The global prevalence of highly pathogenic avian influenza A (H5N8) infection in birds: A systematic review and meta-analysis.

The zoonotic pathogen avian influenza A H5N8 causes enormous economic losses in the poultry industry and poses a serious threat to the public health. Here, we report the first systematic review and meta-analysis of the worldwide prevalence of birds. We filtered 45 eligible articles from seven databases. A random-effects model was used to analyze the prevalence of H5N8 in birds. The pooled prevalence of H5N8 in birds was 1.6%. In the regions, Africa has the highest prevalence (8.0%). Based on the source, village (8.3%) was the highest. In the sample type, the highest prevalence was organs (79.7%). In seasons, the highest prevalence was autumn (28.1%). The largest prevalence in the sampling time was during 2019 or later (7.0%). Furthermore, geographical factors also were associated with the prevalence. Therefore, we recommend site-specific prevention and control tools for this strain in birds and enhance the surveillance to reduce the spread of H5N8.

Characterization and pathogenicity of the porcine epidemic diarrhea virus isolated in China.

Piglet diarrhea caused by the porcine epidemic diarrhea virus (PEDV) is a common problem on pig farms in China associated with high morbidity and mortality rates. In this study, three PEDV isolates were successfully detected after the fourth blind passage in Vero cells. The samples were obtained from infected piglet farms in Jilin (Changchun), and Shandong (Qingdao) Provinces of China and were designated as CH/CC-1/2018, CH/CC-2/2018, and CH/QD/2018. According to the analysis of the complete S protein gene sequence, the CH/CC-1/2018 and CH/CC-2/2018 were allocated to the G2b branch, while CH/QD/2018 was located in the G1a interval and was closer to the vaccine strain CV777. Successful detection and identification of the isolated strains were carried out using electron microscopy and indirect immunofluorescence. Meanwhile, animal challenge experiments and viral RNA copies determination were used to compare the pathogenicity. The results showed that CH/CC-1/2018 in Changchun was more pathogenic than CH/QD/2018 in Qingdao. In conclusion, the discovery of these new strains is conducive to the development of vaccines to prevent the pandemic of PEDV, especially that the CH/CC-1/2018, and CH/CC-2/2018 were not related to the classical vaccine strain CV777.

Lactobacillus rhamnosus GG Promotes Early B Lineage Development and IgA Production in the Lamina Propria in Piglets.

Gut microbes play an important role in the development of host B cells. It has been controversial whether GALT is the development site of B cells in pigs. By investigating the relationship between gut microbes and the development of B cells in the GALT of piglets, we found, to our knowledge for the first time, that early B cells exist in the gut lamina propria (LP) in pigs at different ages. We further used Lactobacillus rhamnosus GG (LGG) to treat piglets. The results showed that LGG promotes the development of the early B lineage, affects the composition of the Ig CDR3 repertoires of B cells, and promotes the production of IgA in the intestinal LP. Additionally, we found that the p40 protein derived from LGG can activate the EGFR/AKT and NF-κB signaling pathways, inducing porcine intestinal epithelial cells (IPEC-J2) to secrete a proliferation-inducing ligand (APRIL), which promotes IgA production in B cells. Finally, we identified ARF4 and DIF3 as candidates for p40 receptors on IPEC-J2 by GST pull-down, liquid chromatography-mass spectrometry/mass spectrometry analysis, and coimmunoprecipitation. In conclusion, LGG could promote early B cell differentiation and development in the intestinal LP in piglets and might contribute to promoting IgA production via secretion of p40, which interacts with the membrane receptors on IPEC-J2 and induces them to secrete APRIL. Our study will provide insight to aid in better utilization of probiotics to increase human health.

Improved pathogenicity of H9N2 subtype of avian influenza virus induced by mutations occurred after serial adaptations in mice.

