Mechanism exploration and biomarker identification of glycemic deterioration in patients with diseases of the exocrine pancreas.

The damage to the endocrine pancreas among patients with diseases of the exocrine pancreas (DP) leads to reduced glycemic deterioration, ultimately resulting in diabetes of the exocrine pancreas (DEP). The present research aims to investigate the mechanism responsible for glycemic deterioration in DP patients, and to identify useful biomarkers, with the ultimate goal of enhancing clinical practice awareness. Gene expression profiles of patients with DP in this study were acquired from the Gene Expression Omnibus database. The original study defines DP patients to belong in one of three categories: non-diabetic (ND), impaired glucose tolerance (IGT) and DEP, which correspond to normoglycemia, early and late glycemic deterioration, respectively. After ensuring quality control, the discovery cohort included 8 ND, 20 IGT, and 12 DEP, while the validation cohort included 27 ND, 15 IGT, and 20 DEP. Gene set enrichment analysis (GSEA) employed differentially expressed genes (DEGs), while immunocyte infiltration was determined using single sample gene set enrichment analysis (ssGSEA). Additionally, correlation analysis was conducted to establish the link between clinical characteristics and immunocyte infiltration. The least absolute shrinkage and selection operator regression and random forest combined to identify biomarkers indicating glycemic deterioration in DP patients. These biomarkers were further validated through independent cohorts and animal experiments. With glycemic deterioration, biological processes in the pancreatic islets such as nutrient metabolism and complex immune responses are disrupted in DP patients. The expression of ACOT4, B2M, and ACKR2 was upregulated, whereas the expression of CACNA1F was downregulated. Immunocyte infiltration in the islet microenvironment showed a significant positive correlation with the age, body mass index (BMI), HbA1c and glycemia at the 2-h of patients. It was a crucial factor in glycemic deterioration. Additionally, B2M demonstrated a significant positive correlation with immunocyte infiltration and clinical features. Quantitative real-time PCR (qRT-PCR) and western blotting confirmed the upregulation in B2M. Immunofluorescent staining suggested the alteration of B2M was mainly in the alpha cells and beta cells. Overall, the study showed that gradually increased immunocyte infiltration was a significant contributor to glycemic deterioration in patients with DP, and it also highlighted B2M as a biomarker.

Early results of high tibial osteotomy versus combined arthroscopic surgery.

Objective: To investigate the early effect of high tibial osteotomy (HTO) compared with combined arthroscopic surgery. Methods: A retrospective study was conducted on patients who underwent HTO at The First Affiliated Hospital of Shandong First Medical University from January 2018 to January 2022. 138 patients (163 knees) with knee osteoarthritis (KOA) treated with HTO were selected. The medial proximal tibial angle (MPTA), joint line convergence angle (JLCA), femoral tibial angle (FTA), hip-knee-ankle (HKA) angle, weight-bearing line (WBL) ratio of the knee joint, opening gap, opening angle, American Knee Society score (KSS), US Hospital for Special Surgery (HSS) score, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score were measured to determine the different effects between HTO and HTO combined with arthroscopic by logistic regression analysis. Results: Patients with HTO combined with arthroscopic surgery have improved functional scores as well as imaging perspectives compared to preoperative. By multivariate logistic analysis, it was concluded that arthroscopic surgery and gender are influential factors in the outcome of HTO surgery. The postoperative KSS score was 2.702 times more likely to be classified as excellent in the HTO combined with arthroscopy group than in the HTO group [Exp (ß) = 2.702, 95% CI (1.049-6.961), P = 0.039]; the postoperative KSS score was 0.349 times more likely to be classified as excellent in women than in men [Exp (ß) = 0.349, 95% CI (0.138-0.883), P = 0.026]. Conclusion: Better results with HTO combined with arthroscopic surgery. HTO combined with arthroscopy is a better choice in the surgical treatment of KOA.

Safety and Toxicology Study of Hu7691, a Novel AKT Inhibitor, following Oral Administration in Rats.

