains for industrial and therapeutic applications.

Characterization of a highly virulent Avibacterium paragallinarum isolate.

Infectious coryza (IC) is an important respiratory infectious disease in chickens. In this study, an Avibacterium paragallinarum Page serovar C strain, named ZJ-C, was isolated from a local layer flock that was routinely vaccinated with an inactivated trivalent vaccine, using reference strain Modesto as the serovar C immunogen. The pathogenicity, immunogenicity, and genetic characteristics of ZJ-C were studied. The minimum pathogenic dose of the isolate was 100 CFU, which was 1/1,000 of the dose of the serovar C reference strain Modesto. The vaccination-challenge trial in specific pathogen-free (SPF) chickens showed that the ZJ-C bacterin could provide 100% protection against challenge from both ZJ-C and Modesto strains, whereas Modesto provided 100% protection against challenge from itself, but only 70% protection against ZJ-C. Sequence analysis of the HMTp210 hypervariable region (region 2) showed that the homology of region 2 between ZJ-C and Modesto was 96.14%, whereas the homology between ZJ-C and the Kume serovar C-4 reference strain HP60 was 99.83%. Phylogenetic analysis of region 2 showed that ZJ-C was most closely related to cluster C-4, represented by HP60. The experimental data obtained in this study will help the selection of optimal vaccine strains and assist serotyping studies of Av. paragallinarum.

Vaccination with inactivated multivalent vaccines is a primary strategy to control Infectious coryza. Avibacterium paragallinarum serotyping is important for effective protection as inactivated whole-cell vaccines provide protection against only the serogroup or serovar from which the vaccine was derived. In this study, a novel serovar within the serogroup C Avibacterium paragallinarum isolate ZJ-C has been characterized first time in China. It was highly virulent and induced 100% cross-protection to Modesto bacterin vaccinated chickens, but not the other way around.

Toosendanin-induced apoptosis of CMT-U27 is mediated through the mitochondrial apoptotic pathway.

Toosendanin (TSN) is an active compound from the fruit of Melia toosendan Sieb et Zucc. TSN has been shown to have broad-spectrum anti-tumour activities in human cancers. However, there are still many gaps in the knowledge of TSN on canine mammary tumours (CMT). CMT-U27 cells were used to select the optimal acting time and best concentration of TSN to initiate apoptosis. Cell proliferation, cell colony formation, cell migration and cell invasion were analysed. The expression of apoptosis-related genes and proteins were also detected to explore the mechanism of action of TSN. A murine tumour model was established to detect the effect of TSN treatments. The results showed that TSN decreased cell viability of migration and invasion, altered CMT-U27 cell morphology, and inhibited DNA synthesis. TSN-induced cell apoptosis by upregulating BAX, cleaved caspase-3, cleaved caspase-9, p53 and cytochrome C (cytosolic) protein expression, and downregulating Bcl-2 and cytochrome C (mitochondrial) expression. In addition, TSN increased the mRNA transcription levels of cytochrome C, p53 and BAX, and decreased the mRNA expression of Bcl-2. Furthermore, TSN inhibited the growth of CMT xenografts by regulating the expression of genes and proteins activated by the mitochondrial apoptotic pathway. In conclusion, TSN effectively inhibited cell proliferation, migration and invasion activity, as well as induced CMT-U27 cell apoptosis. The study provides a molecular basis for the development of clinical drugs and other therapeutic options.

Hausdorff GAN: Improving GAN Generation Quality With Hausdorff Metric.

Data usually resides on a manifold, and the minimal dimension of such a manifold is called its intrinsic dimension. This fundamental data property is not considered in the generative adversarial network (GAN) model along with its its variants; such that original data and generated data often hold different intrinsic dimensions. The different intrinsic dimensions of both generated and original data may cause generated data distribution to not match original data distribution completely, and it certainly will hurt the quality of generated data. In this study, we first show that GAN is often unable to generate simulation data, holding the same intrinsic dimension as the original data with both theoretical analysis and experimental illustration. Next, we propose a new model, called Hausdorff GAN, which removes the issue of different intrinsic dimensions and introduces the Hausdorff metric into GAN training to generate higher quality data. This provides new insights into the success of Hausdorff GAN. Specifically, we utilize a mapping function to map both original and generated data into the same manifold. We then calculate the Hausdorff distance to measure the difference between the mapped original data and the mapped generated data, toward pushing generated data to the side of original data. Finally, we conduct extensive experiments (using MNIST, CIFAR10, and CelebA datasets) to demonstrate the significant performance improvement of the Hausdorff GAN in achieving the largest Inception Score and the smallest Frechet inception distance (FID) score as well as producing diverse generated data at different resolutions.

Comparative Genomics Analysis and Outer Membrane Vesicle-Mediated Horizontal Antibiotic-Resistance Gene Transfer in Avibacterium paragallinarum.