H9N2 subtype, a low pathogenic avian influenza virus, is emerging as a major causative agent circulating poultry workplaces across China and other Asian countries. Increasing case number of interspecies transmissions to mammals reported recently provoked a great concern about its risks inducing global pandemics. In an attempt to understand the underlying mechanism of how the H9N2 virus disrupts the interspecies segregation to transmit to mammals. A mutant H9N2 strain was obtained by passaging the wildtype H9N2 A/chicken/Hong Kong/G9/1997 eight times from lung to lung in BALB/c mice. Our finding revealed that mice manifested severe clinical symptoms including losses of body weight, pathological damages in pulmonary sites and all died within two weeks after infected with the mutated H9N2, whereas all mice survived upon infected with wildtype strain in comparison, which suggested increased pathogenicity of the mutant strain. In addition, mice showed enhanced levels of proinflammatory cytokines in sera, including IL-6, TNF-α and IL-1ß compared to those subjected to wildtype viral infections. Sequence analysis showed that five amino acid substitutions occurred at PB2627, HA87, HA234, NP387 and M156, and a deletion mutation happened in the M gene (M157). Of these mutations, PB2 E627K played key roles in modulating lethality in mice. Taken together, the mutant H9N2 strain obtained by serial passaging of its wildtype in mice significantly increased its virulence leading to death of mice, which might be associated the accumulated mutations occurred on its genome.

Bacillus subtilis BSH has a protective effect on Salmonella infection by regulating the intestinal flora structure in chickens.

Salmonellosis is a worldwide zoonotic disease that poses a serious threat to the reproduction of livestock and poultry and the health of young animals. Probiotics including Bacillus species, have received increasing attention as a substitute for antibiotics. In this study, chicks infected with Salmonella were fed feed supplemented with the BSH to observe the pathological changes in the liver, detect the number of viable bacteria in the liver and spleen, and record the death of the chicks. The results showed that BSH could reduce the pathological changes in the liver and the invasion of Salmonella into the liver and spleen of chicks. In addition, the survival rate of chicks in the BSH experimental group was 60%, while that in the infected control group was 26%, indicating that BSH had a protective effect on chicks infected with Salmonella. Finally, the fecal microflora of 9-day-old chicks was analyzed by 16S rRNA high-throughput sequencing. The results showed that Salmonella infection could cause intestinal flora changes, while BSH could alleviate this change. In addition, BSH also promoted the proliferation of Lactobacillus salivarius in the cecum of chick. This study emphasized that BSH has anti- Salmonella infection effects in chickens and can be used as a candidate microecological preparation strain.

Amplification of DDR2 mediates sorafenib resistance through NF-κB/c-Rel signaling in hepatocellular carcinoma.

Sorafenib was the first systemic therapy approved by the Food and Drug Administration to treat advanced hepatocellular carcinoma (HCC). However, sorafenib therapy is frequently accompanied by drug resistance. We aimed to explore the mechanisms of sorafenib resistance and provide feasible solutions to increase the response to sorafenib in patients with advanced HCC. The expression profile of discoidin domain receptor 2 (DDR2) in HCC tissues and cells was detected using quantitative real-time PCR (qPCR) and western blotting assays. The effects of DDR2 on sorafenib resistance were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, TdT-mediated dUTP nick end labeling, and flow cytometry assays. The effect of DDR2 on the nuclear factor kappa B (NF-κB) signaling pathway was evaluated by luciferase reporter, immunofluorescence, qPCR and flow cytometry assays. We demonstrated that DDR2 expression was dramatically upregulated in sorafenib-resistant HCC tissues relative to sensitive tissues. Downregulation of DDR2 sensitized HCC cell lines to sorafenib cytotoxicity. Further analysis showed that DDR2 could increase the nuclear location of REL proto-oncogene, a NF-κB subunit, to mediate NF-κB signaling. Blocking NF-κB signaling using the NF-κB signaling inhibitor, bardoxolone methyl, increased the response of HCC cells to sorafenib. Further analysis showed that DNA amplification of DDR2 is an important mechanism leading to DDR2 overexpression in HCC. Our results demonstrated that DDR2 is a potential therapeutic target in patients with HCC, and targeting DDR2 represents a promising approach to increase sorafenib sensitivity in patients with HCC.

Immunological evaluation of invasive Lactobacillus plantarum co-expressing EtMIC2 and chicken interleukin-18 against Eimeria tenella.

