[Identification of serum peptide biomarker for ovarian cancer diagnosis by Clin-TOF-II-MS combined with magnetic beads technology].

Objective: To explore the serum cyclic polypeptide biomarkers for ovarian cancer diagnosis. Methods: A total of 54 patients with epithelial ovarian cancer confirmed by pathology in Cancer Hospital, Chinese Academy of Medical Sciences from March 2018 to September 2018 were selected as the study subjects, and 40 healthy women with normal examination results in the cancer screening center were selected as the control. All of the samples were randomly divided into training set and validation set at the ratio of 1∶1 with a random number. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) combined with magnetic bead technology was used for detecting peptide profiling in serum samples to screen significantly differently expressed peptides between ovarian cancer group and control group of the training set (score>5). Receiver operating characteristic (ROC) curve analysis was used to screen differential peptide peaks with area under curve (AUC) ≥0.8, sensitivity and specificity>90% in the training set and validation set. Liquid chromatography-mass spectrometry (LC-MS/MS) was further used to determine the composition of differentially expressed peptides. Results: By comparing the peptide profiles of the two groups, 102 differential peptide peaks were initially detected in the mass-to-charge ratio range of 1 000 to 10 000. ROC curve analysis showed that there were 42 differential peptide peaks with AUC ≥0.8 in both training set and validation set, 19 of which were highly expressed in ovarian cancer group, and 23 were lowly expressed. There were 15 different peptide peaks in highly expressed ovarian cancer group with sensitivity and specificity over 90%. The mass-to-charge ratios were 7 744.27, 5 913.41, 5 329.87, 4 634.21, 4 202.02, 3 879.26, 3 273.35, 3 253.79, 3 234.34, 2 950.33, 2 664.51, 2 018.38, 1 893.37, 1 498.69 and 1 287.55. There were 15 different peptide peaks in lowly expressed ovarian cancer group with sensitivity and specificity over 90%, the mass-to-charge ratios were 9 288.46, 7 759.77, 5 925.24, 4 652.77, 4 210.42, 3 887.02, 3 279.90, 3 240.82, 2 962.15, 2 932.70, 2 022.42, 1 897.16, 1 501.69, 1 337.38 and 1 290.13. No protein composition was identified in 15 different peptide peaks in lowly expressed ovarian cancer group. The two protein compositions identified in 15 different peptide peaks in highly expressed ovarian cancer group were recombinant serglycin (SRGN) and fibinogen alpha chain (FGA), the mass-to-charge ratios of which were 1 498.696 and 5 913.417, respectively. The sensitivity and specificity of the two proteins for ovarian cancer diagnosis were 100%, 100% and 90.9%, 100%, respectively. Conclusion: SRGN and FGA are highly expressed in the serum of ovarian cancer patients, which may be potential diagnostic markers for ovarian cancer.

RNA sequencing, de novo assembly and differential analysis of the gill transcriptome of freshwater climbing perch Anabas testudineus after 6 days of seawater exposure.

To obtain transcriptomic insights into branchial responses to salinity challenge in Anabas testudineus, this study employed RNA sequencing (RNA-Seq) to analyse the gill transcriptome of A. testudineus exposed to seawater (SW) for 6 days compared with the freshwater (FW) control group. A combined FW and SW gill transcriptome was de novo assembled from 169.9 million 101 bp paired-end reads. In silico validation employing 17 A. testudineus Sanger full-length coding sequences showed that 15/17 of them had greater than 80% of their sequences aligned to the de novo assembled contigs where 5/17 had their full-length (100%) aligned and 9/17 had greater than 90% of their sequences aligned. The combined FW and SW gill transcriptome was mapped to 13,780 unique human identifiers at E-value ≤1.0E-20 while 952 and 886 identifiers were determined as up and down-regulated by 1.5 fold, respectively, in the gills of A. testudineus in SW when compared with FW. These genes were found to be associated with at least 23 biological processes. A larger proportion of genes encoding enzymes and transporters associated with molecular transport, energy production, metabolisms were up-regulated, while a larger proportion of genes encoding transmembrane receptors, G-protein coupled receptors, kinases and transcription regulators associated with cell cycle, growth, development, signalling, morphology and gene expression were relatively lower in the gills of A. testudineus in SW when compared with FW. High correlation (R = 0.99) was observed between RNA-Seq data and real-time quantitative PCR validation for 13 selected genes. The transcriptomic sequence information will facilitate development of molecular resources and tools while the findings will provide insights for future studies into branchial iono-osmoregulation and related cellular processes in A. testudineus.

