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Thymus, an important central immune organ in pigs, is the site of T lymphocyte development and maturation and an important target organ for infection and replication of various pathogens. Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) infection results in severe thymic atrophy in piglets. This study aimed to explore the effects of HP-PRRSV on the thymic structure of piglets to elucidate the pathogenesis of thymic atrophy induced by HP-PRRSV. In this study, histopathological techniques and immunofluorescence double staining techniques were used to analyze thymic tissues infected by HP-PRRSV to explore the structural changes of thymus caused by the viral infection and its target cell types. An antibody of cluster of differentiation (CD) 3 (CD3), CD20, CD80, or calgranulin + calprotectin was applied to identify T cells, B cells, dendritic cells (DCs), and macrophages, respectively. The results indicated that a variety of cell components in the thymic tissue were diffusely damaged after viral infection. In the infected thymic tissue, CD80- or calgranulin + calprotectin- -labeled cells supported the HP-PRRSV infection, whereas CD3-labeled T cells and CD20- -labeled B cells did not support the viral infection. The results showed that HP-PRRSV caused the reduction of visible cell components in the thymic tissue, and the virus attacked CD80- and calgranulin + calprotectin-positive cells (such as DCs and macrophages) in the thymic tissue, which played an important role in the pathogenesis of thymus atrophy. These results lay the foundation for elucidating the immunosuppression of piglets after infection with HP-PRRSV.

To investigate the optimal androgen concentration for culturing Hetian sheep wool follicle and to detect effects of androgen concentration on wool follicle cell proliferation and apoptosis using immunofluorescence labeling and real-time quantitative fluorescence determinations of wool keratin-associated protein gene expression levels. Wool follicles were isolated by microdissection and wool follicles and skin pieces were cultured in various concentrations of dihydrotestosterone (DHT) in culture medium. Next, daily lengthwise growth measurements of wool follicles were obtained using a microscopic micrometer. Cultured Hetian wool follicles were stained using the SACPIC method to reveal wool follicle structure, while sheep skin slices were used to observe cell proliferation by immunostaining and cell apoptosis using the TUNEL method. At the molecular biological level, keratin-associated protein (Kap) gene expression was studied using wool follicles cultured for various numbers of days in vitro. Effects of androgen concentrations on Hetian wool follicle growth and development were experimentally studied. EdU proliferation assays revealed that androgen promoted cell proliferation within wool follicle dermal papillae. TUNEL apoptosis detection demonstrated that androgen treatment could delay cell apoptosis. Quantitative reverse transcription polymerase chain reaction (qPCR) results demonstrated that gene expression level patterns of Hetian mountain sheep super-high sulfur protein. Kap1.1, KIF1.2, Kap2.12 and Kap4.2 gene expression level of the mountainous experimental group was significantly higher than plains Hetian sheep. An androgen concentration of 100 nM can promote the growth of Hetian wool follicle cells in vitro, resulting in overexpression of some genes of the Kap family. Resumo Investigar a concentração ideal de andrógenos em cultura de folículos pilosos de carneiro Hetiano e detectar os efeitos da concentração de andrógenos na proliferação e apoptose de células foliculares, por meio de imunofluorescência e de determinação quantitativa, em tempo real, da fluorescência dos níveis de expressão gênica de proteína associada à queratina. Folículos pilosos foram isolados por microdissecção, e folículos de lã e pedaços de pele foram cultivados em várias concentrações de di-hidrotestosterona (DHT) em meio de cultura. Em seguida, medições diárias de crescimento longitudinal dos folículos capilares foram obtidas usando um micrômetro microscópico. Folículos de lã cultivados de Hetianos foram corados pelo método SACPIC para revelar a estrutura do folículo piloso, enquanto fatias de pele de carneiro foram usadas para observar a proliferação celular por imunocoloração e apoptose celular por meio do método TUNEL. Em âmbito da biologia molecular, a expressão gênica da proteína associada à queratina (Kap) foi estudada usando folículos capilares cultivados por vários dias, in vitro. Os efeitos das concentrações de andrógenos no crescimento e desenvolvimento dos folículos de lã de Hetianos foram estudados experimentalmente. Ensaios de proliferação de EdU revelaram que o andrógeno promoveu a proliferação celular dentro das papilas dérmicas do folículo piloso. A detecção de apoptose por TUNEL demonstrou que o tratamento com andrógeno poderia atrasar a apoptose celular. Os resultados da reação em cadeia da polimerase transcrição reversa quantitativa (qPCR) demonstraram que os padrões de expressão gênica da proteína de enxofre Kap1.1, KIF1.2, Kap2.12 e Kap4.2 foram significativamente maiores no grupo de ovinos Hetianos de montanha. Uma concentração de androgênio de 100 nM pode promover o crescimento de células foliculares de lã de Hetianos in vitro, resultando na superexpressão de alguns genes da família Kap.

Aims Riemerella anatipestifer infections of goslings and ducklings can result in high mortality. Since there are at least 21 serotypes of R. anatipestifer, cross-protection is an important goal for vaccine development. Methods and results In this study, we evaluated the immunostimulatory effect of different immunization regimens - the traditional inactivated vaccine vs prime-boost regimens using DNA and protein subunit vaccines (DNA+subunit, subunit+subunit, subunit+inactivated and DNA+DNA). Results showed that, when compared to the inactivated vaccine, prime-boost regimens induced higher and up to 16-week longer lasting levels of antibody responses, significantly elevated the percentage of the cytotoxic CD8+ T cell and higher expression levels of IFN-γ, IL-6 and IL-12 mRNAs. Furthermore, as an indication of cross-protection, sera from prime-boost regimens were able to recognize lysates of R. anatipestifer serotypes 1, 2 and 6. Conclusions Prime-boost regimens especially DNA-prime and protein-boost, induce strong long-term immune response and may prove protective for breeder ducks requiring long-term protection. Significance and impact of the study It is worth mentioning that the subunit+inactivated regimen group also elicited strong immune response. The cost of this regimen may only be half of the other prime-boost regimens, making this subunit + inactivated combination an attractive option.

