CD317 maintains proteostasis and cell survival in response to proteasome inhibitors by targeting calnexin for RACK1-mediated autophagic degradation.

Unbalanced protein homeostasis (proteostasis) networks are frequently linked to tumorigenesis, making cancer cells more susceptible to treatments that target proteostasis regulators. Proteasome inhibition is the first licensed proteostasis-targeting therapeutic strategy, and has been proven effective in hematological malignancy patients. However, drug resistance almost inevitably develops, pressing for a better understanding of the mechanisms that preserve proteostasis in tumor cells. Here we report that CD317, a tumor-targeting antigen with a unique topology, was upregulated in hematological malignancies and preserved proteostasis and cell viability in response to proteasome inhibitors (PIs). Knocking down CD317 lowered Ca2+ levels in the endoplasmic reticulum (ER), promoting PIs-induced proteostasis failure and cell death. Mechanistically, CD317 interacted with calnexin (CNX), an ER chaperone protein that limits calcium refilling via the Ca2+ pump SERCA, thereby subjecting CNX to RACK1-mediated autophagic degradation. As a result, CD317 decreased the level of CNX protein, coordinating Ca2+ uptake and thus favoring protein folding and quality control in the ER lumen. Our findings reveal a previously unrecognized role of CD317 in proteostasis control and imply that CD317 could be a promising target for resolving PIs resistance in the clinic.

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.

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.

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.

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.

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.