Hu7691 represents a novel Pan-Akt kinase inhibitor, demonstrating excellent selectivity towards non-AGC kinase families and pronounced inhibitory effects on the proliferation of multiple tumor cell lines. However, there is currently a notable absence of in vivo toxicological research evidence concerning Hu7691. This study represents the first investigation into the 14-day repeated-dose toxicity of Hu7691 in male and female Sprague Dawley (SD) rats. Male rats were administered daily doses of 12.5, 50, 100, and 150 mg/kg/day, while female rats received doses of 12.5, 25, 50, and 75 mg/kg/day for 14 consecutive days. Hematological assessments, organ weights, and histopathological examinations revealed corresponding alterations, suggesting potential target organs for toxicity including the spleen, thymus, and gastrointestinal tract. It is worth noting that the test substance may also impact the liver, kidneys, heart, and ovaries. The No Observed Effect Level (NOAEL) was determined to be no greater than 12.5 mg/kg/day. Based on the observed gender-related toxicity differences in preliminary trials, it is recommended that the high dose reference dose for male animals in formal experiments should not be less than 100 mg/kg/day, while for female animals, it should be less than 50 mg/kg/day.

Di (2-ethyl) hexyl phthalate induces liver injury in chickens by regulating PTEN/PI3K/AKT signaling pathway via reactive oxygen species.

Di (2-ethyl) hexyl phthalate (DEHP) is a common environmental endocrine disruptor that induces oxidative stress, posing a significant threat to human and animal health. Oxidative stress can activate the PTEN/PI3K/AKT pathway, which is closely related to cell apoptosis. However, it is unclear whether DEHP induces apoptosis of chicken liver cells by regulating the PTEN/PI3K/AKT pathway through oxidative stress. In this experiment, male laying hens were continuously exposed to 400 mg/kg, 800 mg/kg, and 1600 mg/kg DEHP for 14 d, 28 d, and 42 d. The results showed that liver injury was aggravated with the dose of DEHP gavage, and the ROS/MDA levels in L, M, and H DEHP exposure groups were significantly increased, while the T-AOC/T-SOD/GSH-PX levels were decreased. Meanwhile, DEHP exposure up-regulated the mRNA and protein expression levels of PTEN/Bax/Caspase-9/Caspase-3 and down-regulated the mRNA and protein expression levels of PI3K/AKT/BCL-2, indicating that DEHP may lead to hepatocyte apoptosis through ROS regulation of PTEN/PI3K/AKT axis. In order to further clarify the relationship between oxidative stress and liver injury, we treated chicken hepatocellular carcinoma cell line (LMH) with 2.5 mM N-acetylcysteine (NAC). NAC attenuated these phenomena. In summary, our study suggests that DEHP can induce apoptosis of chicken liver through ROS activation of the PTEN/PI3K/AKT axis.

Oxidative stress and ferroptosis involved in 2-ethylhexyl diphenyl phosphate -induced hepatotoxicity in chicken.

Organophosphorus flame retardants (OPFRs) are increasingly being used in many industries since brominated flame retardants (BFRs) have been phase-out. However, OPFRs are associated with environmental pollution and animal health risks, especially in the farming industry. Nevertheless, no study has evaluated the toxicity of OPFRs, as a new flame retardant, on avian species. In order to investigate the specific toxic effects of 2-ethylhexyl diphenyl phosphate (EHDPHP) exposure on chickens and the molecular biological mechanisms that cause damage to the organism, the chicken liver has been studied as a potential target organ for toxic effects. In this study, 7-day-old male chickens were treated with different concentrations of EHDPHP to further investigate the toxicity and mechanisms of OPRs on birds. The samples were taken at 14 d, 28 d, and 42 d for analysis. EHDPHP exposure affected the growth and development of chickens. Furthermore, the microstructural and ultrastructural observations clearly reflected the damage caused by EHDPHP exposure to the livers. The levels of the liver tissue asparate aminotransferase (AST) and alanine aminotransferase (ALT) decreased with increasing gavage dose. In contrast, the levels of oxidative stress in chicken liver and the mRNA expression of related factors increased with increasing gavage dose. In addition, EHDPHP exposure increased liver tissue iron content and affected mRNA expression and protein levels of ferroptosis-related factors in livers. Besides, ferroptosis causes inflammation, thus promoting the synthesis and release of inflammatory factors. This research indicates that EHDPHP can damage chicken livers through oxidative stress and ferroptosis.