To explore the effect of rehabilitation training on cognitive impairment after cerebrovascular accident and its potential mechanism.100 patients of cerebrovascular accident treated in our hospital from August 2018 to August 2019 were selected as the subjects, and 50 patients with physical examination were selected as healthy control group. The patients with cerebrovascular accident were randomly divided into control group (50 patients) and research group (50 patients). The patients in the control group were given routine medication, the patients in research group were given rehabilitation training on the basis of routine drug therapy. The blood samples were collected on admission and 6 months after admission to detect the molecular markers related to inflammation, nerve cell nutrition and function and apoptosis in the serum. The cognitive function was evaluated by scales. We established a rat cerebral ischemia model, compared the differences in the evasive latency, serum CRP, BNDF, Bcl-2, BAX, Glu, NE levels and BNDF, TrkB, pTrkB, JNK levels in hippocampus, amygdala, and prefrontal tissue between model rats after rehabilitation training and model rats without rehabilitation training.On admission, there were no significant differences in the scores of Barthel index (BI), Fugl-Meyer motor function scale (FM), Montreal cognitive assessment scale (MoCA) and mini-mental state examination (MMSE) (p0.05). 6 months later, the above scores and BNDF, Bcl-2, and norepinephrine were significantly higher in the research group (p0.05), while CRP, Bax, 5-HT and glutamate in the research group were significantly lower than those in the control group (p0.05).Rehabilitation training can improve the motor function, mental state and cognitive level of patients, reduce the levels of neurotoxic factors, pro-inflammatory factors and pro-apoptotic factors, and improve the levels of inhibiting apoptotic factors, neurotrophic factors and neurotransmitters. In animal experiments, rehabilitation training can increase BDNF and its activated receptors in hippocampus, amygdala and prefrontal lobe of rats, and decrease JNK of apoptotic protein, suggesting that rehabilitation training may regulate the expression of apoptotic proteins Bcl-2 and Bax by upregulating BDNF and its receptors and acting on JNK pathway, thereby inhibiting cell apoptosis and improving cognitive impairment after cerebrovascular accident.

AIMS To evaluate the effect of a DNA priming and protein boosting immunization scheme in ducks. METHODS AND RESULTS Pekin ducks were immunized with pTCY/VP2 DNA vaccine; on day 14 (D14) after primary immunization, the ducks were boosted with either the same vaccine (DNA + DNA) or the rVP2 vaccine (DNA + rVP2). CpG oligodeoxynucleotides containing three copies of GACGTT motifs were used as the adjuvant in the vaccines. Compared with unimmunized controls, both immunization schemes significantly increased the titre of antigen-specific antibodies, lymphocyte proliferation index, percentage of CD4+ and CD8+ cells in peripheral blood mononuclear cells (PBMCs) and mRNA expression of interferon (IFN)-α, IFN-γ, interleukin (IL)-6 and IL-12 in antigen-stimulated PBMCs. Furthermore, compared with the DNA + DNA homologous scheme, the DNA + rVP2 heterologous scheme significantly increased lymphocyte proliferation, percentage of CD4+ and CD8+ cells in PBMCs and upregulation of mRNA expression of cytokines 2 weeks after the boost (D28). CONCLUSIONS The DNA + rVP2 immunization scheme enhanced immune responses, mainly Th1 type, against parvovirus in ducks. SIGNIFICANCE AND IMPACT OF THE STUDY The DNA priming and protein boosting heterologous immunization strategy can be applied to develop vaccines against viral infections in ducks. It can potentially be used in breeding ducks because of long-term immunity may confer protection for ducklings.

Alveolar macrophages (AMs) are not only important immune cell of the host, but also important target cell of a variety of respiratory pathogens. They play an important role in defense against pathogen invasion and in maintaining tissue balance. Interferon (IFN)-γ is a well known multipotent cytokine that has antiviral and antibacterial immune activity and enhances antigen presentation. To better reveal the biological processes of porcine AMs activated by IFN-γ, transcriptomic analysis was performed using Illumina HiSeqTM technique. The results identified 2,248 differentially expressed genes (DEGs), of which 753 were upregulated and 1,495 were downregulated, in porcine AMs 12 h after IFN-γ stimulation, compared with mock-treated porcine AMs. A gene ontology function enrichment analysis of these DEGs indicated that these genes were significantly enriched in functional clusters such as immune response, defense response, and intracellular signaling cascades. Analyzing the Kyoto Encyclopedia of Genes and Genomes pathways of the DEGs showed that these genes are mainly involved in cytokine-cytokine receptor interactions, alpha linolenic acid metabolism, and the RIG-I-like receptor signaling pathway. This study shows that a massive gene expression change occurred in porcine AMs following IFN-γ stimulation, which is critical for understanding the mechanisms of IFN-γ-mediated macrophage activation and immune regulation.

The aim of this study was to explore the effects of micro ribonucleic acid (miR)-195 on diabetic nephropathy (DN) rats through targeting Toll-like receptor 4 (TLR4) and inhibiting the nuclear factor-κB (NF-κB) signaling pathway.The model of DN was first successfully established in rats. All rats were randomly divided into six groups, including control group (n=20), model group (n=20), 25 nM miR-195 mimics group (25 nM M group, n=20), 50 nM M group (n=20), 25 nM miR-195 inhibitor group (25 nM I group, n=20), and 50 nM I group (n=20). Urine volume, proteins and inflammatory factors were detected in each group, respectively. Subsequently, macrophages were cultured and transfected in vitro. The mRNA expressions of miR-195 and TLR4 in control group and model groups were determined using fluorescence quantitative polymerase chain reaction (qPCR). The protein expressions of TLR4 and NF-κB in macrophages were determined using Western blotting. Furthermore, the proliferation of macrophages was detected via cell counting kit-8 (CCK-8) assay.Compared with model group, 24-h urine volume, urine protein, creatinine, urea nitrogen, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 levels declined significantly in 25 nM M group and 50 nM M group (p0.05). However, they increased significantly in 25 nM I group and 50 nM I group (p0.05). It could be suggested that miR-195 mimics might relieve the symptoms of DN rats. In kidney tissues in DN, miR-195 was lowly expressed, whereas TLR4 was highly expressed (p0.01). This suggested that there was a negative correlation between the mRNA expressions of miR-195 and TLR4 (r2=0.4836, p=0.0007). After overexpression of miR-195, the protein expression of TLR4 was significantly reduced (p0.01), indicating that miR-195 could negatively regulate the protein expression of TLR4. Besides, the protein expressions of TLR4 and NF-κB in si-TLR4 group were evidently lower than those in NC group (p0.01). Meanwhile, they also had significant differences in si-TLR4 group compared with si-TLR4 + miR-195 inhibitor group (p0.05). The above results demonstrated that the protein expressions of TLR4 and NF-κB in macrophages could be markedly inhibited by si-TLR4, but be promoted by si-TLR4 + miR-195 inhibitor. CCK-8 assay demonstrated that the proliferation ability of macrophages was remarkably weaker in miR-195 mimics group than NC group (p0.001). Furthermore, it was also significantly weaker in si-TLR4 + miR-195 inhibitor group than si-TLR4 group (p0.05).MiR-195 reduces the release of inflammatory factors and inhibits the proliferation of macrophages through targeting TLR4 and blocking the NF-κB pathway, thereby alleviating the symptoms of DN rats.