Chicken coccidiosis is a protozoan parasitic disease that leads to considerable economic losses in the poultry industry. In this study, we used invasive Lactobacillus plantarum (L.P) expressing the FnBPA protein as a novel bacterial carrier for DNA delivery into epithelial cells to develop a live oral DNA vaccine. A fusion DNA vaccine co-expressing EtMIC2 and chicken IL-18 (chIL-18) was constructed and then delivered to the host by invasive L.P. Its efficacy against Eimeria tenella challenge was evaluated in chickens by examining the relative weight gain rate; caecal lesion score; OPG; anti-coccidial index (ACI); levels of EtMIC2 antibody, FnBPA, IL-4, IL-18, IFN-γ and SIgA; and proliferation ability and percentages of CD4+ and CD8+ splenocytes. The experimental results showed that chickens immunized with invasive L.P carrying the eukaryotic expression vector pValac-EtMIC2 (pValac-EtMIC2/pSIP409-FnBPA) had markedly improved immune protection against challenge compared with that of chickens immunized with non-invasive L.P (pValac-EtMIC2/pSIP409). However, invasive L.P co-expressing EtMIC2 with the chIL-18 vector exhibited the highest protection efficiency against E. tenella. These results indicate that invasive Lactobacillus-expressing FnBPA improved humoural and cellular immunity and enhanced resistance to E. tenella. The DNA vaccine delivered by invasive Lactobacillus provides a new concept and method for the prevention of E. tenella.

Immune responses induced by recombinant Lactobacillus plantarum expressing the spike protein derived from transmissible gastroenteritis virus in piglets.

Transmissible gastroenteritis coronavirus (TGEV) is one of the most severe threats to the swine industry. In this study, we constructed a suite of recombinant Lactobacillus plantarum with surface displaying the spike (S) protein coming from TGEV and fused with DC cells targeting peptides (DCpep) to develop an effective, safe, and convenient vaccine against transmissible gastroenteritis. Our research results found that the recombinant Lactobacillus plantarum (NC8-pSIP409-pgsA-S-DCpep) group expressing S fused with DCpep could not only significantly increase the percentages of MHC-II+CD80+ B cells and CD3+CD4+ T cells but also the number of IgA+ B cells and CD3+CD4+ T cells of ileum lamina propria, which elevated the specific secretory immunoglobulin A (SIgA) titers in feces and IgG titers in serum. Taken together, these results suggest that NC8-pSIP409-pgsA-S-DCpep expressing the S of TGEV fused with DCpep could effectively induce immune responses and provide a feasible original strategy and approach for the design of TGEV vaccines.

Immune response characterization of mice immunized with Lactobacillus plantarum expressing spike antigen of transmissible gastroenteritis virus.

The highly infectious porcine transmissible gastroenteritis virus (TGEV), which belongs to the coronaviruses (CoVs), causes diarrhea and high mortality rates in piglets, resulting in severe economic losses in the pork industry worldwide. In this study, we used Lactobacillus plantarum (L. plantarum) to anchor the expression of TGEV antigen (S) to dendritic cells (DCs) via dendritic cell-targeting peptides (DCpep). The results show that S antigen could be detected on the surface of L. plantarum by different detection methods. Furthermore, flow cytometry and ELISA techniques were used to measure the cellular, mucosal, and humoral immune responses of the different orally gavaged mouse groups. The obtained results demonstrated the significant effect of the constructed L. plantarum expressing S-DCpep fusion proteins in inducing high expression levels of B7 molecules on DCs, as well as high levels of IgG, secretory IgA, and IFN-γ and IL-4 cytokines compared with the other groups. Accordingly, surface expression of DC-targeted antigens successfully induced cellular, mucosal, and humoral immunity in mice and could be used as a vaccine.

Lactobacillus plantarum displaying conserved M2e and HA2 fusion antigens induces protection against influenza virus challenge.