Ammonia exposure affects the mRNA and protein expression levels of certain Rhesus glycoproteins in the gills of climbing perch.

The freshwater climbing perch, Anabas testudineus, is an obligate air-breathing and euryhaline teleost capable of active ammonia excretion and tolerant of high concentrations of environmental ammonia. As Rhesus glycoproteins (RhGP/Rhgp) are known to transport ammonia, this study aimed to obtain the complete cDNA coding sequences of various rhgp isoforms from the gills of A. testudineus, and to determine their mRNA and protein expression levels during 6 days of exposure to 100 mmol l-1 NH4Cl. The subcellular localization of Rhgp isoforms in the branchial epithelium was also examined in order to elucidate the type of ionocyte involved in active ammonia excretion. Four rhgp (rhag, rhbg, rhcg1 and rhcg2) had been identified from the gills of A. testudineus They had conserved amino acid residues for NH4+ binding, NH4+ deprotonation, channel gating and lining of the vestibules. Despite inwardly directed NH3 and NH4+ gradients, there were significant increases in the mRNA expression levels of the four branchial rhgp in A. testudineus at certain time points during 6 days of ammonia exposure, with significant increases in the protein abundances of Rhag and Rhcg2 on day 6. Immunofluorescence microscopy revealed a type of ammonia-inducible Na+/K+-ATPase α1c-immunoreactive ionocyte with apical Rhag and basolateral Rhcg2 in the gills of fish exposed to ammonia for 6 days. Hence, active ammonia excretion may involve NH4+ entering the ionocyte through the basolateral Rhcg2 and being excreted through the apical Rhag, driven by a transapical membrane electrical potential generated by the apical cystic fibrosis transmembrane conductance regulator Cl- channel, as suggested previously.

Deep sequencing-based analysis of gene expression in bovine mammary epithelial cells after Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae infection.

The goal of this study was to characterize the transcriptome of primary bovine mammalian epithelial cells (pBMECs) and to identify candidate genes for response and resistance to Staphylococcus aureus (strain S108), Escherichia coli (strain E23), and Klebsiella pneumoniae (strain K96) infection. Using Solexa sequencing, approximately 4.9 million total sequence tags were obtained from each of the three infected libraries and the control library. Gene Ontology (GO) analysis of the S108-infected pBMECs showed differentially expressed genes (DEGs) were significantly involved in metabolic processes. In E23-infected pBMECs, DEGs were predominantly associated with cell death and programmed cell death GO terms, while in K96-infected pBMECs, DEGs were primarily involved in metabolic processes and in utero embryonic development. Analysis of the cluster of orthologous groups of proteins showed that the S108-infected, E23-infected and K96-infected pBMECs were significantly involved in "Translation, ribosomal structure and biogenesis", "General function prediction only" and "Replication, recombination and repair". The transcriptome sequences were also annotated for KEGG orthology, and it was found that DEGs in S108-infected pBMECs were significantly involved in oxidative phosphorylation and Parkinson's disease. The clustered pathway terms of the DEGs of the E23-infected pBMECs were found to involve the NOD-like receptor signaling pathway and oxidative phosphorylation, while those of the K96-infected pBMECs were primarily involved in oxidative phosphorylation and apoptosis. Our results have identified a number of immune-related genes that showed changes in gene expression after bacterial infection, and provided insight into the interactions between pBMECs and the bacteria.

Irreversible inhibitors of the epidermal growth factor receptor: thienopyrimidine core with α,ß-unsaturated amide side chain.