Novel and Alternative Therapeutic Strategies for Controlling Avian Viral Infectious Diseases: Focus on Infectious Bronchitis and Avian Influenza.

The growth of poultry farming has enabled higher spread of infectious diseases and their pathogens among different kinds of birds, such as avian infectious bronchitis virus (IBV) and avian influenza virus (AIV). IBV and AIV are a potential source of poultry mortality and economic losses. Furthermore, some pathogens have the ability to cause zoonotic diseases and impart human health problems. Antiviral treatments that are used often lead to virus resistance along with the problems of side effects, recurrence, and latency of viruses. Though target hosts are being vaccinated, the constant emergence and re-emergence of strains of these viruses cause disease outbreaks. The pharmaceutical industry is gradually focusing on plant extracts to develop novel herbal drugs to have proper antiviral capabilities. Natural therapeutic agents developed from herbs, essential oils (EO), and distillation processes deliver a rich source of amalgams to discover and produce new antiviral drugs. The mechanisms involved have elaborated how these natural therapeutics agents play a major role during virus entry and replication in the host and cause inhibition of viral pathogenesis. Nanotechnology is one of the advanced techniques that can be very useful in diagnosing and controlling infectious diseases in poultry. In general, this review covers the issue of the poultry industry situation, current infectious diseases, mainly IB and AI control measures and, in addition, the setup of novel therapeutics using plant extracts and the use of nanotechnology information that may help to control these diseases.

Comparative transcriptomic analysis of porcine epidemic diarrhea virus epidemic and classical strains in IPEC-J2 cells.

In recent years, porcine epidemic diarrhea (PED) has become widespread and caused huge economic losses for the global pig industry. There is growing evidence that frequent outbreaks of diarrhea are caused by the variants of porcine epidemic diarrhea virus (PEDV) with high pathogenicity. Herein, an epidemic strain of PEDV HLJ strain was isolated and characterized from Heilongjiang Province of China, and the whole genomic expression profile of intestinal porcine epithelial cells (IPEC-J2) infected with HLJ strain was investigated in comparison with classical CV777 strain. A total of 26,851 genes were identified, of these, 25,880 were known genes and 971 were novel genes. There were 258 differentially expressed genes (DEGs) identified between PEDV HLJ-infected and uninfected cells at 24 h post infection (hpi), and 201 DEGs between PEDV HLJ and CV777 infection. A comparative analysis revealed that 258 DEGs were enriched in 468 gene ontology (GO) terms and mapped to 179 KEGG pathways, and 201 DEGs in 1120 GO terms and mapped to 115 KEGG pathways for HLJ-infected cells in contrast to the uninfected and CV777-infected cells, respectively. Specifically, PEDV HLJ strain could activate anti-viral innate immune response and inflammation more intensively than CV777, in which mRNA levels of interferon (IFN-ß), chemokines (CCL5 and CXCL10) and pro-inflammatory cytokines (IL-8 and TNF-α) were induced earlier and more strongly. Subsequently, 20 DEGs and 5 proteins were selected and validated by real-time fluorescence quantitative PCR (RT-qPCR) and western blot, and the results were consistent with the transcriptomic analysis. Overall, this study may be helpful for understanding the pathogenesis mechanism of PEDV variants, and contribute to the effective prevention and control of PEDV infection.

Astragalus Polysaccharide Protects Against Cadmium-Induced Autophagy Injury Through Reactive Oxygen Species (ROS) Pathway in Chicken Embryo Fibroblast.