Avian influenza virus (AIV) can infect poultry, mammals, and other hosts and causes enormous economic losses to the global poultry industry. In this study, to develop a novel and potent oral vaccine based on Lactobacillus plantarum (L. plantarum) for controlling the spread of AIV in the poultry industry, we constructed a recombinant L. plantarum strain displaying the 3M2e-HA2 protein of the influenza virus and determined the effect of N/pgsA'-3M2e-HA2 against AIV in chicks. We first confirmed that the 3M2e-HA2 fusion protein was expressed on the surface of L. plantarum via flow cytometry and immunofluorescence experiments. Our experimental results demonstrated that chicks immunized with N/pgsA'-3M2e-HA2 could induce specific humoral, mucosal, and T cell-mediated immune responses, eliciting the host body to protect itself against AIV. Additionally, compared to oral administration, the intranasal immunization of chicks with N/pgsA'-3M2e-HA2 provided a stronger immune response, resulting in a potent protective effect that hindered the loss of body weight, decreasing pulmonary virus titers and reducing lung and throat pathological damages. Thus, our results indicate that our novel approach is an effective method of vaccine design to promote mucosal immunity.

Effect of apigenin on apoptosis induced by renal ischemia/reperfusion injury in vivo and in vitro.

OBJECTIVES: This study aims to investigate the effects and molecular mechanisms of apigenin (ApI) on renal ischemia/reperfusion (I/R) injury in vivo and in vitro. METHODS: In vivo, the left renal artery was clamped for 45 min and the right kidney was removed to study renal I/R injury on Sprague-Dawley (SD) rats. ApI was injected at 60 min before renal ischemia. In vitro, renal tubular epithelial cells (HK-2) were pretreated with or without ApI (20 uM) for 60 min and then treated with hypoxia/reoxygenation (H/R). Renal function, histology, cells apoptosis, and cell viability were tested. Furthermore, the potential molecular mechanisms were assessed. RESULTS: ApI pretreatment could significantly alleviated the renal function and the pathological damage as well as cells apoptosis after I/R injury. Meanwhile, ApI treatment protects H/R induced HK-2 cell apoptosis in vitro. The results of Western blot showed that ApI significantly increased the expressions of B-cell lymphoma 2 (Bcl-2) and phosphor-AKt (p-AKt), Phosphoinositide 3-kinase (PI3K), while down-regulated the expressions of Caspase3 and Bax induced by H/R injury. CONCLUSIONS: ApI pretreatment can protect renal function against I/R injury and prevent renal tubular cells from apoptosis in vivo and in vitro which might through PI3K/Akt mediated mitochondria-dependent apoptosis signaling pathway.

A genetic variant in long non-coding RNA MALAT1 associated with survival outcome among patients with advanced lung adenocarcinoma: a survival cohort analysis.

BACKGROUND: Recently studies have demonstrated that the long non-coding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 (MALAT1) may participate in the development and progression of lung cancer. In this study, we hypothesized that genetic variant of this lncRNA may affect the prognosis of lung cancer patients. METHODS: We conducted a follow-up study for 538 patients with non-small cell lung carcinoma (NSCLC), including 140 early-staged (stage I and II) and 398 advanced staged (stage III and IV) patients. The genetic variant rs3200401 in MALAT1 was then genotyped among this population by using TaqMan assay. The association of this variant with overall survival of these patients was further analyzed. RESULTS: It was shown that among the advanced lung adenoma patients, subjects carrying rs3200401 CT and CT + TT genotypes had significantly longer median survival time (MST = 29.9, 28.9 vs. 19.3 month, Long-rank P = 0.019 and 0.024, respectively) and decreased death risks [crude HR (95% CI) = 0.65 (0.43-0.98) and 0.64 (0.44-0.95), P = 0.040 and 0.025, respectively], when compared to subjects wtih the MALAT1 rs3200401 CC genotype. However, the beneficial effect of rs3200401 was not seen among early NSCLC and advanced lung squamous cell carcinoma patients. We further tested the TCGA data, and found that a higher expression of MALAT1 was associated with metastatic of advanced lung adenocarcinoma but not with lung squamous cell carcinoma. CONCLUSIONS: The rs3200401 T allele located on the lncRNA MALAT1 was associated with a better survival for advanced lung adenocarcinoma patients, which may offer a novel prognostic biomarker for this patient subgroup. However, these results need to be validated in larger populations of lung cancer and the biological function of this variant still warrants further investigation.

Recombinant Lactobacillus plantarum expressing HA2 antigen elicits protective immunity against H9N2 avian influenza virus in chickens.