To design and evaluate a novel oxyntomodulin (OXM) derivative with albumin-binding helix domain and dual GLP-1 receptor (GLP-1R) and glucagon receptor (GcgR) activation activity to achieve metabolize improvement on the diabetes-related complication.Mutation (D-Ser2) on OXM was performed and then different helix albumin-binding domains were fused to the mutated OXM via a thrombin-cleavable linker to generate seven fusion peptides, named LM01-LM07. Seven LM peptides were synthesized and screened via in vitro receptor activation test, albumin binding measurement and protease cleavage assay to select potent candidate peptide for further in vivo study. Moreover, acute and chronic efficacy studies were conducted to evaluate the efficacy of selected candidate using db/db mice.LM06, as selected OXM derivative, exhibited higher albumin-binding affinity, sustained-release efficiency and balanced activation activities on both GLP-1R and GcgR compared with other ones. Moreover, LM06 was demonstrated with improved hypoglycemic and insulinotropic abilities in receptor-deficient mice via activating GLP-1R. In addition, prolonged anti-diabetic efficacies of LM06 were demonstrated via hypoglycemic duration assay and OGTT in db/db mice. Further pharmacokinetic test of LM06 in both rats and monkeys identified improved half-life and other metabolic characteristics. Nevertheless, 8-week subcutaneously dosed LM06 in db/db mice achieved prominent efficacies on glucostasis, weight-lowering, pancreatic function and adipogenesis via activating GLP-1R and GcgR. Moreover, LM06 also could accelerate diabetic skin wound closure in combination with low-intensity ultrasound.LM06, as a long-acting dual GLP-1R/GcgR agonist, exerts potential as a once-weekly therapeutic candidate against diabetes-related complication in combination with low-intensity ultrasound.

This study aimed at investigating the effect of atorvastatin on the expression of TLR4 and NF-κB in brain tissue of stroke rats and its protective effect on brain.Sixty SD rats were selected. The control group (n=20) was raised without treatment. MCAO was used in the experimental group (n=20) and model group (n=20) for stroke modeling. The experimental group was treated with atorvastatin intragastric administration, while the model group received no treatment. The blood lipid level was detected by a full-automatic biochemical analyzer. The expression levels of TLR4 and NF-κB proteins were detected by Western-Blot (WB), and the mRNA expression of NF-κB and TLR4 in brain tissues was detected by real-time quantitative fluorescence (qtPCR). The neurological deficit score was performed.TC, TG, LDL-C in blood lipid indexes of the experimental group and the model group were remarkably higher than those of control group, while HDL-C was remarkably lower than that of control group (p0.05). After treatment, TC, TG, LDL-C in the experimental group gradually decreased, while HDL-C concentration gradually increased (p0.05). The expression of TLR4 and NF-κB protein in the experimental group was significantly lower than that in the model group (p0.01) but significantly higher than that in the control group (p0.01). The expression of TLR4 and NF-κB mRAN in the experimental group was also considerably lower than that in the model group (p0.01) but higher than that in the control group (p0.01). After atorvastatin treatment, the neurological deficit score of the experimental group was remarkably lower than that of the model group (p0.001).Atorvastatin can effectively reduce the expression of TLR4 and NF-κB in brain tissue of stroke rats, and has a certain protective effect on cerebral nerve function, which is expected to be the first choice for stroke treatment in the future.

1. Paired box (Pax7) gene is a member of the paired box family and plays a critical role in animal growth and muscle development. However, the molecular characterisation of the goose Pax7 gene is unknown. 2. The open-reading frame of goose Pax7 is composed of 1509 bp, which encodes a protein of 503 amino acids and shares high homology with Pax7 of other birds. 3. Ten single-nucleotide polymorphisms were identified in the genomic DNA sequence, 8 located in the intron region and two located in the exon region. 4. Association analysis showed the C122T locus was significantly associated with the body weight of Zhedong-White geese in week 4, 6, 8, 10 and 12. 5. It was concluded that the goose Pax7 gene may be an important candidate gene for goose growth traits and marker-assisted selection.

Aim The cardioprotective effects of sevoflurane postconditioning (SpostC) are eliminated under diabetic conditions, and the underlying mechanism for this phenomenon remains unclear. Many studies have demonstrated that the hypoxia-inducible factor-1 (HIF-1) signalling pathway in the myocardium is impaired under diabetic conditions. This study was to investigate whether deferoxamine (DFO)-induced activation of HIF-1 signalling pathway can restore the cardioprotective effects of SpostC in diabetic rats. Methods A model of myocardial ischaemia/reperfusion (I/R) injury was induced via ligation of the left anterior descending artery. SpostC was conducted by administering 1.0 MAC sevoflurane. After inducing the I/R injury, the following parameters were measured: myocardial infarct size, cardiac function, myocardial ultrastructure, mitochondrial respiratory function, respiratory chain enzyme activity, rate of reactive oxygen species (ROS) generation, and protein expression of HIF-1α, vascular endothelial growth factor (VEGF), cleaved caspase-3, Bcl-2 and Bax. Results After DFO activated HIF-1 in the impaired myocardium of diabetic rats, SpostC significantly upregulated the protein expression of HIF-1α and its downstream mediator VEGF. This improved myocardial mitochondrial respiratory function and respiratory chain enzyme activity and reduced ROS generation as well as the protein expression of cleaved caspase-3 and Bax. As a result, myocardial infarct size decreased, and cardiac function and mitochondrial ultrastructure improved. Conclusion This study demonstrates for the first time that abolishment of the cardioprotective effects of SpostC in diabetic rats is associated with impairment of the HIF-1 signalling pathway and that DFO can activate HIF-1 to restore these cardioprotective effects of SpostC in diabetic rats.