Cadmium (Cd) is a harmful heavy metal pollutant, which can cause oxidative stress in the body and induce cell damage. Reactive oxygen species (ROS) is a general term for substances that contain oxygen and are active in the body. However, excessive ROS can damage the body. Cadmium poisoning can cause a large amount of ROS in cells and autophagy. Astragalus polysaccharide (APS) is a plant polysaccharide with biological functions, such as antioxidant and anti-stress activities. In this study, chicken embryo fibroblasts (CEF) were used to determine the relationship between ROS and autophagy damage of Cd-infected cells and the mechanism of APS on cadmium-induced autophagy damage. The results showed that a 10-µL dose of 10 µmol/L cadmium chloride (CdCl2) can induce CEF autophagy and damage when CEF was added for 36 h. Cadmium induced CEF autophagy damage by increasing ROS production. APS could significantly reduce ROS production and LC3-II and Beclin-1 protein expression, increase the expression of mTOR and the level of antioxidation, and restore the viability and morphological damage of CEF exposed to Cd. Our study suggests that APS can alleviate Cd-induced CEF autophagy damage by reducing the production of ROS.

Selenium Deficiency Causes Inflammatory Injury in the Bursa of Fabricius of Broiler Chickens by Activating the Toll-like Receptor Signaling Pathway.

The aim of our study was to observe the effect of selenium (Se) deficiency on inflammatory injury in the bursa of Fabricius of broiler chickens and to determine the role of the Toll-like receptor (TLR)/myeloid differential protein-88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway during this process. Here, we revealed that severe inflammatory injury occurred in the broiler bursa of Fabricius with Se deficiency via histopathology. Moreover, the ultrastructural pathological results showed that the nuclear, mitochondrial, endoplasmic reticulum and cytomembrane structures were damaged to varying degrees. Additionally, interleukin-2 (IL-2), interleukin-6 (IL-6), and interferon (IFN-γ) mRNA expression was markedly upregulated in the broiler bursa of Fabricius with Se deficiency. Furthermore, TLR, toll-interleukin-1 receptor domain-containing adapter-inducing interferon-ß (TRIF), MyD88, and NF-κB mRNA expression was also markedly elevated in the broiler bursa of Fabricius with Se deficiency. The above results suggested that Se deficiency increases the expression of numerous proinflammatory cytokines and is probably due to the activation of the TLR/MyD88/NF-κB signaling pathway, which causes inflammatory injury in the bursa of Fabricius of broiler chickens. Our findings provide a theoretical reference for further studying the underlying mechanism of Se deficiency-induced inflammatory injury in the bursa of Fabricius of broiler chickens.

Selenium Deficiency Induces Autophagy in Chicken Bursa of Fabricius Through ChTLR4/MyD88/NF-κB Pathway.

To explore the role of ChTLR4/MyD88/NF-κB signaling pathway on autophagy induced by selenium (Se) deficiency in the chicken bursa of Fabricius, autophagosome formation in the bursa of Fabricius was observed by transmission electron microscopy. Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression of ChTLR4 and its signaling pathway molecules (MyD88, TRIF, and NF-κB), inflammatory factors (IL-1ß, IL-8, and TNF-α), and autophagy-related factors (ATG5, Beclin1, and LC3-II) in the Se-deficient chicken bursa of Fabricius at different ages. The results showed that ChTLR4/MyD88/NF-κB signaling pathway was activated in the chicken bursa of Fabricius and autophagy was induced at the same time by Se deficiency. In order to verify the relationship between the autophagy and ChTLR4/MyD88/NF-κB signaling pathway, HD11 cells were used to establish the normal C group, low Se group, and low Se + TLR4 inhibitor (TAK242) group. The results demonstrated that autophagy could be hindered when the TLR4 signaling pathway was inhibited under Se deficiency. Furthermore, autophagy double-labeled adenovirus was utilized to verify the integrity of autophagy flow induced by Se deficiency in HD11 cells. The results showed that it appeared to form a complete autophagy flow under the condition of Se deficiency and could be blocked by TAK242. In summary, we found that Se deficiency was involved in the chicken bursa of Fabricius autophagy occurring by activating the ChTLR4/MyD88/NF-κB pathway.

Hypericin Inhibit Alpha-Coronavirus Replication by Targeting 3CL Protease.

The porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus (α-CoV) that causes high mortality in infected piglets, resulting in serious economic losses in the farming industry. Hypericin is a dianthrone compound that has been shown as an antiviral activity on several viruses. Here, we first evaluated the antiviral effect of hypericin in PEDV and found the viral replication and egression were significantly reduced with hypericin post-treatment. As hypericin has been shown in SARS-CoV-2 that it is bound to viral 3CLpro, we thus established a molecular docking between hypericin and PEDV 3CLpro using different software and found hypericin bound to 3CLpro through two pockets. These binding pockets were further verified by another docking between hypericin and PEDV 3CLpro pocket mutants, and the fluorescence resonance energy transfer (FRET) assay confirmed that hypericin inhibits the PEDV 3CLpro activity. Moreover, the alignments of α-CoV 3CLpro sequences or crystal structure revealed that the pockets mediating hypericin and PEDV 3CLpro binding were highly conserved, especially in transmissible gastroenteritis virus (TGEV). We then validated the anti-TGEV effect of hypericin through viral replication and egression. Overall, our results push forward that hypericin was for the first time shown to have an inhibitory effect on PEDV and TGEV by targeting 3CLpro, and it deserves further attention as not only a pan-anti-α-CoV compound but potentially also as a compound of other coronaviral infections.

Time-dependent viral interference between influenza virus and coronavirus in the infection of differentiated porcine airway epithelial cells.

Coronaviruses and influenza viruses are circulating in humans and animals all over the world. Co-infection with these two viruses may aggravate clinical signs. However, the molecular mechanisms of co-infections by these two viruses are incompletely understood. In this study, we applied air-liquid interface (ALI) cultures of well-differentiated porcine tracheal epithelial cells (PTECs) to analyze the co-infection by a swine influenza virus (SIV, H3N2 subtype) and porcine respiratory coronavirus (PRCoV) at different time intervals. Our results revealed that in short-term intervals, prior infection by influenza virus caused complete inhibition of coronavirus infection, while in long-term intervals, some coronavirus replication was detectable. The influenza virus infection resulted in (i) an upregulation of porcine aminopeptidase N, the cellular receptor for PRCoV and (ii) in the induction of an innate immune response which was responsible for the inhibition of PRCoV replication. By contrast, prior infection by coronavirus only caused a slight inhibition of influenza virus replication. Taken together, the timing and the order of virus infection are important determinants in co-infections. This study is the first to show the impact of SIV and PRCoV co- and super-infection on the cellular level. Our results have implications also for human viruses, including potential co-infections by SARS-CoV-2 and seasonal influenza viruses.

Oral Immunization of Recombinant Lactococcus lactis and Enterococcus faecalis Expressing Dendritic Cell Targeting Peptide and Hexon Protein of Fowl Adenovirus 4 Induces Protective Immunity Against Homologous Infection.

Hepatitis-hydropericardium syndrome (HPS) causes severe economic losses in the global poultry industry. The present study aims to explore oral immunization of recombinant Lactococcus lactis and Enterococcus faecalis expressing Hexon protein of fowl adenovirus 4 (FAdV-4). The bacteria L. lactis NZ9000 and E. faecalis MDXEF-1 were, respectively, modified as host strain to deliver truncated Hexon protein (ΔHexon) or ΔHexon protein fusing with dendritic cell (DC) targeting peptide (DC-ΔHexon) on the surface of bacteria. The expression of target protein in L. lactis NZ9000 and E. faecalis MDXEF-1 were detected by western blot. To evaluate the immune responses and protective efficacies provided by the live recombinant bacteria, chickens were immunized with the constructed ΔHexon-expressing bacteria three times at 2-week intervals, then experimentally challenged with hypervirulent FAdV-4/GX01. The results showed that oral immunizations with the four ΔHexon-expressing bacteria (NZ9000/ΔHexon-CWA, NZ9000/DC-ΔHexon-CWA, MDXEF-1/ΔHexon-CWA, and MDXEF-1/DC-ΔHexon-CWA), especially the two bacteria carrying DC-targeting peptide, stimulated higher levels of ΔHexon-specific sera IgG and secretory IgA (sIgA) in jejunal lavage fluid, higher proliferation of peripheral blood lymphocytes (PBLs) and higher levels of Th1/Th2-type cytokines, along with significantly decreased virus loads in liver and more offered protective efficacies against FAdV infection compared with PBS and empty vector control groups (p < 0.01). For chickens in the group MDXEF-1/DC-ΔHexon-CWA, the levels of aspartate transaminase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH) in sera, and the virus loads in livers were significantly decreased vs. the other three ΔHexon-expressing bacteria (p < 0.01). The pathological changes in the hearts, livers, spleens and kidneys of chickens in MDXEF-1/DC-ΔHexon-CWA group were relatively slight compared to infection control group and other three ΔHexon-expressing bacteria groups. The rate of protection in MDXEF-1/DC-ΔHexon-CWA group was 90%. The present work demonstrated that cell surface-displayed target protein and immune enhancers in L. lactis and E. faecalis might be a promising approach to enhance immunity and immune efficacy against pathogen FAdV-4 infection.

