RESUMO
OBJECTIVE: To investigate the polarization of human acute monocytic leukemia THP-1 cells-derived macrophages induced by Nippostrongylus brasiliensis proteins in vitro, so as to provide insights into the elucidation of the mechanisms underlying host immune responses to hookworm infections. METHODS: The in-vitro culture of N. brasiliensis was established and maintained in the laboratory, and the third- (L3) and fifth-stage larvae (L5) were collected under a sterile condition for preparation of L3 and L5 proteins. The in-vitro culture of THP-1 cells was established, stimulated with 500 ng/mL PMA to yield M0 macrophages that were adherent to the plate wall. The LPS + IFN-γ group, IL-4 + IL-13 group, L3 protein group and L5 protein group were given stimulation with 500 ng/mL LPS plus 100 ng/mL IFN-γ, IL-4 and IL-13 (both 100 ng/mL), L3 protein (5 mg/mL) and L5 protein (5 mg/mL), respectively, while the negative control group was given no stimulation. The cell morphology was observed using microscopy, the mRNA expression of M1/M2 macrophages-specific genes was quantified using a quantitative real-time PCR (qPCR) assay, and the surface markers of M1/M2 macrophages were detected using flow cytometry, while the levels of cytokines secreted by M1/M2 macrophages were measured using enzyme-linked immunosorbent assay (ELISA) following stimulations, so as to examine the polarization of THP-1-derived macrophages induced by N. brasiliensis proteins in vitro. RESULTS: Following stimulation with PMA, THP-1 cells appeared wall-adherent M0 macrophages, and polarized to typical M1 macrophages following stimulation with LPS + IFN-γ, and typical M2 macrophages following stimulation with IL-4 + IL-13, IL-3 protein or L5 protein. There was a significant difference in the proportion of M1 macrophages among the negative control group, the LPS + IFN-γ group, the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (χ2 = 3 721.00, P < 0.001), with the highest proportion detected in the LPS + IFN-γ group, and there was also a significant difference in the proportion of M2 macrophages among groups (χ2 = 105.43, P < 0.001). There were significant differences among groups in terms of the mRNA expression of CCL2 (F = 191.95, P < 0.001), TNF-α (F = 129.95, P < 0.001), IL-12b (F = 82.89, P < 0.001), PPARγ (F = 11.30, P < 0.001), IL-10 (F = 9.51, P < 0.001) and Mrc1 genes (F = 12.35, P < 0.001). In addition, there were significant differences in the proportion of positive CD86 and CD206 expression among groups (χ2 = 24 004.33 and 832.50, P < 0.001). Higher IL-1ß and TNF-α levels were measured in the LPS + IFN-γ group than in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (P < 0.001), and greater TGF-ß1 and IL-10 levels were seen in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group than in the negative control group and the LPS + IFN-γ group (P < 0.05). CONCLUSIONS: Both L3 and L5 proteins of N. brasiliensis may induce the polarization of THP-1-derived macrophages to M2 type in vitro.
Assuntos
Leucemia Monocítica Aguda , Animais , Antígenos de Helmintos/farmacologia , Criança , Humanos , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Nippostrongylus/química , Células THP-1/citologia , Células THP-1/efeitos dos fármacosRESUMO
A multi-generational approach was used to investigate the persistent effects of a sub-lethal dose of spinosad in Plutella xylostella. The susceptibility of various sub-populations of P. xylostella to spinosad and the effects of the insecticide on the gene expression of γ-aminobutyric acid receptor (GABAR) were determined. The results of a leaf dip bioassay showed that the sensitivity of P. xylostella to spinosad decreased across generations. The sub-strains had been previously selected based on a determined LC25 of spinosad. Considering that GABA-gated chloride channels are the primary targets of spinosad, the cDNA of P. xylostella was used to clone GABARα by using reverse transcription-polymerase chain reaction (RT-PCR). The mature peptide cDNA was 1477-bp long and contained a 1449-bp open reading frame encoding a protein of 483 amino acids. The resulting amino acid sequence was used to generate a neighbor-joining dendrogram, and homology search was conducted using NCBI BLAST. The protein had high similarity with the known GABAR sequence from P. xylostella. Subsequent semi-quantitative RT-PCR and real-time PCR analyses indicated that the GABAR transcript levels in the spinosad-resistant strain (RR, 145.82-fold) and in Sub1 strain (selected with LC25 spinosad for one generation) were the highest, followed by those in the spinosad-susceptible strain, the Sub10 strain (selected for ten generations), and the Sub5 strain (selected for five generations). This multi-generational study found significant correlations between spinosad susceptibility and GABAR gene expression, providing insights into the long-term effects of sub-lethal insecticide exposure and its potential to lead to the development of insecticide-resistant insect populations.
