Taurochenodeoxycholic Acid Increases cAMP Content via Specially Interacting with Bile Acid Receptor TGR5

Taurochenodeoxycholic acid (TCDCA) is one of the main components of bile acids (BAs). TCDCA has been reported as a signaling molecule, exerting anti-inflammatory and immunomodulatory functions. However, it is not well known whether those effects are mediated by TGR5. This study aimed to elucidate the interaction between TCDCA and TGR5. To achieve this aim, first, the TGR5 eukaryotic vector was constructed. The expression level of TGR5 in 293T cells was determined by immunofluorescence, real-time quantitative PCR (RT-PCR, qPCR), and Western blot. The luciferase assay, fluorescence microscopy, and enzyme-linked immunosorbent assay (ELISA) were recruited to check the interaction of TCDCA with TGR5. TCDCA treatment in 293T cells resulted in TGR5 internalization coupled with a significant increase in cAMP luciferase expression. Our results demonstrated that TCDCA was able to bind to the TGR5 receptor and activate it. These results provide an excellent potential therapeutic target for TCDCA research. Moreover, these findings also provide theoretical evidence for further TCDCA research.

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Krüppel-Like Factor 2 Regulates Degradation of Type II Collagen by Suppressing the Expression of Matrix Metalloproteinase (MMP)-13

Cellular Physiology and Biochemistry ◽

10.1159/000479991 ◽

2017 ◽

Vol 42 (6) ◽

pp. 2159-2168 ◽

Cited By ~ 10

Author(s):

Yuan Yuan ◽

Honglue Tan ◽

Pengyi Dai

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Macrophage Activation ◽

Type Ii Collagen ◽

Luciferase Assay ◽

Rt Pcr ◽

Potential Therapeutic Target ◽

Primary Chondrocytes ◽

Oa Chondrocytes

Background/Aims: Krüppel-like factor 2 (KLF2) plays an essential role in the inhibition of endothelial cell and macrophage activation during the inflammatory process. However, the roles of KLF2 in chondrocytes and the pathological progression of osteoarthritis (OA) remain unknown. The aim of this study was to investigate the function of KLF2 in the inhibition of cartilage matrix destruction in chondrocytes. Methods: RT-PCR and western blot analysis was used to determine the expression of KLF2 in human chondrocytes. Luciferase assay, ELISA assay and MMP-13 enzymatic activity assays were used to investigate the effects of KLF2 in regulating MMP-13 expression. Western blot analysis was used to examine the effects of KLF2 in suppressing degradation of type Ⅱ collagen. Results: KLF2 is expressed in primary chondrocytes and is downregulated in OA chondrocytes. Expression of KLF2 in primary chondrocytes was reduced in response to IL-1β. Overexpression of KLF2 robustly inhibited IL-1β-induced MMP-13 expression. Conversely, knockdown of KLF2 markedly exacerbated MMP-13 expression. Mechanistically, KLF2 could suppress the activation of MMP-13 promoter. However, knockdown of KLF2 could promote the activation of MMP-13 promoter. Importantly, overexpression of KLF2 ameliorated the degradation of type Ⅱ collagen while silencing of KLF2 exacerbated the degradation of type Ⅱ collagen induced by IL-1β. Conclusions: KLF2 may be a potential therapeutic target for OA treatment.

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MiR-125a-5p Alleviates Dysfunction and Inflammation of Pentylenetetrazol- induced Epilepsy Through Targeting Calmodulin-dependent Protein Kinase IV (CAMK4)

Current Neurovascular Research ◽

10.2174/1567202616666190906125444 ◽

2019 ◽

Vol 16 (4) ◽

pp. 365-372 ◽

Cited By ~ 4

Author(s):

Qishuai Liu ◽

Li Wang ◽

Guizhen Yan ◽

Weifa Zhang ◽

Zhigang Huan ◽

...

Keyword(s):

Protein Kinase ◽

Target Gene ◽

Luciferase Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Inflammatory Factor ◽

Dependent Protein Kinase ◽

Expression Levels ◽

Dependent Protein ◽

Polymerase Chain ◽

The Relationship

Background: MicroRNAs (miRNA) are known to play a key role in the etiology and treatment of epilepsy through controlling the expression of gene. However, miR-125a-5p in the epilepsy is little known. Epilepsy in rat models was induced by Pentylenetetrazol (PTZ) and miR- 125a-5p profiles in the hippocampus were investigated in our experiment. Also, the relationship between miR-125a-5p and calmodulin-dependent protein kinase IV (CAMK4) was identified and the related mechanism was also illustrated. Methods: The miR-125a-5p mRNA expression levels were evaluated by quantitative real time polymerase chain reaction (qRT-PCR). Western Blot (WB) was used to analyze the CAMK4 protein expression levels. Seizure score, latency and duration were determined based on a Racine scale. The enzyme-linked immunosorbent assay (ELISA) was used to analyze the inflammatory factor expression. The relationship between miR-125a-5p and CAMK4 was detected through dual luciferase assay. Results: Downregulation of miR-125a-5p was observed in the hippocampus of PTZ-induced epilepsy rats. The overexpression of miR-125a-5p attenuated seizure and decreased inflammatory factor level in the hippocampus of PTZ-induced rats. The miR-125a-5p alleviated epileptic seizure and inflammation in PTZ-induced rats by suppressing its target gene, CAMK4. Conclusion: miR-125a-5p may represent a novel therapeutic treatment for PTZ-induced epilepsy by preventing the activation of CAMK4.