To explore the effect of low protein diet on nonspecific inflammatory changes in mice during aging and related mechanisms.Thirty-two 14-month-old female KM mice were randomly divided into 4 groups: control group, low protein group, high protein group, high protein + rapamycin group. Hematoxylin-eosin (HE) staining was performed to observe the pathological changes of the liver. Immunohistochemistry of liver sections was performed to detect the expression of CD68 protein. HE staining of colon sections was performed to observe intestinal lymphocyte infiltration. The percentage of spleen CD4+ T and CD8+ T cells was detected by flow cytometry. The mTOR expression in the liver was detected by Western blot and immunohistochemistry.Compared with the control group, HE staining of liver tissue sections in high-protein group showed the cytoplasm of hepatocytes was loose and disordered, and the hepatic sinus was significantly expanded. Immunohistochemistry of the liver showed a significant increase in CD68 protein expression. Colorectal HE staining showed extensive lymphocyte infiltration. The number of CD4+ T and CD8+ T cells in spleen flow cytometry was significantly decreased (*p0.05). Western blot and immunohistochemistry detected a significant increase in mTOR expression in the liver (*p0.05, *p0.05). In the High Protein+Rapamycin group and Low-protein group, the time-dependent changes were reduced, the numbers of CD4+ T cell and CD8+ T cell in the spleen were significantly increased (*p0.05) and the expression of mTOR was significantly reduced (*p0.05).Low-protein diet is beneficial for delaying the non-specific inflammatory changes of liver and intestines in middle-aged and aged mice, and this effect may be achieved through down-regulation of mTOR.

Interferon- (IFN-)λ1 is regarded as a potent bio-active molecule in innate immunity. However, little is known about its role in chronic spontaneous urticaria (CSU). We therefore investigated expression of IFN-λ1 in CSU, its cellular location, and its influence on inflammatory cell accumulation by using flow cytometry analysis, skin tissue dispersion, immunohistochemical stain, and a mouse peritoneal inflammation model. The results showed that level of IFN-λ1 was 2.0-fold higher in plasma of the patients with CSU than the level in healthy control (HC) subjects. Among leukocytes examined, only CD8+T cells expressed more IFN-λ1 in CSU blood. Double labeling immunohistochemical staining revealed that IFN-λ1+inflammatory cells such as mast cells, eosinophils, B cells, neutrophils, and macrophages were mainly located in dermis, whereas epidermis tissue highly expressed IFN-λ1. IFN-λ1 induced a dose-dependent increase in number of eosinophils, lymphocytes, mast cells, macrophages, and neutrophils in the peritoneum of mice at 6 h following injection, which was inhibited by pretreatment of the animals with anti-intercellular adhesion molecule- (ICAM-) 1 and/or anti-L-selectin antibodies. In conclusion, IFN-λ1 is likely to play a role in the pathogenesis of CSU. Blocking IFN-λ1 production may help to reduce the accumulation of inflammatory cells in the involved CSU skin.

To investigate the effects of an inhibitor of NF-κB, PDTC (pyrrolidine dithiocarbamate), on TLR4 (Toll-like receptor 4) expression in the left ventricle of Goldblatt hypertension rats.Goldblatt rat model of two-kidney, one-clip (2K1C) hypertension was established in 70 healthy male rats. The rats were randomly divided into sham operation group (S group, n=20), non-drug intervention hypertension group (H group, n=25), and PDTC intervention group (P group, n=25). P group was injected with PDTC. The clip was inserted in the left renal artery of H group and P group (2K1C). Eight weeks after the operation, the rats were sacrificed and the samples of the left ventricle were collected. The concentration of AngII in the left ventricle was assessed by radioimmunoassay. RT-PCR was used to examine the mRNA expression of TLR4 in the left ventricle. Immunohistochemistry was adopted to examine the location of TLR4 and NF-κB in the myocardium. Victoria blue-Ponceau staining of Cardiac collagen was used to evaluate the degree of myocardial fibrosis.Eight weeks after the operation, caudal SBP, meridional end-systolic stress, left ventricular mass index, relative wall thickness, cardiac fibrosis degree, and the concentration of AngII in the left ventricle in P group were significantly lower than those in H group (p0.01). In cardiac myocytes of S group and P group, TLR4 expression was diffused and presumably cytoplasmic. TLR4 mRNA expression in P group was significantly lower than that of H group (p0.01).PDTC not only inhibited the activation of NF-κB, but decreased TLR4 expression and AngII content, indicating that the inflammatory signals and oxidative stress mediated by TLR4/NF-κB are involved in the occurrence and development of left ventricular remodeling. Intervention with TLR4/NF-κB and anti-inflammatory and anti-oxidative therapy may be a new target to reverse left ventricular remodeling.

1. Vasoactive intestinal peptide (VIP) is involved in the control of prolactin (PRL) release and plays a pivotal role as a regulator of reproductive behaviour and neuroendocrine secretion in birds. 2. In this study, a 941-bp cDNA fragment covering the complete coding region (CDS) of goose VIP gene was identified. The cDNA contains a 32-bp 5'-untranslated region (UTR), a 603-bp CDS and a 306-bp 3'-UTR containing two ATTTA sequence elements, two polyadenylation signals (AATAAA) and a 25-bp poly (A) tail. 3. Seven exons and 6 introns were identified, and both the cDNA and genomic DNA sequences showed high identity with those of other species. 4. The sequence analysis indicated that there were two alternatively spliced transcripts the long transcript (VIP-1) encoded both VIP and peptide histidine isoleucine exons and the short one (VIP-2) only encoded VIP. 5. RT-PCR analysis indicates that the expression level of the VIP-1 is much lower than that of VIP-2, and that VIP-1 is negligible or absent in muscle, abdominal fat, ovary and spleen, whereas VIP-2 is widely distributed in all the examined tissues. 6. A total of 12 single nucleotide polymorphisms (SNPs), including 2 SNPs located in the coding region and 10 variations in intron regions, were identified in goose VIP gene.