Overexpression of epidermal growth factor receptor (EGFR) tyrosine kinases has been found in a variety of cancers such as breast, ovarian, colon, and non-small-cell lung cancers, which is associated with poor prognosis in patients. In an effort to find effective irreversible inhibitors of the EGFR tyrosine kinase family (mainly HER2), two series of HER2 tyrosine kinase inhibitors with thieno[3,2-d]pyridine and thieno[2,3-d]pyridine as central part and with a basic α,ß-unsaturated amide side chain were developed. The α,ß-unsaturated amide side chain (the Michael acceptor) at the 6-position, which forms a covalent bond to Cys773 located in the ATP binding pocket of the EGFR enzyme, is a major factor in the generation of irreversible inhibition. In our study, thienopyrimidine instead of quinazoline was used as the central structure, and different substituents were introduced at the 4-position to investigate the structure-activity relationships. The thieno[2,3-d]pyrimidine derivatives 16a-d showed potent HER2 enzyme inhibition and anti-proliferative activity against SK-BR-3 cells. Especially, (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)thieno[2,3-d]pyrimidin-6-yl)-4-(dimethylamino)but-2-enamide 16d was identified as a potential irreversible HER2 inhibitor. Both its catalytic enzyme activity profile and its cellular efficacy were found to be superior to those of the marketed drug lapatinib.

Clinical significance of phospholipase A2 group IIA (PLA2G2A) expression in primary resected esophageal squamous cell carcinoma.

AIM: The aim of this study was to clarify the clinico-pathological outcome and prognostic significance of phospholipase A2 group IIA (PLA2G2A) in esophageal squamous cell carcinoma (ESCC). PATIENTS AND METHODS: Immunohistochemical staining for PLA2G2A was performed on surgical specimens obtained from 132 patients with ESCC, and 43 from matched adjacent non-malignant sites. Differences in PLA2G2A expression and clinical characteristics were compared by χ2 test. Correlations between prognostic outcomes and with PLA2G2A expression were investigated using Kaplan-Meier analysis and the Cox proportional hazards model. RESULTS: Immunoreactivity of PLA2G2A was observed in 32% (42 of 132) of ESCC tissues compared with negative staining in matched adjacent non-malignant sites. In addition, PLA2G2A expression inversely correlated with pathological classification (p < 0.05 for T, N, and M classifications) and clinical staging (p = 0.03). Furthermore, patients with positive PLA2G2A had prolonged overall survival (p < 0.01). CONCLUSIONS: Reduced PLA2G2A expression may be a risk factor for advanced clinicopathological classification and poor patient survival. These findings suggest that PLA2G2A may serve as a useful marker for the prognostic evaluation of ESCC patients.

Temporospatial expression of Dmrt1 in chicken urogenital system (Gallus gallus) using whole mount in situ hybridization.

Doublesex and mab-3-related transcription factor 1 (Dmrt1) is a Z-linked gene that putatively determines the phenotype of gonads in birds. The sex differential expression of Dmrt1 was examined using wholemount in situ hybridization (WISH) in the urogenital systems during embryogenesis. The results revealed that Dmrt1 showed dimorphic expression in chicken gonads, which increased from day 6.5 to day 10.5. The expression of Dmrt1 in male (ZZ) gonads was not twice as much as in female (ZW) gonads, suggesting the existence of other regulatory mechanisms in addition to Z chromosome dosage effect.

Roles of three branchial Na(+)-K(+)-ATPase α-subunit isoforms in freshwater adaptation, seawater acclimation, and active ammonia excretion in Anabas testudineus.

Three Na(+)-K(+)-ATPase (nka) α-subunit isoforms, nka α1a, nka α1b, and nka α1c, were identified from gills of the freshwater climbing perch Anabas testudineus. The cDNA sequences of nka α1a and nka α1b consisted of 3,069 bp, coding for 1,023 amino acids, whereas nka α1c was shorter by 22 nucleotides at the 5' end. In freshwater, the quantity of nka α1c mRNA transcripts present in the gills was the highest followed by nka α1a and nka α1b that was almost undetectable. The mRNA expression of nka α1a was downregulated in the gills of fish acclimated to seawater, indicating that it could be involved in branchial Na(+) absorption in a hypoosmotic environment. By contrast, seawater acclimation led to an upregulation of the mRNA expression of nka α1b and to a lesser extent nka α1c, indicating that they could be essential for ion secretion in a hyperosmotic environment. More importantly, ammonia exposure led to a significant upregulation of the mRNA expression of nka α1c, which might be involved in active ammonia excretion. Both seawater acclimation and ammonia exposure led to significant increases in the protein abundance and changes in the kinetic properties of branchial Na(+)-K(+)-ATPase (Nka), but they involved two different types of Nka-immunoreactive cells. Since there was a decrease in the effectiveness of NH(4)(+) to substitute for K(+) to activate branchial Nka from fish exposed to ammonia, Nka probably functioned to remove excess Na(+) and to transport K(+) instead of NH(4)(+) into the cell to maintain intracellular Na(+) and K(+) homeostasis during active ammonia excretion.

