An 192 bp ERV insertion in the first intron of TLR6 acts as an enhancer to increase TLR6 and TLR1 expression

Abstract Background Toll-like receptors (TLRs) play important roles in building innate immune and inducing adaptive immune responses. Associations of the TLR gene polymorphisms with diseases susceptibility, which are the basis of molecular breeding for disease resistant animals, have been reported extensively. Retrotransposon insertion polymorphisms (RIPs) were developed recently as a new type of molecular marker having great potential in population genetics and quantitative trait locus (QTL) mapping analysis. In this study, bioinformatics prediction combined with the PCR-based amplification was employed to screen for RIPs in porcine TLR genes. Their population distribution and impact on gene activity and phenotype of one RIP was further evaluated. Results Totally, five RIPs, located at the 3' flank of TLR3, 5' flank of TLR5, intron 1 of TLR6, intron 1 of TLR7, and 3' flank of TLR8 respectively, were identified. These RIPs were detected in different breeds with an uneven distribution among them. By using the dual luciferase activity assay a 192 bp endogenous retrovirus (ERV) in the intron 1 of TLR6 was proven to act as an enhancer increasing the activities of TLR6 promoter and multiple mini-promoters. Furthermore, the real-time quantitative polymerase chain reaction (qPCR) analysis demonstrated that the ERV insertion significantly enhances the mRNA expressions of TLR6, the neighboring gene TLR1, and the downstream genes MyD88 (Myeloid differentiation factor 88), Rac1 (Rac family small GTPase 1 ), TIRAP (TIR domain containing adaptor protein), Tollip (Toll interacting protein) of TLR signaling pathway and the inflammatory factors IL6 (Interleukin 6), IL8 (Interleukin 8), and TNFα (Tumor necrosis factor alpha) in 30-day piglet tissues. In addition, the serum IL-6 and TNFα were also significantly upregulated by ERV insertion. Conclusions Overall, five RIPs were identified in several TLRs, and the 192 bp ERV insertion in the first intron of TLR6 can improve the expressions of TLR6, TLR1, their downstream genes, and the inflammatory factors by acting as an enhancer affecting the regulation of TLR pathways, which may be applicable in the molecular breeding of disease resistant animals.

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An 192 bp ERV Insertion in the First Intron of TLR6 Acts as an Enhancer to Increase TLR6 and TLR1 Expression

10.21203/rs.3.rs-105672/v1 ◽

2020 ◽

Author(s):

xiaoyan wang ◽

Zixuan Chen ◽

Eduard Murani ◽

Enrico D'alessandro ◽

Yalong An ◽

...

Keyword(s):

Molecular Breeding ◽

Population Distribution ◽

Quantitative Polymerase Chain Reaction ◽

Adaptor Protein ◽

Small Gtpase ◽

Inflammatory Factors ◽

Neighboring Gene ◽

First Intron ◽

Disease Resistant ◽

Intron 1

Abstract BackgroundToll-like receptors (TLRs) play important roles in building innate immune and inducing adaptive immune responses. Associations of the TLR gene polymorphisms with diseases susceptibility, which are the basis of molecular breeding for disease resistant animals, have been reported extensively. Retrotransposon insertion polymorphisms (RIPs) were developed recently as a new type of molecular marker having great potential in population genetics and quantitative trait locus (QTL) mapping analysis. In this study, bioinformatics prediction combined with the PCR-based amplification was employed to screen for RIPs in porcine TLR genes. Their population distribution and impact on gene activity and phenotype of one RIP was been further evaluated. ResultsThe results showed that five RIPs, located at the 3' flank of TLR3, 5' flank of TLR5, intron 1 of TLR6, intron 1 of TLR7, and 3' flank of TLR8 respectively, was identified. These RIPs were detected in different breeds with an uneven distribution among them. By using the dual luciferase activity assay a 192 bp endogenous retrovirus (ERV) in the intron 1 of TLR6 was proven to act as an enhancer increasing the activities of TLR6 promoter and multiple mini-promoters. Furthermore, the real-time quantitative polymerase chain reaction (qPCR) analysis demonstrated that the ERV insertion significantly enhances the mRNA expressions of TLR6, the neighboring gene TLR1, and the downstream genes MyD88 (Myeloid differentiation factor 88), Rac1 (Rac family small GTPase 1), TIRAP (TIR domain containing adaptor protein), Tollip (Toll interacting protein) of TLR signaling pathway and the inflammatory factors IL6 (Interleukin 6), IL8 (Interleukin 8), and TNFα (Tumor necrosis factor alpha) in 30-day piglet tissues. In addition, the serum IL-6 and TNFα was also significantly upregulated by ERV insertion. ConclusionsOverall, five RIPs were been identified in several TLRs, and the 192 bp ERV insertion in the first intron of TLR6 can improve the expressions of TLR6, TLR1, their downstream genes, and the inflammatory factors by acting as an enhancer affecting the regulation of TLR pathways, which may be applicable in the molecular breeding of disease resistant animals.