Myocarditis, an acute inflammation disease of the heart, is a potentially lethal disease and can lead to sudden death. This study aims to investigate the therapeutic effect of lipoxin A4 (LXA4) on experimental autoimmune myocarditis (EAM) and to explore the underlying mechanism.EAM was induced in BALB/c mice by injection of porcine cardiac myosin and LAX4 at doses of 10 or 50 μg/kg was administrated from day 1 to 21. The severity of myocarditis was evaluated by detection of heart weight/body weight (HW/BW) ratio and histopathological examination of the heart. Cardiac function and heart structure were assessed by echocardiography. Serum levels of Th1 and Th2 cytokines were determined by ELISA. Protein expression was detected by Western blot analysis.The results demonstrated that LXA4 mitigated the severity of myocarditis by decreasing HW/BW ratio and reducing infiltration of inflammatory cells. Echocardiographic analysis indicated that cardiac function of LXA4-treated rats was significantly improved compared with non-treated group. LXA4 treatment significantly increased the levels of Th1 cytokines (TNF-α and IL-6) and decreased Th2 cytokines (IL-4 and IL-10). Furthermore, LXA4 administration effectively inhibited NF-κB nuclear translocation and deactivated PI3K/Akt pathway.LXA4 has a protective effect against EAM by reducing the inflammatory response and inhibiting NF-κB and PI3K/Akt signaling pathway.

It has been shown that hematological traits can act as important indicators of immune function in both humans and livestock. T lymphocytes are key components of the adaptive immune system, playing a critical role in immune response. To identify genomic regions affecting hematological traits and T lymphocyte subpopulations, we performed both a SNP-based genomewide association study (GWAS) and a haplotype analysis for 20 hematological traits and 8 T cell subpopulations at 3 different time points (d 20, 33, and 35) in a Duroc × Erhualian F intercross population. Bonferroni correction was used to calculate the threshold -values for suggestive and 5% genomewide significance levels. In total, for SNP-based GWAS, we detected 96 significant SNP, including 15 genomewide-significant SNP, associated with 23 hematological traits and 234 significant SNP, including 27 genomewide-significant SNP, associated with 8 T cell subpopulations. Meanwhile, we identified 563 significant SNP, including 7 genomewide-significant SNP, associated with 5 hematological traits and 2,407 significant SNP, including 1,261 genomewide-significant SNP, associated with 8 T cell subpopulations by haplotype analysis. Among the significant regions detected, we propose both the () gene and the () gene on SSC3 as plausible candidate genes associated with CD/CD T lymphocytes at d 20. The findings provide insights into the basis of molecular mechanisms that are involved with immune response in the domestic pig and would aid further identification of causative mutations.

1. The low-density lipoprotein receptor-related protein 8 (LRP8), a member of the low-density lipoprotein receptor (LDLR) gene family, participates in the supplying of lipid during follicular development. The objective of the study was to identify and characterise the LRP8 gene in goose. 2. A 2867 bp fragment that covered the complete coding region (CDS) of goose (Anser cygnoides) LRP8 gene was cloned. It encoded a protein of 917 amino acid residues containing a 24-amino acid signal peptide and 5 functional domains. The goose LRP8 showed high nucleic acid and amino acid identities with those in other species. 3. Similarly to duck LRP8 gene, two splice variants of LRP8, LRP8-1 (containing 8 ligand-binding repeats) and LRP8-2 (containing 7 ligand-binding repeats), were identified in goose. 4. Semi-quantitative RT-PCR analysis indicates that the LRP8-1 transcript is expressed in heart, liver, spleen, lung, kidney, breast muscle, duodenum, hypothalamus, pituitary and ovary, negligible or absent in sebum and oviduct, and the LRP8-2 transcript is widely expressed in all examined tissues. 5. A total of 7 SNPs were identified in the coding region of the goose LRP8 gene.

To investigate the pathological changes and related factors leading to failure of bioprosthetic valves.The surgical explanted bioprosthetic valves in Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College from January 2006 to December 2014 were retrospectively reviewed. Calcification, cusp tears, pannus and infectious endocarditis of bioprosthetic valves were evaluated by gross examination, X ray photography and histopathological analysis. Meanwhile, the age and the site at implantation, and the tissue type of bioprosthetic valves, the implantation duration were obtained by chart review and used to be analyzed with the modes of valve failure. U test, χ(2) test and Fisher exact test were used to analyze the data.Total 48 bioprosthetic valves were collected from 40 cases with 26 females. Their age at the time of implantation was 12 to 71 years (median 58 years). The tissue types of bioprosthesis included 24 bovine pericardial valves and 24 porcine aortic valves respectively. The implantation sites included mitral in 29 valves, aortic in 14 valves and tricuspid in 5 valves. The implantation duration ranged from 5 days to 27 years (mean (111±77) months). The study showed that the commonest mode of valve failure was structural valve deterioration (32 valves, 66.7%). The fewer modes of valve failure were infectious endocarditis (9 valves, 18.7%) and non-structural valve deterioration (7 valves, 14.6%). No thrombosis was found in this series. The degree of calcification was correlated positively with implantation duration (r=0.52, P=0.000). The implantation duration of bovine pericardial valves was longer than that of porcine aortic valves ((146±74) months vs. (77±68) months, Z=-3.24, P=0.001). Porcine aortic valves had a higher incidence of cusp tears than bovine pericardial valves (66.7% vs. 16.7%, χ(2)=12.34, P=0.000) despite they had a relatively lower incidence of calcification (41.7% vs. 75.0%, χ(2)=5.37, P=0.019). For the age at the time of implantation, the patients ≤60 years old had more calcification of bioprosthetic valves compared with those60 years old (45.1% vs. 2/17, χ(2)=5.51, P=0.019) whereas the patients ≤60 years old had fewer cusp tears (12.9% vs. 58.8%, P=0.002). Infectious endocarditis was found more frequently in the valves failure within 5 years than those failure after 5 years (6/14 vs. 8.8%, P=0.006).Structural valve deterioration is the principle cause of bioprosthetic valve failure while infectious endocarditis mainly involves the valves failure in early and middle terms after surgery. The mode of valve failure in the patients ≤60 years old is different from that in the patients60 years old at the time of implantation. More calcification would occur in bioprosthetic valve in the younger patients.