A bursal pentapeptide (BPP-I), a novel bursal-derived peptide, exhibits antiproliferation of tumor cell and immunomodulator activity.

The bursa of Fabricius (BF) is the central humoral immune organ unique to birds. Here, we isolated a novel bursal pentapeptide I (BPP-I), LGPGP, from BF. BPP-I could play inhibition effect on MCF-7 but not on CEF or Vero cell proliferation in vitro, and enhance antitumor factor p53 protein expression. Also, BPP-I stimulated antibody production in a dose-dependent manner in hybridoma cell. Furthermore, BPP-I could induce various immune responses in mice immunization experiments, including increase antibody production and cytokines IL-4 and IFN-γ level, and induce T-cell immunophenotyping. These results suggest that BPP-I is a potential immunomodulator of antitumor and immunity. The study could provide some novel insights on the probable candidate reagent for the antitumor and immune improvement.

Gene expression profiling of hybridoma cells after bursal-derived bioactive factor BP5 treatment.

Bursa of Fabricius is the acknowledged vital humoral immune system for B cell differentiation and antibody production. To study the molecular mechanism underlying the effect of bursal-derived BP5, we used gene microarray to analyze the genomic expression profiling of BP5-treated hybridoma cells. BP5 exhibited an immunomodulatory effect on antibody production in hybridoma cells and induced alterations in the gene expression profiles related to the immune-related biological processes, such as T cell activation and proliferation, B cell activation, B cell-mediated immunity, and cytokines cytokine production involved in immune response. In addition, 26 biological pathways associated with immunomodulatory functions were regulated in BP5-treated hybridoma cells, in which p53 signal pathway played an important role in antitumor. Among these regulated genes, 12 differentially expressed genes were verified by qRT-PCR. The activation of p53 activity by BP5 was further confirmed by p53 luciferase reporter assay and p53 expression. Our data revealed that bursal-derived BP5 could regulate various immune-related cellular processes, including antitumor factor p53 signal pathway, perhaps partially accounting for the reported immunomodulatory roles and novel antiproliferation on tumor cells functions of bursal-derived bioactive factor BP5.

Different gene transfer methods at the very early, early, late and whole embryonic stages in chicken.

New technologies in gene transfer combined with experimental embryology make the chicken embryo an excellent model system for gene function studies. The techniques of in ovo electroporation, in vitro culture for ex ovo electroporation and retrovirus-mediated gene transfer have already been fully developed in chicken. Yet to our knowledge, there are no definite descriptions on the features and application scopes of these techniques. The survival rates of different in vitro culture methods were compared and the EGFP expression areas of different gene transfer techniques were explored. It was that the optimal timings of removing embryo for EC culture and Petri dish system was at E1.5 and E2.5, respectively; and optimal timing of injecting retrovirus is at E0. Results indicated that the EC culture, in ovo electroporation, the Petri dish system and retrovirus-mediated method are, respectively, suitable for the very early, early, late and whole embryonic stages in chicken. Comparison of different gene transfer methods and establishment of optimal timings are expected to provide a better choice of the efficient method for a particular experiment.

The Prevalence and Progression of Myopia in Elementary School Students in Shanxi Province, China During the COVID-19 Pandemic.