Analysis of chicken macrophage functions and gene expressions following infectious bronchitis virus M41 infection.

Infectious bronchitis virus (IBV) is a pathogenic coronavirus with high morbidity and mortality in chicken breeding. Macrophages with normal biofunctions are essential for host immune responses. In this study, the HD11 chicken macrophage cell line and chicken peripheral blood mononuclear cell-derived macrophages (PBMCs-Mφ) were infected with IBV at multiplicity of infection (MOI) of 10. The dynamic changes of their biofunctions, including cell viability, pathogen elimination function, phagocytic ability, and gene expressions of related proteins/mediators in innate and acquired immunity, inflammation, autophagy and apoptosis were analyzed. Results showed that IBV infection decreased chicken macrophage viability and phagocytic ability, and increased pathogen elimination function. Moreover, IBV augmented the gene expressions of most related proteins in macrophages involved in multiple host bioprocesses, and the dynamic changes of gene expressions had a close relationship with virus replication. Among them, MHCII, Fc receptor, TLR3, IFN-α, CCL4, MIF, IL-1ß, IL-6, and iNOS showed significantly higher expressions in IBV-infected cells. However, TLR7, MyD88, MDA5, IFN-γ, MHCII, Fc receptor, MARCO, CD36, MIF, XCL1, CXCL12, TNF-α, iNOS, and IL-10 showed early decreased expressions. Overall, chicken macrophages play an important role in host innate and acquired immune responses to resist IBV infection, despite early damage or suppression. Moreover, the IBV-induced autophagy and apoptosis might participate in the virus-host cell interaction which is attributed to the biological process.

Infectious bronchitis virus: Identification of Gallus gallus APN high-affinity ligands with antiviral effects.

Infectious bronchitis virus (IBV) is a coronavirus, causes infectious bronchitis (IB) with high morbidity and mortality, and gives rise to huge economic losses for the poultry industry. Aminopeptidase N (APN) may be one of the IBV functional receptors. In this study, Gallus gallus APN (gAPN) protein was screened by phage-displayed 12-mer peptide library. Two high-affinity peptides H (HDYLYYTFTGNP) and T (TKFSPPSFWYLH) to gAPN protein were selected for in depth characterization of their anti-IBV effects. In vitro, indirect ELISA showed that these two high-affinity ligands could bind IBV S1 antibodies. Quantitative real-time PCR (qRT-PCR) assay, virus yield reduction assay and indirect immunofluorescence assay results revealed 3.125-50 µg/ml of peptide H and 6.25-50 µg/ml of peptide T reduced IBV proliferation in chicken embryo kidney cells (CEKs). In vivo, high-affinity phage-vaccinated chickens were able to induce specific IBV S1 antibodies and IBV neutralizing antibodies. QRT-PCR results confirmed that high-affinity phages reduced virus proliferation in chicken tracheas, lungs and kidneys, and alleviated IBV-induced lesions. By multiple sequence alignment, motif 'YxYY' and 'FxPPxxWxLH' of high-affinity peptides were identified in IBV S1-NTD, while another motif 'YxFxGN' located in S2. These results indicated that high affinity peptides of gAPN could present an alternative approach to IB prevention or treatment.