To observe the expression of neural axon guidance molecules Slit3 and Robo receptors in mouse aortic smooth muscle cell(MASMC) and investigate the effect of exogenous Slit3 protein on migration and proliferation of MASMC.The primary cultured MASMC were identified by immunofluorescent assay. The expression of Slit3/Robo signal pathway was detected by RT-PCR and immunocytochemical staining. MASMC were divided into 6 groups: the negative control group (DMEM medium containing bovine serum albumin 86 μg/L), Slit3 0 μg/L group (DMEM medium without Slit3), Slit3 24 μg/L group (DMEM medium containing Slit3 24 μg/L), Slit3 40 μg/L group (DMEM medium containing Slit3 40 μg/L), Slit3 80 μg/L group (DMEM medium containing Slit3 80 μg/L) and the positive control group (DMEM medium containing platelet derived growth factor 10 μg/L). The effects of exogenous Slit3 on MASMC proliferation and migration were detected by CCK-8 and scratched cells and transwell chambers respectively.(1) The mRNA and protein expressions of Slit2, Slit3, Robo1 and Robo4 were detected in MASMC. mRNA level of Slit2 was lower than Slit3 (P0.05) and there were no significant difference between mRNA level of Robo1 and Robo4. (2) The mitogenic responses of MASMC were significantly enhanced in Slit3 24 μg/L group, Slit3 40 μg/L group and Slit3 80 μg/L group compared with negative control group (1.13±0.04, 1.19±0.02, 1.18±0.08 and 0.64±0.10 respectively, all P0.05). The mitogenic activity of MASMC was the strongest in Slit3 40 μg/L group (compared with positive control group 1.27±0.05, P0.05). (3)The autonomous migration activity of MASMC were significantly increased in Slit3 24 μg/L group, Slit3 40 μg/L group, Slit3 80 μg/L group compared with negative control group (cell scratch width were (0.40±0.03)cm, (0.32±0.03)cm, (0.30±0.02)cm and (0.49±0.01)cm respectively, all P0.05). The autonomous migration activity of MASMC was the strongest in Slit3 80 μg/L group (compared with positive control group (0.22±0.01)cm, P0.05). The transmembrane migration activity of MASMC were significantly increased in Slit3 24 μg/L group, Slit3 40 μg/L group, Slit3 80 μg/L group compared with negative control group (the number of cell migration were 46.67±2.23, 65.33±3.43, 81.67±4.22 and 39.33±2.03 respectively, all P0.05). The transmembrane migration activity of MASMC was the strongest in Slit3 80 μg/L group (compared with positive control group 84.00±2.02, P0.05).Slit2, Slit3, Robo1 and Robo4 were expressed in MASMC, and exogenous Slit3 could promote proliferation and migration of MASMC in vitro.

Propofol is a widely used intravenous general anesthetic. Acetylcholine (ACh) is critical in controlling epithelial ion transport. This study was to investigate the effects of propofol on ACh-evoked secretion in rat ileum epithelium.The Ussing chamber technique was used to investigate the effects of propofol on carbachol (CCh)-evoked short-circuit currents (Isc).Propofol (10Propofol inhibits CCh-induced intestinal secretion by directly targeting basolateral K

Agouti signalling protein (ASIP) is an endogenous antagonist of melanocortin-1 receptor (MC1R) and is involved in the regulation of pigmentation in mammals. The objective of this study was to identify and characterise the ASIP gene in domestic goose. The goose ASIP cDNA consisted of a 44-nucleotide 5'-terminal untranslated region (UTR), a 390-nucleotide open-reading frame (ORF) and a 45-nucleotide 3'-UTR. The length of goose ASIP genomic DNA was 6176 bp, including three coding exons and two introns. Bioinformatic analysis indicated that the ORF encodes a protein of 130 amino-acid residues with a molecular weight of 14.88 kDa and an isoelectric point of 9.73. Multiple sequence alignments and phylogenetic analysis showed that the amino-acid sequence of ASIP was conserved in vertebrates, especially in the avian species. RT-qPCR showed that the goose ASIP mRNA was differentially expressed in the pigment deposition tissues, including eye, foot, feather follicle, skin of the back, as well as in skin of the abdomen. The expression level of the ASIP gene in skin of the abdomen was higher than that in skin of the back. Those findings will contribute to further understanding the functions of the ASIP gene in geese plumage colouring.

Objectives: To compare blood counts between arterial and venous blood to and from visceral organs and indirectly look into the function of the organs. Methods: Splenic, renal and superior mesenteric arterial and venous blood samples were obtained from the arteries and veins in 38 post-pubertal rabbits and blood profile, including complete and differential blood counts, haemoglobin concentration and haematocrit, were measured with an automatic haematology analyser. Results and conclusions: The rabbit spleen released a large amount of leucocytes (both lymphocytes and granulocytes) into the splenic venous blood (a venous increase of 33% in total leucocyte count), and also received more leucocytes (36–58% more in terms of concentration) from the artery than the kidney or intestine. Significantly fewer red blood cells were present in the renal venous blood than in the arterial blood (a venous reduction of 5% in erythrocyte count), but it remains to be clarified why and how the reduction was induced. More than 3–4% of water might be taken into the mesenteric venous blood during microcirculation (a venous reduction of 3–4% in erythrocyte-related parameters) and a significant number of leucocytes (mainly large leucocytes) in the mesenteric blood capillaries might migrate into the surrounding intestinal tissue (a venous reduction of 13% in leucocyte count).

Oncoprotein p28(GANK), overexpressed in hepatocellular carcinomas (HCC), binds to RelA and retains NF-κB in the cytoplasm to suppress NF-κB transactivation. However, the mechanism has not yet been elucidated. In this study, we clarified the mechanism of NF-κB regulated by p28(GANK). p28(GANK) reduced TNF-α-induced nuclear translocation of RelA/NF-κB independent of HDAC3. p28(GANK) interacted with p300 to attenuate assembly of RelA with p300, which lessened acetylation of RelA on the lysine 310 sites. Moreover, overexpression of p28(GANK) attenuated the capability of NF-κB binding to the target gene IκBα promoter, but also weakened adriamycin-induced NF-κB pro-apoptotic gene Fas and FasL expression, which subsequently made p53-deficient tumor cells resistance to adriamycin. These results present mechanistic insight into the key role of p28(GANK) in post-translational regulation of RelA/NF-κB.

