scholarly journals RNA-Seq Analysis Reveals the Role of Omp16 in Brucella-Infected RAW264.7 Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Dong Zhou ◽  
Feijie Zhi ◽  
Jiaoyang Fang ◽  
Weifang Zheng ◽  
Junmei Li ◽  
...  
Keyword(s):  
Proinflammatory Cytokines ◽  
Cellular Response ◽  
Outer Membrane Proteins ◽  
Raw264.7 Cells ◽  
Economic Losses ◽  
Rna Seq ◽  
Protective Antigens ◽  
Cytokines And Chemokines ◽  
Relevant Role

Brucellosis is an endemic zoonotic infectious disease in the majority of developing countries, which causes huge economic losses. As immunogenic and protective antigens at the surface of Brucella spp., outer membrane proteins (Omps) are particularly attractive for developing vaccine and could have more relevant role in host–pathogen interactions. Omp16, a homolog to peptidoglycan-associated lipoproteins (Pals), is essential for Brucella survival in vitro. At present, the functions of Omp16 have been poorly studied. Here, the gene expression profile of RAW264.7 cells infected with Brucella suis vaccine strain 2 (B. suis S2) and ΔOmp16 was analyzed by RNA-seq to investigate the cellular response immediately after Brucella entry. The RNA-sequence analysis revealed that a total of 303 genes were significantly regulated by B. suis S2 24 h post-infection. Of these, 273 differentially expressed genes (DEGs) were upregulated, and 30 DEGs were downregulated. These DEGs were mainly involved in innate immune signaling pathways, including pattern recognition receptors (PRRs), proinflammatory cytokines, and chemokines by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. In ΔOmp16-infected cells, the expression of 52 total cells genes was significantly upregulated and that of 9 total cells genes were downregulated compared to B. suis S2-infected RAW264.7 cells. The KEGG pathway analysis showed that several upregulated genes were proinflammatory cytokines and chemokines, such as interleukin (IL)-6, IL-11, IL-12β, C–C motif chemokine (CCL2), and CCL22. All together, we clearly demonstrate that ΔOmp16 can alter macrophage immune-related pathways to increase proinflammatory cytokines and chemokines, which provide insights into illuminating the Brucella pathogenic strategies.

scholarly journals YAP1 Is a Potential Predictive Molecular Biomarker for Response to SMO Inhibitor in Medulloblastoma Cells

Cancers ◽  
2021 ◽  
Vol 13 (24) ◽  
pp. 6249
Author(s):  
Gustavo Alencastro Veiga Cruzeiro ◽  
Taciani de Almeida Magalhães ◽  
Graziella Ribeiro de Sousa ◽  
Ricardo Bonfim Silva ◽  
Carlos Alberto Oliveira de Biagi Junior ◽  
...  
Keyword(s):  
Sonic Hedgehog ◽  
Cellular Response ◽  
Molecular Subtypes ◽  
In Vitro Models ◽  
Rna Seq ◽  
Preclinical Models ◽  
Molecular Biomarker ◽  
Shh Pathway ◽  
Smo Inhibitor

Advances in genomics have led to the identification of twelve relevant molecular subtypes within medulloblastoma (MB). The alpha subtype of Sonic hedgehog-driven MB is resistant to therapy (including smoothened inhibitors) due to activation of genes from the non-canonical SHH pathway, such as MYCN, YAP1, or TP53. Using retrospective cohort microarray data, we found that YAP1 is overexpressed in SHH alpha MB and patients profiled as resistant to SMO inhibitors compared to good responders. Here, we performed YAP1 depletion via CRISPR/Cas9 in two in vitro models of SHH-like MB cells and found that this protein is involved in responsiveness to the SMO inhibitor regarding proliferation, apoptosis, and colony formation. Further, considering the synergic combination of YAP1 depletion with SMO inhibition, we assessed single-cell RNA-seq data from five patients and found that SMO and YAP1 are enriched within cells of SHH MB. Importantly, our data suggest that YAP1 is not only a reliable biomarker for cellular response to SMOi but may indicate prospective testing of combination therapy using YAP1 and SMO inhibitors in preclinical models of SHH MB.