Low pathogenic H9N2 subtype avian influenza virus (AIV) can lead to moderate respiratory symptoms and low egg production rates in poultry. Due to its immunologic suppression, other various infectious pathogens give rise to the co-infection of hosts, causing heavy economic losses in the commercial poultry industry in both China and worldwide. Therefore, it is time to explore a novel, safe, and effective vaccine. We have already made use of the surface of Lactobacillus plantarum to display AIV HA2 (NC8-pSIP409-pgsA'-HA2), which demonstrated that it has a good immunogenicity. In this study, by evaluating the immune protection effect of NC8-pSIP409-pgsA'-HA2 on chickens, we found that the hemagglutination inhibition (HI) antibodies, specificity IgG antibody in chickens, the sIgA titer in broncho alveolar lavage fluids (BALF), and the T cell response were increased notably after oral vaccination with NC8-pSIP409-pgsA'-HA2. In addition, weight loss, lung titers, and lung pathologies were improved when chickens were orally vaccinated with NC8-pSIP409-pgsA'-HA2 after challenge with H9N2 AIV. This strategy lays the foundation for the development of recombinant L. plantarum oral vaccines in the prevention of AIV.

Protection of chickens against H9N2 avian influenza virus challenge with recombinant Lactobacillus plantarum expressing conserved antigens.

Avian influenza virus (AIV) is spreading worldwide and is a serious threat to the health of poultry and humans. In many countries, low pathogenic AIVs, such as H9N2, have become an enormous economic burden on the commercial poultry industry because they cause mild respiratory disease and decrease egg production. A recombinant Lactobacillus plantarum NC8 strain expressing NP-M1-DCpep from H9N2 AIV has been studied in a mouse model. However, it remains unknown whether this L. plantarum strain can induce an immune response and provide protection against H9N2 AIV in chickens. In this study, chickens that were orally vaccinated with NC8-pSIP409-NP-M1-DCpep exhibited significantly increased T cell-mediated immune responses and mucosal sIgA and IgG levels, which provided protection against H9N2 AIV challenge. More importantly, compared with oral administration of NC8-pSIP409-NP-M1-DCpep, intranasal administration induced stronger immune responses and provided effective protection against challenge with the H9N2 virus by reducing body weight loss, lung virus titers, and throat pathology. Taken together, these findings suggest that L. plantarum expressing NP-M1-DCpep has potential as a vaccine to combat H9N2 AIV infection.

Expression and purification of swine RAG2 in E. coli for production of porcine RAG2 polyclonal antibodies.

Recombination activating gene 2 (RAG2) is necessary for immature B cell differentiation. Antibodies to human and rabbit RAG2 are currently commercially available, but antibodies to swine RAG remain unavailable to date. In this study, the swine RAG2 genes sequence was synthesized and then cloned into a pET-28a vector. The recombinant fusion protein was successfully expressed in E. coli, purified through nickel column chromatography, and further digested with Tobacco Etch Virus protease. The cleaved protein was purified by molecular-exclusion chromatography and named pRAG2. We used pRAG2 to immunize rabbits, collected the serum and purified rabbit anti-pRAG2 polyclonal antibodies. The rabbit anti-pRAG2 polyclonal antibodies were tested via immunofluorescence on eukaryotic cells overexpressing pRAG2 and also able to recognize pig natural RAG2 and human RAG2 protein in western blotting. These results indicated that the prepared rabbit anti-pRAG2 polyclonal antibodies may serve as a tool to detect immature B cell differentiation of swine.

Bisbenzylisoquinoline alkaloids from the roots of cyclea tonkinensis.

Cissampentine A (1), an enantiomer of cissampentin, three new cycleatjehenine-type bisbenzylisoquinoline alkaloids, cissampentine B-D (2-4), and five known alkaloids were isolated from the roots of Cyclea tonkinensis. Their structures were established by interpretation of NMR, high-resolution ESI-MS data, and CD spectra. In vitro studies indicated that compounds 1 and 4 exhibited cytotoxicity against the HCT-8 tumor cell line (IC50 values of 8.97 and 9.73 µM, respectively), and compound 4 was also active against the Bel-7402 tumor cell line (IC50 value of 5.36 µM).

[Treatment of Chronic Heart Failure Patients with Qi-Yang Deficiency and Blood Stasis Resistance Syndrome by Xnmallong Injection: a Multi-center Randomized Control Study].