Avibacterium paragallinarum is the etiological agent of infectious coryza, an acute respiratory disease of chickens that is globally distributed and causes serious economic losses for chicken production. A. paragallinarum is a Gram-negative bacterium that releases outer membrane vesicles (OMVs). In this study, a comparative genomic analysis of A. paragallinarum isolate P4chr1 and its OMVs was carried out, and the ability to transfer antibiotic resistance genes (ARGs) via the OMVs was studied. Sequencing and data analyses demonstrated that the genomic size of A. paragallinarum P4chr1 was approximately 2.77 Mb with a 25 kb tolerance island that covered six types of antibiotics and 11 ARGs. The genomic size of its OMVs was approximately 2.69 Mb, covering 97% of the genomic length and almost all the gene sequences of P4chr1. Purified and DNase-treated A. paragallinarum P4chr1 OMVs were cocultured with the antibiotic-sensitive A. paragallinarum Modesto strain on an antibiotic (chloramphenicol, erythromycin, tetracycline, or streptomycin)-containing plate, and the corresponding ARGs were detected in the colonies grown on the plates. However, using an antimicrobial susceptibility test, we found that ARGs delivered by OMVs were not persistent but only appeared transiently on the antibiotic-containing plates. Antibiotic resistance and ARGs were lost by the second bacterial passage. IMPORTANCE The functions and roles of OMVs on ARG and virulent gene transfer and dissemination have been reported in numerous Gram-negative bacteria. However, the role of OMVs in mediating antibiotic resistance in A. paragallinarum has not been reported. This study is the first report to compare the genomic characteristics of OMVs with its parent A. paragallinarum strain and to study A. paragallinarum ARG transfer via OMVs. This work has provided useful data for further studies focusing on nonplasmid ARG transfer mediated by A. paragallinarum OMVs.

In Situ Generation of Ultrathin MoS2 Nanosheets in Carbon Matrix for High Energy Density Photo-Responsive Supercapacitors.

Stimuli-responsive supercapacitors have attracted broad interest in constructing self-powered smart devices. However, due to the demand for high cyclic stability, supercapacitors usually utilize stable or inert electrode materials, which are difficult to exhibit dynamic or stimuli-responsive behavior. Herein, this issue is addressed by designing a MoS2 @carbon core-shell structure with ultrathin MoS2 nanosheets incorporated in the carbon matrix. In the three-electrode system, MoS2 @carbon delivers a specific capacitance of 1302 F g-1 at a current density of 1.0 A g-1 and shows a 90% capacitance retention after 10 000 charging-discharging cycles. The MoS2 @carbon-based asymmetric supercapacitor displays an energy density of 75.1 Wh kg-1 at the power density of 900 W kg-1 . Because the photo-generated electrons can efficiently migrate from MoS2 nanosheets to the carbon matrix, the assembled photo-responsive supercapacitor can answer the stimulation of ultraviolet-visible-near infrared illumination by increasing the capacitance. Particularly, under the stimulation of UV light (365 nm, 0.08 W cm-2 ), the device exhibits a ≈4.50% (≈13.9 F g-1 ) increase in capacitance after each charging-discharging cycle. The study provides a guideline for designing multi-functional supercapacitors that serve as both the energy supplier and the photo-detector.

PEDV nsp16 negatively regulates innate immunity to promote viral proliferation.

Type-I IFNs (IFN-I) provide a key mediator of innate antiviral response during virus proliferation. Porcine epidemic diarrhea virus (PEDV), which causes diarrhea in swine of all ages, is a worldwide-distributed alphacoronavirus with economic importance. Here, we screened PEDV RNA modification enzymes involved in regulating antiviral response. Whereas the PEDV nsp13 barely regulates type I IFN, inflammatory cytokines (IL-6, TNF-a) and MHCII, nsp16 and nsp14 (to a lesser extent) down-regulate these antiviral effectors. Importantly, we found nsp16 KDKE tetrad appears to play a key role in interferon inhibition by mutating the D129 catalytic residue. Mechanistically, nsp16 down-regulates the activities of RIG-I and MDA5 mediated IFN-ß and ISRE. In turn, the mRNA levels of IFIT family members (IFIT1, IFIT2, IFIT3) was inhibited in cells overexpressing nsp16. In addition, nsp10 enhanced the inhibitory effect of nsp16 on IFN-ß. Altogether these results indicate PEDV nsp16 negatively regulates innate immunity to promote viral proliferation. Findings from this study provides novel perspective to advance the understanding in the pathogenesis of PEDV.

Rapid detection of food-borne Salmonella contamination using IMBs-qPCR method based on pagC gene.

Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10h, which is a promising rapid method to detect Salmonella in emergency.

Rapid detection of food-borne Salmonella contamination using IMBs-qPCR method based on pagC gene

Abstract Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1 mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18 CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10 h, which is a promising rapid method to detect Salmonella in emergency.

Rapid detection of food-borne Salmonella contamination using IMBs-qPCR method based on pagC gene

Abstract Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1 mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18 CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10 h, which is a promising rapid method to detect Salmonella in emergency.

Rapid detection of food-borne Salmonella contamination using IMBs-qPCR method based on pagC gene

Abstract Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1 mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18 CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10 h, which is a promising rapid method to detect Salmonella in emergency.