Assuntos
Inseticidas , Macrolídeos , Mariposas/genética , Receptores de GABA/genética , Sequência de Aminoácidos , Animais , Combinação de Medicamentos , Expressão Gênica , Proteínas de Insetos/biossíntese , Proteínas de Insetos/genética , Resistência a Inseticidas , Mariposas/metabolismo , Receptores de GABA/biossínteseRESUMO
Ebola hemorrhagic fever is a fatal disease caused by the negative-strand RNA of the Ebola virus. A high-intensity outbreak of this fever was reported in West Africa last year; however, there is currently no definitive treatment strategy available for this disease. In this study, we analyzed the molecular evolutionary history and attempted to determine the positive selection sites in the Ebola genes using multiple-genomic sequences of the various Ebola virus subtypes, in order to gain greater clarity into the evolution of the virus and its various subtypes. Only the glycoprotein (GP) gene was positively selected among the 8 Ebola genes, with the other genes remaining in the purification stage. The positive selection sites in the GP gene were identified by a random-site model; these sites were found to be located in the mucin-like region, which is associated with transmembrane protein binding. Additionally, different branches of the phylogenetic tree displayed different positive sites, which in turn was responsible for differences in the cell adhesion ability of the virus. In conclusion, the pattern of positive sites in the GP gene is associated with the epidemiology and prevalence of Ebola in different areas.
Assuntos
Ebolavirus/genética , Ebolavirus/patogenicidade , Glicoproteínas/metabolismo , Doença pelo Vírus Ebola/virologia , Surtos de Doenças , Ebolavirus/classificação , Evolução Molecular , Glicoproteínas/genética , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/metabolismo , Humanos , Filogenia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
The evolutionary significant units (ESUs) of the salamander Pachyhynobius shangchengensis (Hynobiidae) in the Dabieshan mountains, southeastern China, were identified based on mitochondrial DNA data. We used methods for detecting cryptic species, such as the minimum spanning tree, the automatic barcode gap discovery, and the generalized mixed Yule-coalescent model; geographical partitioning was also used to identify the ESUs. A total of four ESUs were identified.
Assuntos
DNA Mitocondrial , Evolução Molecular , Urodelos/genética , Animais , Evolução Biológica , China , Genes Mitocondriais , Haplótipos , Filogenia , Filogeografia , Urodelos/classificaçãoRESUMO
This study used DNA microarray data to identify differentially expressed genes of osteoporosis and provide useful information for treatments of the disease. We downloaded gene expression data of Osteoporosis GSE35956 from the Gene Expression Omnibus database, which included five normal and five osteoporosis samples. We then identified the differentially expressed genes between normal and disease samples using the R language software, and constructed the protein interaction network. DAVID was used to perform the biological process enrichment and KEGG pathway cluster analyses. We used the Cytoscape plug-in unit, Cluster ONE, to perform cluster module analysis to find hub proteins of the network module and to analyze their Gene Ontology (GO) functions. A total of 294 genes were found to be differentially expressed between normal and disease samples, which were used to construct the differential gene-protein interaction network. GO function analysis revealed that the genes' functions were mainly involved in the intracellular signaling cascade. KEGG pathway analysis suggested that the main metabolic pathways of these genes were those of cancer: the neurotrophin/T cell/Fc epsilon RI/B cell/ ErbB/p53 signaling pathway, the cell cycle pathway, and the chronic myeloid leukemia pathway. Screening analysis of hub proteins revealed that KRT18 had the highest hub degree. In conclusion, we found differentially expressed genes related to osteoporosis. GO biological process enrichment and KEGG pathway enrichment analyses identified significant osteoporosis genes and their molecular functions. Finally, module analysis of hub proteins in interaction networks showed that cell death was one of the main biological processes of osteoporosis genes.