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A 192 bp ERV fragment insertion in the first intron of porcine TLR6 may act as an enhancer associated with the increased expressions of TLR6 and TLR1

Mobile DNA ◽

10.1186/s13100-021-00248-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

XiaoYan Wang ◽

Zixuan Chen ◽

Eduard Murani ◽

Enrico D’Alessandro ◽

Yalong An ◽

...

Keyword(s):

Population Distribution ◽

Quantitative Polymerase Chain Reaction ◽

Adaptor Protein ◽

Small Gtpase ◽

Inflammatory Factors ◽

Tlr Signaling ◽

First Intron ◽

Disease Resistant ◽

Intron 1 ◽

The Impact

Abstract Background Toll-like receptors (TLRs) play important roles in building innate immune and inducing adaptive immune responses. Associations of the TLR genes polymorphisms with disease susceptibility, which are the basis of molecular breeding for disease resistant animals, have been reported extensively. Retrotransposon insertion polymorphisms (RIPs), as a new type of molecular markers developed recently, have great potential in population genetics and quantitative trait locus mapping. In this study, bioinformatic prediction combined with PCR-based amplification was employed to screen for RIPs in porcine TLR genes. Their population distribution was examined, and for one RIP the impact on gene activity and phenotype was further evaluated. Results Five RIPs, located at the 3' flank of TLR3, 5' flank of TLR5, intron 1 of TLR6, intron 1 of TLR7, and 3' flank of TLR8 respectively, were identified. These RIPs were detected in different breeds with an uneven distribution among them. By using the dual luciferase activity assay a 192 bp endogenous retrovirus (ERV) in the intron 1 of TLR6 was shown to act as an enhancer increasing the activities of TLR6 putative promoter and two mini-promoters. Furthermore, real-time quantitative polymerase chain reaction (qPCR) analysis revealed significant association (p < 0.05) of the ERV insertion with increased mRNA expression of TLR6, the neighboring gene TLR1, and genes downstream in the TLR signaling pathway such as MyD88 (Myeloid differentiation factor 88), Rac1 (Rac family small GTPase 1), TIRAP (TIR domain containing adaptor protein), Tollip (Toll interacting protein) as well as the inflammatory factors IL6 (Interleukin 6), IL8 (Interleukin 8), and TNFα (Tumor necrosis factor alpha) in tissues of 30 day-old piglet. In addition, serum IL6 and TNFα concentrations were also significantly upregulated by the ERV insertion (p < 0.05). Conclusions A total of five RIPs were identified in five different TLR loci. The 192 bp ERV insertion in the first intron of TLR6 was associated with higher expression of TLR6, TLR1, and several genes downstream in the signaling cascade. Thus, the ERV insertion may act as an enhancer affecting regulation of the TLR signaling pathways, and can be potentially applied in breeding of disease resistant animals.

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Estimating epidemiologic dynamics from cross-sectional viral load distributions

Science ◽

10.1126/science.abh0635 ◽

2021 ◽

pp. eabh0635

Author(s):

James A. Hay ◽

Lee Kennedy-Shaffer ◽

Sanjat Kanjilal ◽

Niall J. Lennon ◽

Stacey B. Gabriel ◽

...

Keyword(s):

Population Distribution ◽

Quantitative Polymerase Chain Reaction ◽

Cross Sectional ◽

Outbreak Management ◽

Viral Loads ◽

Time Estimates ◽

Load Distributions ◽

Random Samples ◽

Combining Data ◽

Polymerase Chain

Estimating an epidemic’s trajectory is crucial for developing public health responses to infectious diseases, but case data used for such estimation are confounded by variable testing practices. We show that the population distribution of viral loads observed under random or symptom-based surveillance, in the form of cycle threshold (Ct) values obtained from reverse-transcription quantitative polymerase chain reaction testing, changes during an epidemic. Thus, Ct values from even limited numbers of random samples can provide improved estimates of an epidemic’s trajectory. Combining data from multiple such samples improves the precision and robustness of such estimation. We apply our methods to Ct values from surveillance conducted during the SARS-CoV-2 pandemic in a variety of settings and offer alternative approaches for real-time estimates of epidemic trajectories for outbreak management and response.

