G-quadruplex in the TMV Genome Regulates Viral Proliferation and Acts as Antiviral Target of Photodynamic Therapy.

Plant viruses seriously disrupt crop growth and development, and classic protein-targeted antiviral drugs could not provide complete protection against them. It is urgent to develop antiviral compounds with novel targets. Photodynamic therapy shows potential in controlling agricultural pests, but nonselective damage from reactive oxygen species (ROS) unexpectedly affects healthy tissues. A G-quadruplex (G4)-forming sequence in the tobacco mosaic virus (TMV) genome was identified to interfere the RNA replication in vitro, and affect the proliferation of TMV in tobacco. N-methyl mesoporphyrin IX stabilizing the G4 structure exhibited inhibition against viral proliferation, which was comparable to the inhibition effect of ribavirin. This indicated that G4 could work as an antiviral target. The large conjugate planes shared by G4 ligands and photosensitizers (PSs) remind us that the PSs could work as antiviral agents by targeting G4 in the genome of TMV. Chlorin e6 (Ce6) was identified to stabilize the G4 structure in the dark and selectively cleave the G4 sequence by producing ROS upon LED-light irradiation, leading to 92.2% inhibition against TMV in vivo, which is higher than that of commercial ningnanmycin. The inhibition of Ce6 was lost against the mutant variants lacking the G4-forming sequence. These findings indicated that the G-quadruplex in the TMV genome worked as an important structural element regulating viral proliferation, and could act as the antiviral target of photodynamic therapy.

Hexavalent chromium disrupts the skin barrier by targeting ROS-mediated mitochondrial pathway apoptosis in keratinocytes.

Hexavalent chromium (Cr(VI)), a toxic heavy metal, is ubiquitous in daily life. Exposure to this toxic substance in occupational settings can cause dermatitis and cancer. As the body's largest organ, the skin plays a crucial role in protecting the organism against external aggressions. While previous studies have focused on the effects of Cr(VI) on skin inflammation, this study investigates the potential toxicity of Cr(VI) from the skin barrier and integrity perspective. The in vivo results of this study showed that mice exposed to Cr(VI) experienced skin deterioration and hemorrhaging, as well as a reduction in the thickness of the collagen fiber layer. TUNEL and Occludin staining results revealed that Cr(VI)'s toxicity primarily targeted keratinocytes. Experiments in vitro demonstrated that Cr(VI) treatment decreased the activity of HaCaT cells, altered cell morphology, and increased LDH secretion. Further research revealed that Cr(VI) could modify membrane permeability, impair membrane integrity, and reduce the protein expression of ZO-1 and Occludin. In addition, it was discovered that Cr(VI) promoted cell apoptosis and inhibited AKT activation. However, the addition of a caspase inhibitor and an AKT activator prevented Cr(VI)-induced injury to the cell membrane barrier, indicating that apoptosis plays a crucial role in this process. The addition of three apoptotic pathway inhibitors, confirmed that Cr(VI) damaged the cell barrier through ROS-mediated mitochondrial pathway apoptosis. Moreover, the use of a ROS inhibitor significantly reduced Cr(VI)-induced apoptosis and cell barrier injury. In conclusion, this study provides an experimental foundation for the treatment of skin injury caused by Cr(VI).

The role of transforming growth factor beta-1 protein in Escherichia coli secondary infection induced by H9N2 avian influenza virus in chickens.

The H9N2 subtype of avian influenza virus (AIV) is common in poultry production. It causes mild clinical signs but rarely leads to poultry mortalities. However, higher mortality can occur in chickens with co-infections, especially avian pathogenic Escherichia coli (APEC), which results in huge economic losses for the poultry industry. Unfortunately, the mechanism of co-infection remains unknown. Our previous studies screened several proteins associated with bacterial adhesion, including transforming growth factor beta-1 (TGF-ß1), integrins, cortactin, E-cadherin, vinculin, and fibromodulin. Herein, we investigated the contribution of TGF-ß1 to APEC adhesion after H9N2 infection. We first infected H9N2 and APEC in chicken, chicken embryo and DF-1 cells, and demonstrated that H9N2 infection promotes APEC adhesion to hosts in vitro and in vivo by plate count method. Through real-time fluorescence quantification and enzyme-linked immunosorbent assay, it was demonstrated that H9N2 infection not only increases TGF-ß1 expression but also its activity in a time-dependent manner. Then, through exogenous addition of TGF-ß1 and overexpression, we further demonstrated that TGF-ß1 can increase the adhesion of endothelial cells to DF-1 cells. Furthermore, the capacity of APEC adhesion to DF-1 cells was significantly decreased either by adding a TGF-ß1 receptor inhibitor or using small interfering RNAs to interfere with the expression of TGF-ß1. To sum up, H9N2 infection can promote the upregulation of TGF-ß1 and then increase the adhesion ability of APEC. Targeting TGF-ß1 and its associated pathway will provide valuable insights into the clinical treatment of E. coli secondary infection induced by H9N2 infection.