scholarly journals Identification of genetic determinants of hemolytic activity of Riemerella anatipestifer using random transposon mutagenesis

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Bingqing Sun ◽  
Yafei Xue ◽  
Xiaoli Du ◽  
Xiaohua He ◽  
Zuocheng Zou ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Hemolytic Activity ◽  
Genetic Basis ◽  
Cytoplasmic Membrane ◽  
Outer Membrane Proteins ◽  
Economic Losses ◽  
Genetic Determinants ◽  
Riemerella Anatipestifer ◽  
Cytoplasmic Proteins

AbstractRiemerella anatipestifer causes epizootic infectious disease in poultry resulting in serious economic losses especially to the duck industry. In our previous study, R. anatipestifer was found to lyse duck erythrocytes in vitro. In the present study, a random Tn4351 mutagenesis library of hemolytic R. anatipestifer strain SX containing 4000 mutants was constructed to investigate the genetic basis of hemolytic activity. Thirty mutants with reduced hemolytic activity and one with increased hemolytic activity were screened and insertions in 24 genes were identified. Of these genes, four were predicted to encode outer membrane proteins, one encoded a cytoplasmic membrane protein, 11 encoded cytoplasmic proteins, and eight encoded proteins with unknown locations. Based on current annotations of the R. anatipestifer genomes, of the 24 genes, 7 (29.17%) were involved in iron utilization. The hemolytic activities of the complemented strains M2 (pRES-Riean_0790) and M18 (pRES-Riean_0653) were restored, indicating that both Riean_0653 and Riean_0790 are involved in the hemolytic activity of strain SX. However, the recombinant proteins rRiean_0317, rRiean_0790, rRiean_0653, rRiean_1027, rRiean_1143, and rRiean_1561 had no hemolytic activity, suggesting that none were hemolysins.

scholarly journals C28-induced autophagy of female germline stem cells in vitro and its potential mechanisms

2019 ◽  
Author(s):  
Rong Hu ◽  
Xiuying Pei ◽  
Huchen Zhou ◽  
Ji Wu ◽  
Ping Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Er Stress ◽  
Cellular Response ◽  
Germline Stem Cells ◽  
Rna Seq ◽  
Sequestosome 1 ◽  
Expression Of Genes ◽  
Female Germline ◽  
Molecular Compounds

Abstract BackgroundThere are few studies indicating that small molecular compounds affect the proliferation, differentiation, apoptosis, and autophagy of female germline stem cells (FGSCs). However, the epigenetic regulatory mechanism of small molecular compounds that induce autophagy in FGSCs remains unknown.ResultsIn this study, we found that C28 reduced the viability and proliferation of FGSCs, respectively. Additionally, western blotting showed that the expression of autophagy marker light chain 3 beta II (LC3B-II) was significantly increased and expression of sequestosome-1 (SQSTM1) was significantly reduced in C28-treated groups. Immunofluorescence showed that, in C28-treated groups, the number of LC3B-II-positive puncta was increased significantly. These results indicated that C28 induced autophagy of FGSCs in vitro. ChIP-seq data showed that autophagy-related biological processes such as regulation of mitochondrial membrane potential, Golgi vesicle transport, and cellular response to reactive oxygen species were enriched. In addition, RNA-Seq showed that the expression of genes (Trib3, DDIT3, and ATF4) related to endoplasmic reticulum (ER) stress was enhanced by C28.ConclusionC28 could induce FGSC autophagy in vitro leading to a decrease in the number of FGSCs. H3K27ac and ER stress might play roles in C28-induced autophagy of FGSCs in vitro.

scholarly journals Bacteriophage inhibits murine norovirus replication after co-incubation in RAW 264.7 cells

2021 ◽  
Author(s):  
Lili Zhang ◽  
Khashayar Shahin ◽  
Ran Wang
Keyword(s):  
Cellular Response ◽  
Antimicrobial Effect ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Murine Norovirus ◽  
Rna Seq ◽  
Qpcr Analysis ◽  
Ifn Γ ◽  
Phage Treatment