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LncRNA XIST regulates atherosclerosis progression in ox-LDL-induced HUVECs

Open Medicine ◽

10.1515/med-2021-0200 ◽

2021 ◽

Vol 16 (1) ◽

pp. 117-127

Author(s):

Hongmei Gao ◽

Zhaohui Guo

Keyword(s):

Smooth Muscle ◽

Muscle Protein ◽

Umbilical Vein ◽

Smooth Muscle Actin ◽

Quantitative Polymerase Chain Reaction ◽

Density Lipoprotein ◽

Inflammatory Factors ◽

Luciferase Reporter ◽

Western Blot Assay ◽

Lactate Dehydrogenase Activity

Abstract Long noncoding RNAs (lncRNAs) have been verified as vital regulators in human disease, including atherosclerosis. However, the precise role of X-inactive-specific transcript (XIST) in atherosclerosis remains unclear. The proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) exposed to low-density lipoprotein (ox-LDL) were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide, and flow cytometry assays, correspondingly. The western blot assay was used to quantify protein expression. Lactate dehydrogenase activity and the concentrations of inflammatory factors were measured by matched kits. The real-time quantitative polymerase chain reaction (qPCR) was used to determine α-smooth muscle actin, smooth muscle protein 22-α, XIST, miR-98-5p, and pregnancy-associated plasma protein A (PAPPA) levels in HUVECs. The relationship among XIST, miR-98-5p, and PAPPA was analyzed by dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays. We found ox-LDL repressed proliferation and induced inflammation and apoptosis in HUVECs. Loss-of-functional experiment suggested that the downregulation of XIST overturned the ox-LDL-induced effects on HUVECs. Additionally, overexpression of miR-98-5p-induced effects on ox-LDL-stimulated HUVECs was abolished by upregulation of XIST. However, silencing of miR-98-5p strengthened the ox-LDL-induced effects on HUVECs by increasing expression of PAPPA. Mechanistically, XIST could regulate PAPPA expression in ox-LDL-induced HUVECs by sponging miR-98-5p, providing understanding for atherosclerosis.

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Lipopolysaccharide induces neuroinflammation in microglia by activating the MTOR pathway and downregulating Vps34 to inhibit autophagosome formation

Journal of Neuroinflammation ◽

10.1186/s12974-019-1644-8 ◽

2020 ◽

Vol 17 (1) ◽

Cited By ~ 8

Author(s):

Xiaoxia Ye ◽

Mingming Zhu ◽

Xiaohang Che ◽

Huiyang Wang ◽

Xing-Jie Liang ◽

...

Keyword(s):

Inflammatory Response ◽

Microglial Activation ◽

Adaptor Protein ◽

Mtor Pathway ◽

Microglial Cells ◽

Inflammatory Factors ◽

Intraventricular Injection ◽

Enzyme Linked Immunosorbent Assay ◽

Autophagic Flux

Abstract Background Microglial activation is a prominent feature of neuroinflammation, which is present in almost all neurodegenerative diseases. While an initial inflammatory response mediated by microglia is considered to be protective, excessive pro-inflammatory response of microglia contributes to the pathogenesis of neurodegeneration. Although autophagy is involved in the suppression of inflammation, its role and mechanism in microglia are unclear. Methods In the present study, we studied the mechanism by which lipopolysaccharide (LPS) affects microglial autophagy and the effects of autophagy on the production of pro-inflammatory factors in microglial cells by western blotting, immunocytochemistry, transfection, transmission electron microscopy (TEM), and real-time PCR. In a mouse model of neuroinflammation, generated by intraventricular injection of LPS (5 μg/animal), we induced autophagy by rapamycin injection and investigated the effects of enhanced autophagy on microglial activation by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Results We found that autophagic flux was suppressed in LPS-stimulated N9 microglial cells, as evidenced by decreased expression of the autophagy marker LC3-II (lipidated form of MAP1LC3), as well as increased levels of the autophagy adaptor protein SQSTM1. LPS significantly decreased Vps34 expression in N9 microglial cells by activating the PI3KI/AKT/MTOR pathway without affecting the levels of lysosome-associated proteins and enzymes. More importantly, overexpression of Vps34 significantly enhanced the autophagic flux and decreased the accumulation of SQSTM1 in LPS-stimulated N9 microglial cells. Moreover, our results revealed that an LPS-induced reduction in the level of Vps34 prevented the maturation of omegasomes to phagophores. Furthermore, LPS-induced neuroinflammation was significantly ameliorated by treatment with the autophagy inducer rapamycin both in vitro and in vivo. Conclusions These data reveal that LPS-induced neuroinflammation in N9 microglial cells is associated with the inhibition of autophagic flux through the activation of the PI3KI/AKT/MTOR pathway, while enhanced microglial autophagy downregulates LPS-induced neuroinflammation. Thus, this study suggests that promoting the early stages of autophagy might be a potential therapeutic approach for neuroinflammation-associated diseases.