PURPOSE: To investigate the prevalence of myopia and the risk factors associated with its progression in elementary school students during the COVID-19 pandemic in Shanxi Province, China. METHODS: The investigation included 960 students spanning first to sixth grade from six elementary schools in Shanxi Province, China. All participants received non-cycloplegic refraction and vision tests in December of 2019 (before the COVID-19 pandemic) and in June of 2020 (after classes resumed). Information concerning the students' eye-use behaviors, physical activities, diet and sleep during the pandemic was collected using a questionnaire survey. A total of 913 students (457 males) completed all tests and the questionnaire. RESULTS: The overall prevalence rate of myopia was 16.6% in December of 2019, and it increased with age. There was no gender difference in the prevalence of myopia (χ2 = 3.210, P = .073), but females exhibited a lower average spherical equivalent (SE) (P = .026). When the classes were resumed 6 months later, the overall prevalence rate of myopia was found to be 39.4%, which was significantly higher than it before the pandemic (χ2 = 117.425, P < .001). The average SE of the participants was -0.95D, which was significantly lower than the average SE (-0.43D) before the pandemic (P < .001). SE variation (ΔSE) in grade 6 was significantly higher than that in grade 1. No significant difference in ΔSE was found between males and females. Analyses of ordinary least squares (OLS)-estimated linear, natural logarithmic and quadratic functions revealed that the progression of myopia during the COVID-19 pandemic was significantly correlated with screen time, types of electronic devices, the amount of sleep, age, and the number of parents with myopia. CONCLUSIONS: The prevalence rate and progression of myopia among elementary school students in Shanxi Province increased significantly during the COVID-19 pandemic, which was likely related to China's home-based online learning programs. Therefore, it is necessary to optimize the educational programs for elementary school students when they study at home. We recommend increased time for outdoor activities and limiting screen time.

Transcriptome Analysis of Genes Responding to Infection of Leghorn Male Hepatocellular Cells With Fowl Adenovirus Serotype 4.

Fowl adenovirus serotype 4 (FAdV-4) is a highly pathogenic virus with a broad host range that causes huge economic losses for the poultry industry worldwide. RNA sequencing has provided valuable and important mechanistic clues regarding FAdV-4-host interactions. However, the pathogenic mechanism and host's responses after FAdV-4 infection remains limited. In this study, we used transcriptome analysis to identify dynamic changes in differentially expressed genes (DEGs) at five characteristic stages (12, 24, 36, 48, and 60 h) post infection (hpi) with FAdV-4. A total of 8,242 DEGs were identified based on comparison of five infection stages: 0 and 12, 12 and 24, 24 and 36, 36 and 48, and 48 and 60 hpi. In addition, at these five important time points, we found 37 common upregulated or downregulated DEGs, suggesting a common role for these genes in host response to viral infection. The predicted function of these DEGs using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these DEGs were associated with viral invasion, host metabolic pathways and host immunosuppression. Interestingly, genes involved in viral invasion, probably EGR1, SOCS3, and THBS1, were related to FAdV-4 infection. Validation of nine randomly selected DEGs using quantitative reverse-transcription PCR produced results that were highly consistent with those of RNA sequencing. This transcriptomic profiling provides valuable information for investigating the molecular mechanisms underlying host-FAdV-4 interactions. These data support the current molecular knowledge regarding FAdV-4 infection and chicken defense mechanisms.

The association of antihypertensive medication use with risk of cognitive decline and dementia: a meta-analysis of longitudinal studies.

The association of antihypertensive medication use with cognitive decline (including mild cognitive impairment) or dementia (including Alzheimer's disease (AD), vascular dementia (VD) and any dementia) has still been an area of controversy. This study examined the association of antihypertensive medication use with cognitive decline or dementia using a quantitative meta-analysis of longitudinal studies. EMBASE and MEDLINE were searched for articles published up to Feb 2011. All studies that examined the relationship between antihypertensive medication use and the incidence of dementia or cognitive decline were included. Pooled relative risk (RR) was calculated using fixed and random-effects models. Fourteen studies met our inclusion criteria for this meta-analysis. All subjects were without dementia or cognitive impairment at baseline. There were subjects with (32,658) and without (36,905) antihypertensive medication use. The quantitative meta-analysis showed that there was no significant difference in incidence of AD (RR: 0.90, 95% confidence interval (CI): 0.79-1.03), cognitive decline (RR: 0.97; 95% CI: 0.92-1.03) and cognitive impairment (RR: 0.97; 95% CI: 0.92-1.03). The quantitative meta-analysis showed that the subjects with antihypertensive medication use were lower incidence of VD (RR: 0.67, 95% CI: 0.52-0.87) and any dementia (RR: 0.87; 95% CI: 0.77-0.96) than those without. The quantitative meta-analysis showed that antihypertensive medication use could decrease risk of the development of VD and any dementia, but could not decrease that of AD, cognitive decline and cognitive impairment.