OBJECTIVE: To evaluate the efficacy and safety of Xinmailong Injection (XI) in treatment of chronic heart failure (CHF) patients with qi-yang deficiency and blood stasis resistance syndrome (QY-DBSRS). METHODS: Totally 238 CHF patients with QYDBSRS were assigned to the treatment group (118 cases) and the control group (120 cases) by randomized, double-blind, placebo parallel controlled method. Patients in the treatment group received routine therapy and XI (100 mg/2 mL, by dripping at 5 mg/kg, twice per day for 5 consecutive days), while those in the control group received routine therapy and XI mimetic agent (100 mg/2 mL, by dripping at 5 mg/kg, twice per day for 5 consecutive days). The heart function classification of New York Heart Association (NYHA), 6-min walking distance, left ventricular ejection fraction (LVEF), scores for Chinese medical symptoms were observed before and after treatment, and safety assessed. RESULTS: Totally 235 patients actually entered full analysis set (FAS), including 120 cases in the control group and 115 cases in the treatment group. The total effective rate of heart function, 6-min walking distance and increased post-pre-treatment distance in the experimental group were superior to those of the control group with statistical difference (all P < 0.05). Compared with the control group, increased value of post-pre-treatment LVEF, the total effective rate of Chinese medical syndrome efficacy, scores for Chinese medical symptoms and decreased post-pre-treatment value of Chinese medical syndrome scores were obviously improved (all P < 0.05). There was no significant difference in the incidence of adverse events between the two groups (P > 0.05). CONCLUSIONS: XI could improve the heart function of CHF patients, improve Chinese medical symptoms, elevate exercise tolerance, and improve LVEF. It had no obvious toxic and side effects.

[The Cross-sectional Study on the Influence Factors for Encrustation of Double J Stent in Patients with Urolithiasis].

OBJECTIVE: To study the influence factors for encrustation of double J stent in patients with urolithiasis. METHODS: In this study, there were 84 urolithiasis patients with double J stent included from February to July 2014 in our hospital. The encrustation on double J stent was evaluated by a PC stereo microscope. The nterrelated clinical data were obtained, then the factors which may affect the encrustation were studied by logistic regression analysis. RESULTS: The mean indwelling time was (17. 0±6. 0) d, and a thin encrustation formed on the stents for most cases [67/84(79. 8%)]. Compared with the cases who did not form a thin encrustation, those having a thin encrustation formation on the stent were younger [(44. 9±11. 5) vs. (54. 4±12. 6), P=0. 004]; The patients with proteinuria got a higher rate of encrustation [62/73(84. 9%) vs. 5/11 (45. 5%), P=0. 002]. The patients with urinary tract infection had a higher rate of encrustation [26/28(92. 9%) vs. 41/56(73. 2%), P 3. 035]. The patients with hematuriaalso got a higher rate of encrustation [67/80(83. 8%) vs. 0/4, P=0. 001]. Different sex, retention time,serum calcium,inorganic phosphorus, uric acid, urine pH,lithiasis component had no effects on encrustation (P>0. 05). Logistic regression analysis showed that age and proteinuria was retained as idependent correlated factors with encrustation (P<0. 05), while hematuria and urinary tract infections had a low ntensity correlation with encrustation (P>0. 05). CONCLUSION: For encrustation of double J stentin patients with urolithiasis, younger age, increased urinary protein, hematuria and infections are important risk-factors.

[Chemical constituents of Cocculus orbiculatus var. mollis root].

OBJECTIVE: To study the chemical constituents in the root of Cocculus orbiculatus var. mollis. METHODS: The compounds were isolated by silica gel chromatography, their structures were established by spectroscopic methods. RESULTS: Eleven compounds were isolated and identified as wattisine A (I), O-methylcocsoline (II), (+) cocsoline (III), (+) cocsuline (IV), magnoflorine (V), sino-coculine (VI), isosinococuline (VII), (-) coclaurine (VIII), daucosterol (IX), beta-sitosterol (X) and 1-oleioyl-3-(9Z, 12Z-arachoyl) glycerol (XI). CONCLUSION: Compound I is isolated from this genus for the first time,and compound II - XI are isolated from this plant for the first time.