Selenium Deficiency via the TLR4/TRIF/NF-κB Signaling Pathway Leading to Inflammatory Injury in Chicken Spleen.

The aim of the present study was to investigate the effect of selenium (Se) deficiency on the expression of the toll-like receptor (TLR) signal transduction pathway in the spleen of chickens and explore the relationship between the TLR4/TRIF/NF-κB signaling pathway and inflammatory spleen injury. A total of 200 one-day-old healthy broilers were allocated to two groups. The experimental group was fed a self-made low-Se diet (0.004 mg/kg) while the control group was fed a complete formula feed (0.2 mg/kg) for 15, 25, 35, 45, and 55 days, respectively. We observed histopathological changes in the chicken spleens. The messenger RNA(mRNA) expression levels of 8 kinds of ChTLRs, myeloid differential protein-88 (MyD88), toll-interleukine-1 receptor domain-containing adapter-inducing interferon-ß (TRIF), nuclear factor-κB (NF-κB), and cytokine (IL-6, TNF-α, IL-2, and IFN-γ) were detected via quantitative real-time PCR. Western blotting was used to detect the protein expression level of TLR4. Then principal component analysis (PCA) was used to analyze the correlation between the ChTLRs, MyD88, TRIF, and NF-κB. The results showed that the boundary between red pulp and white pulp was unclear, the number of lymphocytes decreased, and the nucleus was fragmented and dissolved in the experimental group at 25-55 days. At 15-45 days, the relative expression of TLR4 mRNA was higher than in the control group, and the difference was extremely significant on day 15 (P < 0.01).The relative expression of TRIF mRNA in the experimental group was higher than in the control group at 25-55 days, and the relative expression of NF-κB mRNA in the experimental group was higher than in the control group at 15-45 days. The relative expression of IL-6 mRNA in the experimental group was higher than in the control group at 15-45 days. The protein expression level of TLR4 in the experimental group was higher than in the control group at 15-45 days. The PCA results showed that there was a strong correlation between TLR4, TRIF, and component 1. The results suggest that TLR4 plays an important role in regulating the expression of inflammatory cytokines in the spleens of Se-deficient chickens, and Se deficiency may cause inflammatory injury through the TLR4/TRIF/NF-κB signaling pathway in chicken spleen.

Molecular Characterization of Infectious Bronchitis Virus Strain HH06 Isolated in a Poultry Farm in Northeastern China.

Spike (S) glycoprotein is an important virulent factor for coronaviruses (CoVs), and variants of CoVs have been characterized based on S gene analysis. We present phylogenetic relationship of an isolated infectious bronchitis virus (IBV) strain with reference to the available genome and protein sequences based on network, multiple sequence, selection pressure, and evolutionary fingerprinting analysis in People's Republic of China. One hundred and elven strains of CoVs i.e., Alphacoronaviruses (Alpha-CoVs; n = 12), Betacoronaviruses (Beta-CoVs; n = 37), Gammacoronaviruses (Gamma-CoVs; n = 46), and Deltacoronaviruses (Delta-CoVs; n = 16) were selected for this purpose. Phylogenetically, SARS-CoV-2 and SARS-CoVs clustered together with Bat-CoVs and MERS-CoV of Beta-CoVs (C). The IBV HH06 of Avian-CoVs was closely related to Duck-CoV and partridge S14, LDT3 (teal and chicken host). Beluga whale-CoV (SW1) and Bottlenose dolphin-CoVs of mammalian origin branched distantly from other animal origin viruses, however, making group with Avian-CoVs altogether into Gamma-CoVs. The motif analysis indicated well-conserved domains on S protein, which were similar within the same phylogenetic class and but variable at different domains of different origins. Recombination network tree indicated SARS-CoV-2, SARS-CoV, and Bat-CoVs, although branched differently, shared common clades. The MERS-CoVs of camel and human origin spread branched into a different clade, however, was closely associated closely with SARS-CoV-2, SARS-CoV, and Bat-CoVs. Whereas, HCoV-OC43 has human origin and branched together with bovine CoVs with but significant distant from other CoVs like SARS CoV-2 and SARS-CoV of human origin. These findings explain that CoVs' constant genetic recombination and evolutionary process that might maintain them as a potential veterinary and human epidemic threat.