Tripterygium wilfordii Hook F is a medicinal plant used for the treatment of glomerulonephritis in China. We studied the effect of Tripterygium wilfordii multiglycoside (TWG) on glomerular albumin permeability (Palbumin) in vitro.Isolated rat glomeruli were incubated with protamine (600 micrograms/ml) for 30 min, or with human recombinant tumour necrosis factor (TNF-alpha 0.4 ng/ml), superoxide (10 units/ml), or serum from a focal segmental glomerular sclerosis (FSGS) patient for 10 min at 37 degrees C. TWG, 1 mg/ml, was added in parallel tubes to study the effect on Palbumin. Control glomeruli were incubated under identical conditions. The albumin reflection coefficient (sigma albumin) was calculated from the change in glomerular volume in response to an applied oncotic gradient. Convectional permeability (Palbumin) was calculated as (1 - sigma albumin).Compared with controls, protamine increased the Palbumin of glomeruli (0.83 +/- 0.05, n = 25, vs 0.18 +/- 0.03, n = 20); pretreatment with TWG blocked this effect (0.13 +/- 0.04, n = 25). TNF-alpha also increased the Palbumin (0.79 +/- 0.04, n = 24 vs 0.04 +/- 0.07, n = 19); preincubation with TWG blocked this effect (0.03 +/- 0.09, n = 24). Palbumin of glomeruli incubated with xanthine and xanthine oxidase, resulting in the production of superoxide, also increased as compared to controls (0.85 +/- 0.04, n = 15 vs 0.08 +/- 0.05, n = 14); TWG blocked this effect as well (0.21 +/- 0.08, n = 14). FSGS serum also increased Palbumin of glomeruli significantly (0.88 +/- 0.02, n = 49 vs 0.00 +/- 0.02, n = 49); preincubation with TWG blocked this effect (0.05 +/- 0.07, n = 30). TWG by itself had no effect on Palbumin (0.19 +/- 0.10, n = 15).Our results show that TWG blocks protamine, TNF-alpha, superoxide, and FSGS serum-mediated increase in glomerular albumin permeability in vitro. We conclude that reduction of proteinuria by Tripterygium wilfordii multiglycoside in various kinds of glomerular diseases in vivo might be due to protection of the glomerular filtration barrier.

Many natural proteins have been developed into drugs and produced for direct application. Identifying improved hosts to achieve high-level heterologous protein production is a challenge in the study of heterologous protein expression in recombinant yeast. In this study, a novel high-throughput assay to screen such overproducing Saccharomyces cerevisiae strains was systematically developed. The protocol designed was based on screening host strain derivatives with increased superoxide dismutase dependent resistance to oxidative stress. Yeast cells transformed with recombinant plasmid carrying SOD1 gene as a reporter responded exquisitely to oxidative stress induced by elevated concentrations of paraquat. Improved yeast strains resulting from screening clones subjected to genome shuffling through selective pressure argue for a more effective screening system compared with traditonal selection. Moreover, this approach can be employed in general biochemical analysis without utilization of flow cytometry or well plate reader. Therefore, it is expected that the high-throughput assay would make superior strains producing heterologous proteins.

The phosphoprotein scaffold Dishevelled is an essential component of both Wnt signalling and of the signalsome that constitutes the supermolecular 'punctae' of assembled proteins often observed in fluorescence microscopy. The C-terminal region beyond the DEP domain displays unique and interesting character, exploited herein by careful analysis of the primary structure. Human Dishevelled-1, -2, -3 and fly Dishevelled (Dsh) sequences were downloaded and interrogated in silico. The C-terminus of Dishevelled-3 is revealed by FoldIndex(®) to be rich in ordered structure. It displays primary sequence that is unique and divergent in important ways from vertebrate isoforms as well as from the fly Dsh. The region is amphipathic, high in prolyl content, and harbours polyprolines. Dishevelled-3 displays some regions, where the proline content is40%. Polyprolyl sequences (2-4 residues) likely constitute important sites of interaction with other Dishevelled isoforms. Several histidine-single amino acid repeats are notable. The 637,638/647,648 repeats of Dvl3 are essential for Wnt non-canonical, but not canonical signalling. Mutagenesis reveals that the C-terminal sequence is essential for the formation of punctae, made visible by fluorescence microscopy. These Dvl3-based signalsomes are very large (25-35 MDa-MW), supermolecular complexes that display dynamic reorganization in response to Wnt stimulation. Dishevelled-3 C-terminus is rich in structure and unique motifs, worthy of detailed analysis with modern molecular tools.

The 3-hydroxyl-3-methylglutaryl Coenzyme A reductase (HMGCR) gene was examined for polymorphisms in Beijing-you chickens. A "T" base insert was detected at nucleotide 2749 of the 3-UTR region of the HMGCR gene and was used as the basis for distinguishing a B allele, distinct from the A. Serum and muscle contents of total cholesterol. LDL-cholesterol in serum was significantly lower in AB birds and lowest in BB birds. Real-time PCR showed that the same trends across genotypes occurred in an abundance of HMGCR transcripts in liver, but there was no difference in contents of HMGCR mRNA in breast or thigh muscles. Hepatic expression and serum LDL-cholesterol were meaningfully correlated (partial, with total serum cholesterol held constant, r = 0.923). In muscle, similar genotypic differences were found for the abundance of the LDL receptor (LDLR) transcript. Cholesterol content in breast muscle related to LDLR expression (partial correlation with serum LDL-cholesterol held constant, r = 0.719); the equivalent partial correlation in thigh muscle was not significant. The results indicated that the B allele significantly reduces hepatic abundance of HMGCR transcripts, probably accounting for genotypic differences in serum cholesterol. In muscle, the cholesterol content appeared to reflect differences in LDLR expression with apparent mechanistic differences between breast and thigh.

The complete genome sequence of a Chinese very virulent infectious bursal disease virus (vvIBDV) strain, Harbin-1, was determined. Based on the sequence analysis, the molecular characteristics and potential virulence determinants and origin of vvIBDV strains were identified. Phylogenetic analysis indicated that a reassortment and/or recombination event may have occurred in the emergence of Chinese vvIBDV strains.