Evaluation of the antiviral effect of four plant polysaccharides against duck circovirus.

Recently, outbreaks of duck circovirus (DuCV) are frequently occurring worldwide due to secondary infections caused by post infection-induced immunosuppression. Due to a lack of preventive drugs and vaccines, the waterfowl industry losses are ever increasing. In this study, we extracted Astragalus polysaccharides (APS), pine pollen polysaccharides (PPPS), Aloe vera polysaccharides (AVE), and Ficus carica polysaccharides (FCPS) from Astragalus, pine pollen, aloe, and F. carica leaves, respectively. We randomly divided 150 one-day-old Cherry Valley ducks into five groups, which were inoculated with the DuCV solution and orally administered APS, PPPS, AVE, FCPS, and phosphate buffer saline (PBS), respectively. We collected the duck immune organs and serum samples at 8, 16, 24, 32, 40, and 48 days post-infection (dpi). Using clinical symptom analysis, molecular biology experiments, and serological experiments, we proved that plant polysaccharides could (a) improve the duck immunity, (b) reduce the viral load, and (c) mitigate DuCV-induced damage to immune organs, with both APS and PPPS having significant effects. Moreover, we detected viral load and cytokines within the first 8 dpi. Since the body's innate immunity could inhibit viral replication within five days of virus infection, 1-5 dpi was the best treatment time. Among the four polysaccharides showing in vitro anti-apoptotic activity, APS and PPPS significantly inhibited the DuCV infection-induced apoptosis of peripheral blood lymphocytes. Overall, since our findings show APS and PPPS having significant anti-DuCV effects both in vivo and in vitro, they can be promising candidates for preventing DuCV infection in ducks.

Anti-tumor activity of polysaccharides extracted from Pinus massoniana pollen in colorectal cancer- in vitro and in vivo studies.

The prevalence and mortality rate of colorectal cancer (CRC) have been increasing dramatically worldwide. Pinus massoniana pollen, a well-known natural food, is one of the most commonly consumed traditional medicines in China. P. massoniana pollen polysaccharides (PPPS) have antitumor effects, but it remains unclear whether they can inhibit CRC. Here, we have demonstrated that PPPS inhibited CRC cell proliferation effectively, induced morphology changes, triggered apoptosis by upregulating key apoptosis-related proteins, and arrested the cell cycle at the G0/G1 phase. Moreover, PPPS markedly inhibited CRC cell metastasis by downregulating MMP-9 and inhibiting epithelial-mesenchymal transition. In vivo, PPPS exhibited potent antitumor activity and no observable toxicity in BALB/c nude mice bearing HCT-116 tumors. Most strikingly, PPPS pre-treatment dramatically inhibited the growth of incipient tumors, although not as effectively as in the PPPS-Ther group. Thus, our results suggest that PPPS can be a potential anti-CRC agent, paving the way for developing complex carbohydrates for tumor prevention and treatment.

Pine pollen polysaccharides promote cell proliferation and accelerate wound healing by activating the JAK2-STAT3 signaling pathway.

Natural medicine can be used to develop wound healing agents due to its excellent characteristics of promoting rapid wound healing. Pine pollen polysaccharides (PPPS), a water-soluble polysaccharide with hydrophilicity and viscosity, which is suitable for the development of wound dressing. The purpose of this study is to explore the role and mechanism of PPPS in the process of wound healing. The results showed that PPPS could accelerate the wound healing, promote cell proliferation, transform the cell cycle from G1 phase to S and G2 phase, and increase the expression of Cyclin B1 in vitro. These effects of PPPS were achieved by activating JAK2-STAT3 signaling pathway. Similarly, PPPS could accelerate the healing of mouse cutaneous wounds, and could promote the growth of chicken embryo chorioallantoic vessels. In conclusion, this study indicates that PPPS is a new promising natural agent for promoting wound healing.