AbstractBacteriophages (phages) are viruses of bacteria. Despite the growing progress in research on phage interactions with eukaryotic cells, our understanding of the roles of phages and their potential implications remains incomplete. The objective of this study was to investigate the effects of the Staphylococcus aureus phage vB_SauM_JS25 on murine norovirus (MNV) replication. Experiments were performed using the RAW 264.7 cell line. After phage treatment, MNV multiplication was significantly inhibited, as indicated by real-time quantitative PCR (RT-qPCR) analysis, western blot, and immunofluorescence assay. Furthermore, we demonstrated the transcriptional changes in phage–MNV co-incubated RAW 264.7 cells through RNA sequencing (RNA-Seq) and bioinformatic analysis. Our subsequent analyses revealed that the innate immune response may play an important role in the restriction of MNV replication, such as the cellular response to IFN-γ and response to IFN-γ. In addition, the gene expression of IL-10, Arg-1, Ccl22, GBP2, GBP3, GBP5, and GBP7 was proven to increase significantly by RT-qPCR, showing a strong correlation between RT-qPCR and RNA-Seq results. Furthermore, phage treatment activated guanylate binding proteins (GBPs), as revealed by RT-qPCR analysis, western blotting, and confocal microscopy. Taken together, these data suggest that the phage affects the innate response (such as the IFN-inducible GTPases and GBPs), reflecting their direct antimicrobial effect on the membrane structure of the MNV replication complexes, and therefore, exerts an antiviral effect in vitro. Collectively, our findings provide insights into the interactions of immune cells and phages, which improve our understanding of the actual role and potential of phages.

scholarly journals MLN4924 protects against interleukin-17A-induced pulmonary inflammation by disrupting ACT1-mediated signaling

2019 ◽  
Vol 316 (6) ◽  
pp. L1070-L1080 ◽  
Author(s):  
Rui Hao ◽  
Yunduan Song ◽  
Runsheng Li ◽  
Yaxian Wu ◽  
Xinyi Yang ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Proinflammatory Cytokines ◽  
Inflammatory Diseases ◽  
Pulmonary Inflammation ◽  
Tumor Necrosis Factor Receptor ◽  
Inhibitory Effect ◽  
Chronic Obstructive ◽  
Cytokines And Chemokines ◽  
Interleukin 17A

An excessive inflammatory response in terminal airways, alveoli, and the lung interstitium eventually leads to pulmonary hypertension and chronic obstructive pulmonary disease. Proinflammatory cytokine interleukin-17A (IL-17A) has been implicated in the pathogenesis of pulmonary inflammatory diseases. MLN4924, an inhibitor of NEDD8-activating enzyme (NAE), is associated with the treatment of various types of cancers, but its role in the IL-17A-mediated inflammatory response has not been identified. Here, we report that MLN4924 can markedly reduce the expression of proinflammatory cytokines and chemokines such as IL-1β, IL-6, and CXCL-1 and neutrophilia in a mouse model of IL-17A adenovirus-induced pulmonary inflammation. MLN4924 significantly inhibited IL-17A-induced stabilization of mRNA of proinflammatory cytokines and chemokines in vitro. Mechanistically, MLN4924 significantly blocked the activation of MAPK and NF-κB pathways and interfered with the interaction between ACT1 and tumor necrosis factor receptor-associated factor proteins (TRAFs), thereby inhibiting TRAF6 ubiquitination. Taken together, our data uncover a previously uncharacterized inhibitory effect of MLN4924 on the IL-17A-mediated inflammatory response; this phenomenon may facilitate the development of MLN4924 into an effective small-molecule drug for the treatment of pulmonary inflammatory diseases.

scholarly journals Anti-inflammatory Effects of Hydrogen in LPS-induced RAW264.7 Cells via Inhibiting NF-κB and MAPKs Signaling Pathways

2021 ◽  
Author(s):  
Tao Zhang ◽  
Fuping Wang ◽  
Guoqiang Jiang ◽  
Guobao Chen ◽  
Lili Han ◽  
...  
Keyword(s):  
Inflammatory Mediators ◽  
Molecular Mechanisms ◽  
Signal Transduction Pathway ◽  
Raw264.7 Cells ◽  
Raw 264.7 Cells ◽  
Mrna Levels ◽  
Rna Seq ◽  
Inflammatory Signaling ◽  
Anti Inflammatory