The mechanisms of inhibition of DNA replication after DNA damage through the activation of the S-phase checkpoint have been the focus of several investigations over the last 40 yr. Recent studies have identified several components of this checkpoint response and there is strong interest in its biochemical characterization. Helpful for the delineation of the mechanism of the S-phase checkpoint is the observation that factors inhibiting DNA replication in vivo can be found in active form in extracts prepared from irradiated cells, when these are tested using the simian virus 40 (SV40) assay for in vitro DNA replication. In this assay, replication of plasmids carrying the minimal origin of SV40 DNA replication is achieved in vitro using cytoplasmic cell extracts and SV40 large tumor antigen (TAg) as the only noncellular protein. Here, we describe protocols developed to measure in vitro DNA replication with the purpose of analyzing its regulation after exposure to DNA damage. The procedures include the preparation of components of the in vitro DNA replication reaction including cytoplasmic extracts from cells that have sustained DNA damage. The assay is powerful but is limited by the fact that initiation steps carried out by the TAg in vitro may have different cellular determinants.

To observe the effect of Astragalus and Angelica on acute renal injury.Using the ischemia/reperfusion model established by blocking blood flow through clamping of bilateral renal artery for 45 min, the changes of glomerular filtration rate (GFR), renal plasma flow (RPF), fractional excretion of sodium (FENa), urinary volume (UV) and mean arterial pressure (MAP) as well as the morphological change of kidney before and after ischemia/reperfusion were observed.Astragalus and Angelica could promote recovery of RPF and GFR after ischemia/reperfusion, prevent the oliguria or shorten the oliguria period, reduce the increment of FENa and improve the histomorphological injury of kidney.Astragalus and Angelica have certain effect in protecting kidney from acute renal injury.

To investigate law of FOS protein induced by myocardial ischemia reperfusion (MI/R) in acute period, a model in 20 anaethetized SD rats was established. Rats with normal and ischemia were used as control groups. Specimens were studied immunohistochemically with c-fos antibody. After ischemia 20 minutes, followed by 30 minutes reperfusion, the area of MI/R showed nuclei of myocytes positive staining in cryosection slides. In C2 group, the area showed nuclei of myocytes (37.76% +/- 9.66%) positive staining weakly. In C3 group, nuclei of cardiac myocytes(40.34% +/- 3.32%) was significantly positive. In C4 group it began to attenuate(35.36% +/- 4.81%). The myocardium in normal and ischemic control groups showed negative staining. No changes were seen with HE staining. Our data indicated that immunohistochemical method may reveal acute MI/R injury of ischemia 20 min and reperfusion 30 min with anti-FOS protein staining and there is problely a peak between 60-120 min after reperfusion. It is possible that the method be used to diagnose sudden cardiac death in forensic medicine.

To determine the preventive and regressive effects of total flavones of metasequosia (TFM) on left ventricular hypertrophy in rats.Left ventricular hypertrophy was inducedin by partial ligation of abdominal aorta. The rats were given ig TFM(4, 40, 400 mg.kg-1.d-1) for six weeks.TFM markedly reduced the HW/BW, LVW/BW, myofibril diameter and Ca2+ content in left ventricles but the systolic blood pressure (SBP) in rats wasn't obviously influenced.TFM can prevent and reverse the left ventricular hypertrophy due to pressure overload in rats. The mechanism may be related to its calcium antagonistic properties.

To testify the inductive osteogenesis of allogeneic bone matrix gelatin (BMG) in promoting intervertebral fusion.The gelatin sponge, allogeneic BMG, decalcified bone matrix (DBM) and alcohol conserved bone were implanted respectively into the intervertebral space of rabbit, whose intervertebral discs were removed before implantation. The intervertebral spaces were evaluated by X-ray and histological examination at 4, 8, and 12 weeks after operation.No obvious immune rejection was observed. Amounts of new bone were formed in the intervertebral spaces at 4 and 8 weeks. And complete infusion of the intervertebral spaces were appeared at 12 weeks.Allogeneic BMG can promote bone fusion of intervertebral spaces through osteoinduction, which suggests that allogeneic BMP is an ideal substitute for bone replacement.

To probe the changes of vascular responsiveness and intracellular signaling during opiate withdrawal syndrome.Morphine withdrawal syndrome in rats was precipitated by iv naloxone following daily injection of increasing dose of morphine for 2 weeks, the changes in mean arterial blood pressure (MBP) caused by acetylcholine (ACh) were recorded. Isolated mesenteric vascular beds were perfused with Kreb's solution containing different concentration of drugs. The cytoplasmic Ca2+ ([Ca2+]i) in Fura 2-loaded cultured bovine aortic endothelial cells (aec) and smooth muscle cells (smc) were assayed. The positive immunoreaction to the phosphorylated cyclic AMP responsive element-binding protein (phospho-CREB) in cultured smc was calculated.Naloxone 2 mg/kg iv following chronic morphine treatment precipitated severe opiate withdrawal signs in company with blunted hypotensive effect of Ach that was the same as chronic morphine treated rats before withdrawal. In the mesenteric vascular beds from chronic morphine treated rats, the EC50 of pressor effect of norepinephrine (NE) was decreased from (2.06 +/- 0.38) to (1.14 +/- 0.21) micromol/L (n = 8, P0.01) after the perfusion solution containing morphine 20 micromol/L was replaced by Kreb's containing naloxone 25 micromol/L. Furthermore, NE-induced perfusion pressure increases were completely prevented by Kreb's contained morphine 40 micromol/L. Morphine acutely applied to control smc produced some variable and naloxone-reversible [Ca2+]i changes, but naloxone did not. However, naloxone increased [Ca2+]i in two thirds of smc preincubated with morphine 0.1 or 0.5 mmol/L for 48 h from (97 +/- 20) to (167 +/- 29) nmol/L (n = 9, P0.01) and from (106 +/- 19) to (225 +/- 48) nmol/L (n = 10, P0.01), respectively, and it also increased the ratio of positive immunoreaction to phospho-CREB from (7.7 +/- 3.2) % to (19.6 +/- 4.7) % (n = 6, P0.01) in smc preincubated with morphine 0.5 mmol/L. In addition, naloxone decreased [Ca2+]i from (146 +/- 34) to (78 +/- 24) nmol/L in one third morphine-preincubated aec (n = 10, P0.01).That naloxone enhances vascular responsiveness to NE in chronic morphine treatment rats may be relevant to [Ca2+]i transient facilitation in company with cAMP-dependent phosphorylation enhancement.