Flavonoids Regulate Inflammation and Oxidative Stress in Cancer.

Cancer is the second leading cause of death globally. Millions of persons die due to cancer each year. In the last two decades, the anticancer effects of natural flavonoids have become a hot topic in many laboratories. Meanwhile, flavonoids, of which over 8000 molecules are known to date, are potential candidates for the discovery of anticancer drugs. The current review summarizes the major flavonoid classes of anticancer efficacy and discusses the potential anti-cancer mechanisms through inflammation and oxidative stress action, which were based on database and clinical studies within the past years. The results showed that flavonoids could regulate the inflammatory response and oxidative stress of tumor through some anti-inflammatory mechanisms such as NF-κB, so as to realize the anti-tumor effect.

The Cell Tropism of Porcine Respiratory Coronavirus for Airway Epithelial Cells Is Determined by the Expression of Porcine Aminopeptidase N.

Porcine respiratory coronavirus (PRCoV) infects the epithelial cells in the respiratory tract of pigs, causing a mild respiratory disease. We applied air-liquid interface (ALI) cultures of well-differentiated porcine airway cells to mimic the respiratory tract epithelium in vitro and use it for analyzing the infection by PRCoV. As reported for most coronaviruses, virus entry and virus release occurred mainly via the apical membrane domain. A novel finding was that PRCoV preferentially targets non-ciliated and among them the non-mucus-producing cells. Aminopeptidase N (APN), the cellular receptor for PRCoV was also more abundantly expressed on this type of cell suggesting that APN is a determinant of the cell tropism. Interestingly, differentiation-dependent differences were found both in the expression of pAPN and the susceptibility to PRCoV infection. Cells in an early differentiation stage express higher levels of pAPN and are more susceptible to infection by PRCoV than are well-differentiated cells. A difference in the susceptibility to infection was also detected when tracheal and bronchial cells were compared. The increased susceptibility to infection of bronchial epithelial cells was, however, not due to an increased abundance of APN on the cell surface. Our data reveal a complex pattern of infection in porcine differentiated airway epithelial cells that could not be elucidated with immortalized cell lines. The results are expected to have relevance also for the analysis of other respiratory viruses.

Metabonomic analysis of hypophosphatemic laying fatigue syndrome in laying hens.

Laying fatigue syndrome (LFS) is a common disease in poultry, which is characterized by low egg laying rate, increased broken and soft shell egg rate and osteoporosis, and even death of poultry. Insufficient phosphorus content in feed is one of the major causes of LFS. In this study, a total of 22-week-old Roman white shell hens were randomly divided into two groups, including control (group C) and low dietary phosphorus (group P) groups. The hens of groups C and P were fed with a full mixed diet and a mixed diet containing 0.18% available phosphorus content, respectively. At 25, 29 and 34 weeks, the production performance of hens was detected and the serum samples of hens were collected to detect the changes of serum phosphorus, calcium, osteopelectin (OPG), parathyroid hormone (PTH), estradiol (E2), tartaric acid-resistant phosphatase (TRACP) and alkaline phosphatase (ALP). The keels were removed and x-rayed. In addition, all serum samples were tested by LC-MS metabolomics. Our results showed that low dietary phosphorus decreased the production performance, phosphorus content, and E2 and OPG levels, while increased calcium and PTH levels, and ALP and TRACP activities in laying hens. The hens of group P had bent keels. Besides, small molecular metabolites in serum were enriched in 10 pathways and 17 metabolites were significantly different according to the area under the receiver operating characteristic curve (AUC) analysis. Our results showed that low phosphorus diet could induce LFS. Also, 17 metabolites detected by metabonomics can be used as biomarkers for clinical diagnosis and early warning of hypophosphatemic laying fatigue syndrome (HLFS). This study provides a scientific basis for the early prevention and treatment of HLFS.