Abstract Hydrogen (H2), a new type of medical gas molecule, which has significant preventive effect on numerous diseases and its anti-inflammatory properties has been proven in previous studies. However, the mechanisms of H2 anti-inflammatory activity in signal transduction pathway or protein level regulation are inadequately inexplicit. In the current study, the effect of H2 on LPS-induced inflammation in RAW 264.7 cells were assessed and its molecular mechanisms were clarified. The in vitro model of inflammation was induced by lipopolysaccharide (LPS) in RAW264.7 cells. Cell viability was evaluated by MTT assay. Protein expression of inflammatory mediators were analyzed by ELISA and Western blot. mRNA levels were detected by RT-qPCR. In addition, RNA sequencing (RNA-seq) was conducted to explore the molecular targets of H2 anti-inflammatory. According to the findings, H2 reversed LPS-induced variety in NO levels and TNF-a production as well as IL-6, IL-10 proteins and related mRNA levels in macrophages. RNA-seq newly discovered that H2 acted on inflammatory signaling molecule protein kinase C 8 (PKC8) and heterodimer activator protein-1 (AP-1). The WB analysis was then used to determine the key proteins in the inflammatory signaling pathway involved in PKC8 and AP-1, which found that H2 inhibited the phosphorylation of key proteins in the NF-kB and MAPKs pathways, thereby the expression of mRNA and inflammatory mediators were affected. The findings of this study show that H2 may serve as a promising anti-inflammatory gas in mitigating inflammatory conditions.

Vibrio harveyi virulence gene expression in vitro and in vivo during infection in black tiger shrimp Penaeus monodon

2020 ◽  
Vol 139 ◽  
pp. 153-160
Author(s):  
S Peeralil ◽  
TC Joseph ◽  
V Murugadas ◽  
PG Akhilnath ◽  
VN Sreejith ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virulence Genes ◽  
Vibrio Harveyi ◽  
Penaeus Monodon ◽  
Challenge Test ◽  
Virulence Gene ◽  
Economic Losses ◽  
Virulence Gene Expression

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.

Apoptosis and proliferation ofperipheral blood T-cells as alternative processes in pathogenesis of diabetes mellitus type 1

2019 ◽  
Vol 1 (4) ◽  
pp. 16-20 ◽  
Author(s):  
A. V. Lugovaya ◽  
N. M. Kalinina ◽  
V. Ph. Mitreikin ◽  
Yu. P. Kovaltchuk ◽  
A. V. Artyomova ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
T Cells ◽  
High Risk ◽  
Peripheral Blood ◽  
Cellular Response ◽  
Proliferative Potential ◽  
Autoreactive T Cells ◽  
Alternative Processes

Apoptosis, along with proliferation, is a form of lymphocyte response to activating stimuli. In the early stages of cell differentiation, the apoptotic response prevails and it results to the formation of tolerance to inductor antigen. Mature lymphocytes proliferate in response to stimulation and it means the initial stage in the development of the immune response. Since in this case apoptosis and proliferation acts as alternative processes, their ratio can serve as a measure of the effectiveness of the cellular response to activating signals. The resistance of autoreactive T-cells to apoptosis is the main key point in the development of type 1 diabetes mellitus (T1DM). Autoreactive T-cells migrates from the bloodstream to the islet tissue of the pancreas and take an active part in b cells destruction. The resistance of autoreactive effector T-cells to apoptosis may suggest their high proliferative potential. Therefore, the comparative evaluation of apoptosis and proliferation of peripheral blood lymphocytes can give a more complete picture of their functional state and thus will help to reveal the causes of ineffective peripheral blood T-ceiis apoptosis in patients with T1DM and will help to understand more deeply the pathogenesis of the disease. in this article, the features of proliferative response of peripheral blood T-cells in patients with T1DM and in individuals with high risk of developing T1DM have been studied. Apoptosis of T-cell subpopulations has been investigated. The correlation between the apoptotic markers and the intensity of spontaneous and activation- induced in vitro T-cells proliferation of was revealed. it was determined, that autoreactive peripheral blood T-cells were resistant to apoptosis and demonstrated the increased proliferative potential in patients with T1DM and in individuals with high risk of developing T1DM.

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