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Sequence requirements for premature transcription arrest within the first intron of the mouse c-fos gene

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2832-2841.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2832-2841

Author(s):

N Mechti ◽

M Piechaczyk ◽

J M Blanchard ◽

P Jeanteur ◽

B Lebleu

Keyword(s):

Rna Polymerase Ii ◽

In Vitro Transcription ◽

Rna Transcripts ◽

First Intron ◽

Nuclear Extracts ◽

Intron 1 ◽

A Cell ◽

Sequence Requirements

A strong block to the elongation of nascent RNA transcripts by RNA polymerase II occurs in the 5' part of the mammalian c-fos proto-oncogene. In addition to the control of initiation, this mechanism contributes to transcriptional regulation of the gene. In vitro transcription experiments using nuclear extracts and purified transcription templates allowed us to map a unique arrest site within the mouse first intron 385 nucleotides downstream from the promoter. This position is in keeping with that estimated from nuclear run-on assays performed with short DNA probes and thus suggests that it corresponds to the actual block in vivo. Moreover, we have shown that neither the c-fos promoter nor upstream sequences are absolute requirements for an efficient transcription arrest both in vivo and in vitro. Finally, we have characterized a 103-nucleotide-long intron 1 motif comprising the arrest site and sufficient for obtaining the block in a cell-free transcription assay.

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Functional analysis of a stable transcription arrest site in the first intron of the murine adenosine deaminase gene

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2718-2729.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2718-2729

Author(s):

S F Kash ◽

J W Innis ◽

A U Jackson ◽

R E Kellems

Keyword(s):

Rna Polymerase Ii ◽

Adenosine Deaminase ◽

Functional Characterization ◽

Gel Filtration ◽

Point Mutations ◽

Elongation Factor ◽

Dependent Manner ◽

First Intron ◽

Intron 1

Transcription arrest plays a role in regulating the expression of a number of genes, including the murine adenosine deaminase (ADA) gene. We have previously identified two prominent arrest sites at the 5' end of the ADA gene: one in the first exon and one in the first intron (J. W. Innis and R. E. Kellems, Mol. Cell. Biol. 11:5398-5409, 1991). Here we report the functional characterization of the intron 1 arrest site, located 137 to 145 nucleotides downstream of the cap site. We have determined, using gel filtration, that the intron 1 arrest site is a stable RNA polymerase II pause site and that the transcription elongation factor SII promotes read-through at this site. Additionally, the sequence determinants for the pause are located within a 37-bp fragment encompassing this site (+123 to +158) and can direct transcription arrest in an orientation-dependent manner in the context of the ADA and adenovirus major late promoters. Specific point mutations in this region increase or decrease the relative pausing efficiency. We also show that the sequence determinants for transcription arrest can function when placed an additional 104 bp downstream of their natural position.

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Ticagrelor and clopidogrel suppress NF-κB to treat acute coronary syndrome

10.21203/rs.2.14619/v1 ◽

2019 ◽

Author(s):

Zhuyin Jia ◽

Yiwei Huang ◽

Xiaojun Ji ◽

Jiaju Sun ◽

Guosheng Fu

Keyword(s):