Effects of duck circovirus on immune function and secondary infection of Avian Pathogenic Escherichia coli.

Duck circovirus (DuCV) infection occurs frequently in ducks in China and is generally believed to lead to immunosuppression and secondary infection, though there has been a lack of detailed research and direct evidence. In this study, one-day-old Cherry Valley ducklings were artificially infected with DuCV alone and co-infected with DuCV and Avian Pathogenic Escherichia coli (APEC). The immune indexes at 32 d old were systematically monitored, including immune organ weight, lymphocyte transformation rate, IL-10, IL-12, soluble CD4 (sCD4), soluble CD8 (sCD8), IFN-γ, viral loads in each organ, APEC colonization, and so on. The results showed the development of immune organs in ducklings was affected, resulting in a decrease in the lymphocyte transformation rate (LTR), IL-12, sCD4, sCD8, IFN-γ and an increase in IL-10 content at 8 to 32 d postinfection (dpi). In the detection of virus loads in some organs, it was found that 8 dpi, DuCV existed stably in various organs, suggesting the importance of preventing and controlling the virus in the early stage of culture. The results of exploring the DuCV infection that shows some influence on secondary infection by APEC. The results showed that DuCV infection could significantly enhance the pathogenicity of APEC and the colonization ability of APEC in vivo. DuCV can induce more serious APEC infection in 24 dpi than in 14 dpi. Based on the above results, it can be concluded that DuCV infection will affect the immune system, cause immunosuppression, and lead to more serious secondary infection.

Establishment and evaluation of an indirect ELISA for detection of antibodies to goat Klebsiella pneumonia.

BACKGROUND: Klebsiella pneumonia, a Gram-negative bacterium belonging to the genus Enterobacter, causes many human and livestock diseases. Notably, infected goats may develop pneumonia, septicemia, which can lead to occasional death, resulting in great economic losses in goat-farming industry. However, there are little systematic methods for detection of goat Klebsiella pneumoniae in livestock production. RESULTS: In this study, we developed a Klebsiella pneumoniae goat polyclonal antibody and established an indirect ELISA method to detect the Klebsiella pneumoniae. After screening and optimizing the conditions for detection, we determined the optimal working dilutions of the coated-bacterial antigen, the polyclonal antibody, and the enzyme-labeled secondary antibody that were 1:800 (2.99 × 107 CFU/ml), 1:6400, and 1:5000, respectively. The optimal condition of coating and blocking were both 4 °C for 12 h. The optimal dilution buffers of bacterial antigen, the antibodies, and the blocking buffer were 0.05 mol/L carbonate buffer, 1% BSA phosphate buffer, and 1.5% BSA carbonate buffer, respectively. The cut-off value was determined to be 0.28, and the analytical sensitivity was 1:800 (dilution of a positive sample). Furthermore, there was no cross-reaction between the coated antigen and goat serum positive for antibodies against other bacteria, indicating that indirect ELISA could detect Klebsiella pneumoniae specifically in most cases. The average coefficients of variation of intra-assay and inter-assay were 4.37 and 5.17% indicating favorable reproducibility of indirect ELISA. In the detection of clinical veterinary samples, the positive rate of indirect ELISA was 6.74%, higher than that of conventional agglutination assays. CONCLUSIONS: Taken together, we successfully established an indirect ELISA method for detecting antibodies against Klebsiella pneumoniae in goats, which can be applied in production.

Polysaccharides from Pinus massoniana pollen improve intestinal mucosal immunity in chickens.

Intestinal mucosa is the largest immune organ in animals, and its immune function is directly related to the resistance against various diseases. Taishan Pinus massoniana pollen polysaccharides (TPPPS) have been recognized as an effective vaccine adjuvant and potential immune enhancer against viral infections. However, little is known about their direct immune-enhancing activity on intestinal mucosa. In this study, we extracted the polysaccharides from Taishan masson pine pollen to investigate its promotive effect on intestinal mucosal immunity. A total of 120 1-day-old chickens were divided into 4 groups and inoculated with PBS or 3 different doses of TPPPS (10 mg/mL, 20 mg/mL, and 40 mg/mL), respectively. Feces, intestinal specimens, and serum samples were collected from the chickens at 7, 14, and 21 d after inoculation. The antibodies in serum, mucosal secretion of IgA, structure of intestinal villi, and expressions of cytokine genes and mucosal immune-related genes in the chickens were all significantly improved by TPPPS treatments. At 21 d after inoculation following the challenge of Newcastle disease virus, the chickens inoculated with 20 and 40 mg/mL TPPPS exhibited decreased weight loss and reduced intestinal pathologic damage and viral loads in the intestine. In summary, our results demonstrate that TPPPS can enhance mucosal immunity and promote intestinal villi development. This study has established the foundation for the development of novel immune-enhancing agent with immune-regulatory effects on intestinal mucosa.