The effects of gastric cancer interstitial fluid on tumors based on traditional Chinese medicine 'phlegm' theory and the investigation on the mechanism through microRNA-21 regulation.

This study aimed to investigate the effects of gastric cancer interstitial fluid (GCIF) on tumors and explore the possible mechanism of Xiaotan Sanjie decoction (XTSJ) on treatment of gastric cancer from the view of regulating microRNA-21 (miR-21) expression. The GCIF was extracted and identified by measuring the levels of interleukin-8 (IL-8), intercellular adhesion molecule 1 (ICAM-1) and miR-21. The effects of GCIF on the proliferation of SGC-7901 cells and tumor growing were assessed by cell counting kit-8 (CCK-8) assay and subcutaneously transplanted tumor-bearing nude mice model, respectively. Additionally, inhibition effect of XTSJ decoction on proliferation of SGC-7901 cells intervened by GCIF were assessed in vitro and anti-cancer effect of it was further assessed using orthotopic transplanted tumor-bearing nude mice model. The concentration of SGC-7901 gastric cancer cells were dependent on the concentration of the added GCIF. After 72 hours of continuous culture, the interstitial fluid had an obvious proliferative effect on the SGC-7901 tumor cells, which was the most significant in the high concentration group. XTSJ decoction could inhibit the growth-promoting effect (P < 0.01) of GCIF on gastric cancer cells. Intervention of the GCIF might promote the growth (P < 0.05) of the subcutaneously transplanted tumors in nude mice and decrease the net weight of the tumor-bearing nude mice (P < 0.05) after tumor removal. The GCIF was able to up-regulate the expression (P < 0.001) of miR-21 in the subcutaneously transplanted tumors. XTSJ decoction could downregulate the expression (P < 0.05) of miR-21 in SGC-7901 orthotopically transplanted tumors. XTSJ decoction can inhibit the multiplicative effect of GCIF on gastric cancer cells, growth of gastric tumor and promotion effect of GCIF on tumors, probably due to the down-regulating miR-21 expression in tumor tissues.

Probiotic and hepatoprotective activity of lactobacillus isolated from Mongolian camel milk products.

The improving-intestinal-microbial-balance properties of lactic acid bacteria (LAB) are well known. Thus, LAB could play a vital role in the pathogenesis of liver diseases. In the present study, 107 LAB strains were isolated from Mongolian camel milk products and identified to species, then screened for their probiotic properties. As a result, we identified 71 Lactobacillus bacteria belonging to 9 different species, and 36 Lactococcus bacteria belonging to 8 different species. Among them, six strains of LAB with strong tolerance and adhesion ability were further studied for their protective effect on acute liver injury induced by lipopolysaccharide (LPS)/D-galactosamine (D-GalN). These six strains of LAB were fed to mice for 7 weeks, and on the final day of the experiment, LPS/D-GalN were used to induce acute liver injury. After challenging, the degree of liver pathological changes, secretion of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum and liver, and the expression of tumour necrosis factor (TNF)-α and interleukin (IL)-6 in the liver and intestines were observed and quantified. The results showed that the degree of liver pathological changes in mice fed with the six LAB strains were relieved to varying degrees compared with the LPS/D-GalN-induced model group, and the expressions of AST, ALT, IL-6, and TNF-α factor were also significantly decreased. Moreover, the expression levels of these factors in mice pretreated with Lactobacillus paracasei subsp. paracasei WXD5 were significantly decreased compared with other experimental groups. This suggests the probiotic potential and pharmacological value of L. paracasei subsp. paracasei as a liver injury inhibitor in the intervention of inflammation-based liver disease.