Cell Cycle ◽

Acute Coronary Syndrome ◽

Cell Migration ◽

Cell Viability ◽

Umbilical Vein ◽

Quantitative Polymerase Chain Reaction ◽

Statistical Significance ◽

Inflammatory Factors ◽

Mrna Levels ◽

Coronary Syndrome

Abstract Background: Inflammatory cytokines are involved in acute coronary syndrome (ACS),and NF-kB is the central regulator of inflammation. Moreover, ticagrelor and clopidogrelcan prevent thrombotic events and improve the care of patients with ACS. Thus, we speculated that ticagrelor and clopidogrel relieve ACS by regulating NF-kB pathway. Methods: After human umbilical vein endothelial cells (HUVECs) were cultured with ticagrelor or clopidogrel and given lipopolysaccharide (LPS) and CD14, the mRNA levels of related inflammatory factors, the protein level changes of molecules in the NF-kB pathway, and the changes in cell viability, apoptosis and the cell cycle, cell migration, vascular formation and other vital activities were detected using quantitative Polymerase chain reaction (qPCR), Western blotting and immunofluorescence assay, CCK8， flow cytometry, transwell assay, matrigel, respectively. All data was expressed as the mean ± S.D. The statistical significance of data was assessedby an unpaired two-tailed t-test. Results: Ticagrelor and clopidogrel can suppress the NF-kB pathway by inhibiting the phosphorylation and entry into the nucleus of p65, restraining the degradation of IKBa, improving cell viability, restoring the cell cycle, cell migration and angiogenic ability, and inhibiting apoptosis. Conclusions: Ticagrelor and clopidogrel alleviate cellular dysfunction through suppressing NF-kB signaling to treat acute coronary syndrome.

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Integrin-linked kinase and PINCH: partners in regulation of cell-extracellular matrix interaction and signal transduction

Journal of Cell Science ◽

10.1242/jcs.112.24.4485 ◽

1999 ◽

Vol 112 (24) ◽

pp. 4485-4489 ◽

Cited By ~ 7

Author(s):

C. Wu

Keyword(s):

Growth Factor ◽

Protein Kinase ◽

Signaling Pathways ◽

Focal Adhesion ◽

Adaptor Protein ◽

Small Gtpase ◽

Beta Catenin ◽

Dependent Manner ◽

Mesenchymal Transition ◽

Integrin Linked Kinase

Integrin-linked kinase (ILK) is a focal adhesion serine/threonine protein kinase that is emerging as a key signaling protein functioning at one of the early convergence points of integrin- and growth factor-signaling pathways. ILK binds to PINCH through the N-terminal ankyrin (ANK) repeat domain and the PINCH binding is crucial for focal adhesion localization of ILK. The ILK-PINCH interaction also connects ILK to Nck-2, an SH2-SH3-containing adaptor protein that interacts with components of growth factor and small GTPase signaling pathways. The kinase activity of ILK is regulated by both cell adhesion and growth factors in a phosphoinositide 3-kinase (PI3K)-dependent manner. ILK phosphorylates downstream targets such as protein kinase B (PKB, also known as Akt) and glycogen synthase kinase 3 (GSK-3) and regulates their activities. Overexpression of ILK in epithelial cells leads to striking morphological changes mimicking epithelial-mesenchymal transition, including upregulation of integrin-mediated fibronectin matrix assembly and downregulation of cell-cell adhesions. Furthermore, ILK regulates nuclear translocation of (beta)-catenin and gene expression, and promotes cell cycle progression and tumor formation. Recent genetic studies in Drosophila melanogaster and Caenorhabditis elegans have shown that lack of expression of ILK or PINCH results in phenotypes resembling those of integrin-null mutants, which demonstrates that ILK and PINCH are indispensable for integrin function during embryonic development.

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Cdc42 Couples T Cell Receptor Endocytosis to GRAF1-Mediated Tubular Invaginations of the Plasma Membrane

Cells ◽

10.3390/cells8111388 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1388 ◽

Cited By ~ 1

Author(s):

Rossatti ◽

Ziegler ◽

Schregle ◽

Betzler ◽

Ecker ◽

...

Keyword(s):

T Cell ◽

T Cell Receptor ◽

T Cell Activation ◽

Cell Activation ◽

Adaptor Protein ◽

Cell Receptor ◽

Small Gtpase ◽

Endocytic Pathway ◽

Bar Domain ◽

Endocytic Vesicles

: T cell activation is immediately followed by internalization of the T cell receptor (TCR). TCR endocytosis is required for T cell activation, but the mechanisms supporting removal of TCR from the cell surface remain incompletely understood. Here we report that TCR endocytosis is linked to the clathrin-independent carrier (CLIC) and GPI-enriched endocytic compartments (GEEC) endocytic pathway. We show that unlike the canonical clathrin cargo transferrin or the adaptor protein Lat, internalized TCR accumulates in tubules shaped by the small GTPase Cdc42 and the Bin/amphiphysin/Rvs (BAR) domain containing protein GRAF1 in T cells. Preventing GRAF1-positive tubules to mature into endocytic vesicles by expressing a constitutively active Cdc42 impairs the endocytosis of TCR, while having no consequence on the uptake of transferrin. Together, our data reveal a link between TCR internalization and the CLIC/GEEC endocytic route supported by Cdc42 and GRAF1.

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