Screening active fractions from Pinus massoniana pollen for inhibiting ALV-J replication and their structure activity relationship investigation.

The objective was to identify the active fractions of polysaccharide against replication of ALV-J and elucidate their structure activity relationship. The optimal extraction conditions were extracting temperature 90℃, pH 9 and the ratio of liquid to solid 30:1. Under these conditions, extraction yield of total polysaccharide was 6.5 % ± 0.19 %. Total polysaccharide was then purified by DEAE-52 cellulose and Sephadex G-200 gel. Three fractions, PPP-1, PPP-2, and PPP-3, were identified with molecular weight of 463.70, 99.41, and 26.97 kDa, respectively. Three polysaccharide fractions were all composed of 10 monosaccharides in different proportions. Compared with PPP-1, which was mainly composed of glucose, PPP-2 and PPP-3 contained a higher proportion of galactose, glucuronic acid and galacturonic acid. The Congo red assay indicated that the PPP-2 may have a triple helical structure, while PPP-1 and PPP-3 were absent. In vitro assay showed that there was no significant cytotoxicity among the polysaccharide fractions under the concentration of 800 µg mL-1 (P > 0.05). The antiviral test showed that PPP-2 had the strongest activity, indicating PPP-2 was the major antiviral component. The structure-activity relationship showed that the antiviral activities of polysaccharide fractions were affected by their monosaccharide composition, molecular weight, and triple helical structure, which was a result of a combination of multiple molecular structural factors. These results showed that the PPP-2 could be exploited as a valued product for replacing synthetic antiviral drugs, and provided support for future applications of polysaccharide from Pinus massoniana pollen as a useful source for antiviral agent.

Effects of Pinus massoniana pollen polysaccharides on intestinal microenvironment and colitis in mice.

The stability of the intestinal microenvironment is the basis for maintaining the normal physiological activities of the intestine. On the contrary, disordered dynamic processes lead to chronic inflammation and disease pathology. Pinus massoniana pollen polysaccharide (PPPS), isolated from Taishan Pinus massoniana pollen, has been reported with extensive biological activities, including immune regulation. However, the role of PPPS in the intestinal microenvironment and intestinal diseases is still unknown. In this work, we initiated our investigation by using 16S rRNA high-throughput sequencing technology to assess the effect of PPPS on gut microbiota in mice. The result showed that PPPS regulated the composition of gut microbiota in mice and increased the proportion of probiotics. Subsequently, we established immunosuppressive mice using cyclophosphamide (CTX) and found that PPPS regulated the immunosuppressive state of lymphocytes in Peyer's patches (PPs). Moreover, PPPS also regulated systemic immunity by acting on intestinal PPs. PPPS alleviated lipopolysaccharide (LPS) -induced Caco2 cell damage, indicating that PPPS has the ability to reduce the damage and effectively improve the barrier dysfunction in Caco2 cells. In addition, PPPS alleviated colonic injury and relieved colitis symptoms in dextran sodium sulfate (DSS)-induced colitis mice. Overall, our findings indicate that PPPS shows a practical regulatory effect in the intestinal microenvironment, which provides an essential theoretical basis for us to develop the potential application value of PPPS further.

Taishan Pinus massoniana pollen polysaccharide inhibits H9N2 subtype influenza virus infection both in vitro and in vivo.