The Roles of Bursal Nonapeptide (BP9) on AIV Vaccine Immune Response in Chick Immunization and on Avian Immature B Cell.

BACKGROUND: Bursa of Fabricius plays the vital functions on B cell development and antibody production in poultry. The bursal-derived peptide plays the essential roles on avian immature B cell development. OBJECTIVES: Here we explored the functions of the recently reported bursal nonapeptide (BP9) on the antibody production and the molecular basis of BP9 on avian immature B cell. METHODS: Chicken were twice immunized with Avian Influenza Virus (AIV) inactivated vaccine plus with BP9 at three dosages, respectively. On two weeks after the second immunization, sera samples were collected from all experimental groups to measure AIV-specific Agglutination Inhibition (HI) antibody titers. Also, on 7th day after the second immunization, spleen lymphocytes were isolated from the immunized chicken to detect the lymphocyte viabilities. DT40 cells were treated with BP9 from 0.02 to 2 µg/mL for 4 and 20h to detect sIgM mRNA levels, and total RNAs from BP9-treated DT40 cells were collected to investigate the gene expression profiles of DT40 cells, and to analyze the enriched pathways and functional biological processes. Finally, nine gene expressions were validated with quantitative PCR (qPCR). RESULTS: Our investigation proved the strong regulatory roles of BP9 on AIV-specific HI antibody titers and lymphocyte viabilities. BP9 promoted sIgM mRNA levels in DT40 cells, and upregulated 598 gene expressions and downregulated 395 gene expressions in DT40 cells with 0.2µg/mL BP9 treatment. Moreover, our findings verified the significantly enriched six pathways and various the biological functional processes of BP9 on avian immature B cell. Also, we found eight signaling pathways in the enriched biological processes of BP9-treated DT40 cells, and the expressions of nine selected genes with qPCR were identical to that of microarray data. CONCLUSION: BP9 promoted the antibody production in the 21-old-day chicken immunization, and stimulated the sIgM expression in DT40 cells. Furthermore, we analyzed the gene expression profile and immune-related biological processes of DT40 cells treated with BP9, which provided some new insights into the mechanism on immature B cell development, and provided important references for adjuvant development on vaccine improvement and clinical application.

The Immunological Functions of Muramyl Dipeptide Compound Adjuvant on Humoral, Cellular-mediated and Mucosal Immune Responses to PEDV Inactivated Vaccine in Mice.

BACKGROUND: The muramyl dipeptide compound adjuvant, CVC1303, was one new resigned adjuvant to PEDV inactivated vaccine. Exploring the effects of CVC1303 on the immune induction to PEDV vaccine was of vital importance to the clinical application. OBJECTIVES: Here we explored the functions of CVC1303 on the humoral, cellular and mucosal immune response to PEDV vaccine in mice immunization. METHODS: Mice were twice subcutaneously injected with PEDV vaccine including high, medium and low dosages CVC1303, respectively. On 30th day after the second immunization, sera samples were collected from the immunized mice to measure PEDV-specific IgG and IgG subclasses levels, and lymphocytes were isolated to detect T cell subtype and intracellular IL-4 and IL-6 cytokine productions, and the expressions of co-stimulatory molecule on dendritic cells in the immunized mice. Small intestinal and lung washings were collected on 30th and 47th day after the second immunization to measure PEDV-specific IgA levels, and SP immunohistochemical method staining was employed to analyze the deviations of IgA+ positive cells in the small intestinal of the immunized mice. RESULTS: Our investigation proved the strong regulatory roles of CVC1303 on PEDV-specific IgG and IgG1 antibody and cytokines productions, and the significant increased CD3+CD4+T cells subpopulation and expressions of co-stimulatory molecules on dendritic cells in the immunized mice. Moreover, our findings verified the significantly enhanced PEDV-specific IgA antibody titers in small intestinal and lung in the mice immunized with PEDV vaccine and CVC1303. CONCLUSION: Compound adjuvant CVC1303 could effectively improve the PEDV-specific immune responses and mucosal immune, which provided an experimental basis for the further clinical application of new adjuvant CVC1303 and the development of improvement on the mucosal immune response.