The H9N2 subtype avian influenza virus (AIV) is one of the most prevalent AIV subtypes that can be found throughout most countries. Currently, due to the neglect of low pathogenic avian influenza virus (LPAIV) and monotonous control technique, an expanding H9N2 virus epizootic have been arisen and causes great economic losses in the poultry industry. Therefore, novel anti-influenza drugs are necessary for the prevention and control of H9N2 AIV. Our previous studies have found that Taishan Pinus massoniana pollen polysaccharides (TPPPS) have antiviral effects, but whether they can inhibit the H9N2 AIV remains unclear. Here, we further investigated the effects of TPPPS on the H9N2 virus and its underlying mechanisms of action. We found that TPPPS significantly inhibited the replication of the H9N2 virus in a dose-dependent manner, especially during the period of virus adsorption in vitro. Transmission electron microscopy demonstrated that TPPPS reduce infection by interfering with virus entry into host cells rather than by interacting with the H9N2 virus particles. A fluorescence quantitative PCR (qPCR) assay and an animal experiment were performed to evaluate the anti-viral effect of TPPPS in vivo. As expected, the lungs of chickens treated with TPPPS had fewer lesions and lower virus contents compared with the PBS group. In addition, pre-treatment with TPPPS clearly enhanced host disease resistance and delayed infection by the H9N2 virus. Taken together, our results reveal that TPPPS suppress H9N2 virus replication both in vitro and in vivo and therefore shows promising as an anti-AIV agent.

Recombinant Lactococcus Lactis Expressing M1-HA2 Fusion Protein Provides Protective Mucosal Immunity Against H9N2 Avian Influenza Virus in Chickens.

H9N2 subtype low pathogenicity avian influenza virus (LPAIV) is distributed worldwide and causes enormous economic losses in the poultry industry. Despite immunization of almost all chickens with inactivated vaccines, the disease still remains widespread. We speculated that improving mucosal or cellular immune responses could contribute to improved control of H9N2 viruses. In this study, we constructed a novel Lactococcus lactis (L. lactis) strain expressing a recombinant fusion protein consisting of the M1 and HA2 proteins derived from an antigenically conserved endemic H9N2 virus strain. The M1-HA2 fusion protein was cloned downstream of a gene encoding a secretory peptide, and we subsequently confirmed that the fusion protein was secreted from L. lactis by Western blotting. We assessed the immunogenicity and protective effects of this recombinant L. lactis strain. Eighty 1-day-old chickens were divided into four groups, and the experimental groups were orally vaccinated twice with the recombinant L. lactis strain. Fecal and intestinal samples, sera, and bronchoalveolar lavage fluid were collected at 7, 14, and 21 days post-vaccination (dpv). Chickens vaccinated with the recombinant L. lactis strain showed significantly increased levels of serum antibodies, T cell-mediated immune responses, and mucosal secretory IgA (SIgA). Following challenge with H9N2 virus at 21 dpv, chickens vaccinated with the recombinant L. lactis strain showed decreased weight loss, lower viral titers in the lung, and reduced lung pathological damage. In summary, our results demonstrated that a recombinant L. lactis strain expressing an H9N2 M1-HA2 fusion protein could induce protective mucosal and systemic immunity. This oral vaccine is H9N2 virus-specific and represents a significant design improvement compared with previous studies. Our study provides a theoretical basis for improving mucosal immune responses to prevent and control H9N2 virus infection.

Screening host proteins required for bacterial adherence after H9N2 virus infection.

H9N2 subtype low pathogenic avian influenza virus (LPAIV) is distributed worldwide and causes great economic losses in the poultry industry, especially when complicated with other bacterial infections. Tissue damages caused by virus infection provide an opportunity for bacteria invasion, but this mechanism is not sufficient for low pathogenic strains. Moreover, although H9N2 virus infection was demonstrated to promote bacterial infection in several studies, its mechanism remained unclear. In this study, infection experiments in vivo and in vitro demonstrated that the adhesion of Escherichia coli (E. coli) to host cells significantly increased after H9N2 virus infection, and this increase was not caused by pathological damages. Subsequently, we constructed a late chicken embryo infection model and used proteomics techniques to analyze the expression of proteins associated with bacterial adhesion after H9N2 virus infection. A total of 279 significantly differential expressed proteins were detected through isobaric tags for relative and absolute quantitation (iTRAQ) coupled with nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis. The results of Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that differentially expressed proteins were enriched in host innate immunity; cell proliferation, differentiation, and apoptosis; and pathogenicity-related signaling pathways. Finally, we screened out several proteins, such as TGF-ß1, integrins, cortactin, E-cadherin, vinculin, and fibromodulin, which were probably associated with bacterial adhesion. The study analyzed the mechanism of secondary bacterial infection induced by H9N2 virus infection from a novel perspective, which provided theoretical and data support for investigating the synergistic infection mechanism between the H9N2 virus and bacteria.