Influence of Feeding Type and Nosema ceranae Infection on the Gut Microbiota of Apis cerana Workers.

The gut microbiota plays an essential role in the health of bees. To elucidate the effect of feed and Nosema ceranae infection on the gut microbiota of honey bee (Apis cerana), we used 16S rRNA sequencing to survey the gut microbiota of honey bee workers fed with sugar water or beebread and inoculated with or without N. ceranae. The gut microbiota of A. cerana is dominated by Serratia, Snodgrassella, and Lactobacillus genera. The overall gut microbiota diversity was show to be significantly differential by feeding type. N. ceranae infection significantly affects the gut microbiota only in bees fed with sugar water. Higher abundances of Lactobacillus, Gluconacetobacter, and Snodgrassella and lower abundances of Serratia were found in bees fed with beebread than in those fed with sugar water. N. ceranae infection led to a higher abundance of Snodgrassella and a lower abundance of Serratia in sugar-fed bees. Imputed bacterial Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed the significant metagenomics functional differences by feeding and N. ceranae infections. Furthermore, A. cerana workers fed with sugar water showed lower N. ceranae spore loads but higher mortality than those fed with beebread. The cumulative mortality was strongly positive correlated (rho = 0.61) with the changes of overall microbiota dissimilarities by N. ceranae infection. Both feeding types and N. ceranae infection significantly affect the gut microbiota in A. cerana workers. Beebread not only provides better nutrition but also helps establish a more stable gut microbiota and therefore protects bees in response to N. ceranae infection. IMPORTANCE The gut microbiota plays an essential role in the health of bees. Scientific evidence suggests that diet and infection can affect the gut microbiota and modulate the health of the gut; however, the interplay between those two factors and the bee gut microbiota is not well known. In this study, we used a high-throughput sequencing method to monitor the changes of gut microbiota associated with both feeding types and Nosema ceranae infection. Our results showed that the gut microbiota composition and diversity of Asian honey bee were significantly associated with both feeding types and the N. ceranae infection. More interestingly, bees fed with beebread showed higher microbiota stability and lower mortality rates than those fed with sugar water when infected by N. ceranae. Those data suggest that beebread has the potential not only to provide better nutrition but also help to establish a more stable gut microbiota to protect bees against N. ceranae infection.

The critical role of 15-lipoxygenase-1 in colorectal epithelial cell terminal differentiation and tumorigenesis.

Terminal differentiation is an important event for maintaining normal homeostasis in the colorectal epithelium, and the loss of apoptosis is an important mechanism underlying colorectal tumorigenesis. The very limited current data on the role of lipoxygenase (LOX) metabolism in tumorigenesis suggests that the oxidative metabolism of linoleic and arachidonic acid possibly shifts from producing antitumorigenic 15-LOX-1 and 15-LOX-2 products to producing protumorigenic 5-LOX and 12-LOX products. We examined whether this shift occurs in vitro in the human colon cancer cell line Caco-2 in association with the loss of terminal differentiation and apoptosis, or in vivo during the formation of colorectal adenomas in patients with familial adenomatous polyposis (FAP). Restoring terminal differentiation and apoptosis of Caco-2 cells increased the mRNA levels of 5-LOX, 15-LOX-2, and 15-LOX-1, but the only significant increases in protein expression and enzymatic activity were of 15-LOX-1. In FAP patients, 15-LOX-1 expression and activity were significantly down-regulated in adenomas (compared with paired nonneoplastic epithelial mucosa), whereas 5-LOX and 15-LOX-2 protein expressions and enzymatic activities were not. We conducted a validation study with immunohistochemical testing in a second group of FAP patients; 15-LOX-1 expression was down-regulated in colorectal adenomas (compared with nonneoplastic epithelial mucosa) in 87% (13 of 15) of this group. We confirmed the mechanistic relevance of these findings by demonstrating that ectopically restoring 15-LOX-1 expression reestablished apoptosis in Caco-2 cells. Therefore, 15-LOX-1 down-regulation rather than a shift in the balance of LOXs is likely the dominant alteration in LOX metabolism which contributes to colorectal tumorigenesis by repressing apoptosis.