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Epitope Mapping of Monoclonal Antibodies to Calreticulin Reveals That Charged Amino Acids Are Essential for Antibody Binding

Antibodies ◽

10.3390/antib10030031 ◽

2021 ◽

Vol 10 (3) ◽

pp. 31

Author(s):

Ann Christina Bergmann ◽

Cecilie Kyllesbech ◽

Rimantas Slibinskas ◽

Evaldas Ciplys ◽

Peter Højrup ◽

...

Keyword(s):

Amino Acids ◽

Monoclonal Antibodies ◽

Epitope Mapping ◽

Biological Function ◽

Specific Binding ◽

Enzyme Linked Immunosorbent Assay ◽

Potential Therapeutic Target ◽

Charged Amino Acids ◽

Chaperone Protein ◽

Terminal Amino

Calreticulin is a chaperone protein, which is associated with myeloproliferative diseases. In this study, we used resin-bound peptides to characterize two monoclonal antibodies (mAbs) directed to calreticulin, mAb FMC 75 and mAb 16, which both have significantly contributed to understanding the biological function of calreticulin. The antigenicity of the resin-bound peptides was determined by modified enzyme-linked immunosorbent assay. Specific binding was determined to an 8-mer epitope located in the N-terminal (amino acids 34–41) and to a 12-mer peptide located in the C-terminal (amino acids 362–373). Using truncated peptides, the epitopes were identified as TSRWIESK and DEEQRLKEEED for mAb FMC 75 and mAb 16, respectively, where, especially the charged amino acids, were found to have a central role for a stable binding. Further studies indicated that the epitope of mAb FMC 75 is assessable in the oligomeric structure of calreticulin, making this epitope a potential therapeutic target.

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Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽

10.1159/000517003 ◽

2021 ◽

pp. 1-6

Author(s):

Salman Khan ◽

Syed Asad Ali Shah ◽

Syed Muhammad Jamal

Keyword(s):

Polymerase Chain Reaction ◽

Viral Genome ◽

Foot And Mouth Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Chain Reaction ◽

Kappa Value ◽

Polymerase Chain ◽

Pcr Assays ◽

Level Of Agreement

Background: Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. Methods: A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. Results: S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (p = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. Conclusions: The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

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Sini-San Regulates the NO-cGMP-PKG Pathway in the Spinal Dorsal Horn in a Modified Rat Model of Functional Dyspepsia

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/3575231 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Zhenyu Wu ◽

Xiaofang Lu ◽

Shengsheng Zhang ◽

Chunyang Zhu

Keyword(s):

Gene Expression ◽

Dorsal Horn ◽

Functional Dyspepsia ◽

Rat Model ◽

Spinal Dorsal Horn ◽

Visceral Hypersensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

Gastric Distention ◽

Real Time Quantitative Pcr ◽

Spinal Dorsal

The present study investigated the effect of Chinese medicine Sini-San (SNS) on visceral hypersensitivity in a rat model of functional dyspepsia (FD), and it explored related underlying mechanisms. The rat model of FD was developed by combining neonatal iodoacetamide (IA) treatment and adult tail-clamping. After SNS treatment, the behavior and electromyographic testing were performed to evaluate the visceromotor responses of rats to gastric distention. Immunofluorescence was used to detect the distribution of iNOS-positive cells in the spinal dorsal horn, while the real-time quantitative PCR and western blot were used for detection of the gene expression of c-fos, iNOS, and GABAb and protein levels of iNOS and GABAb in the spinal dorsal horn, respectively. The protein concentration of cGMP and PKG proteins in the spinal dorsal horn were quantified by enzyme-linked immunosorbent assay. In this study, SNS treatment significantly reduced the behavioral score and electromyographic response to graded intragastric distension pressure. The middle-dose of SNS treatment significantly reduced the distribution of iNOS-positive cells in the spinal dorsal horn of FD model rats. The gene expression of c-fos, iNOS, and GABAb and the protein contents of iNOS, GABAb, cGMP, and PKG in the spinal dorsal horn of FD model rats were restored to a normal level by middle-dose of SNS treatment. Our results suggest that Sini-San may alleviate the visceral hypersensitivity in FD model rats via regulation of the NO/cGMP/PKG pathway in the spinal dorsal horn.

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Identification, Characterization, and Molecular Detection of Alfalfa mosaic virus in Potato

Phytopathology ◽

10.1094/phyto-96-1237 ◽

2006 ◽

Vol 96 (11) ◽

pp. 1237-1242 ◽

Cited By ~ 34

Author(s):

H. Xu ◽

J. Nie

Keyword(s):

Mosaic Virus ◽

Gene Sequence ◽

Alfalfa Mosaic Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Polymorphism Analysis ◽

Rt Pcr ◽

Potato Leaf ◽

Simple Method ◽

Cp Gene ◽

Nucleotide Sequence Alignment

Alfalfa mosaic virus (AMV) was detected in potato fields in several provinces in Canada and characterized by bioassay, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction (RT-PCR). The identity of eight Canadian potato AMV isolates was confirmed by sequence analysis of their coat protein (CP) gene. Sequence and phylogenetic analysis indicated that these eight AMV potato isolates fell into one strain group, whereas a slight difference between Ca175 and the other Canadian AMV isolates was revealed. The Canadian AMV isolates, except Ca175, clustered together among other strains based on alignment of the CP gene sequence. To detect the virus, a pair of primers, AMV-F and AMV-R, specific to the AMV CP gene, was designed based on the nucleotide sequence alignment of known AMV strains. Evaluations showed that RT-PCR using this primer set was specific and sensitive for detecting AMV in potato leaf and tuber samples. AMV RNAs were easily detected in composite samples of 400 to 800 potato leaves or 200 to 400 tubers. Restriction analysis of PCR amplicons with SacI was a simple method for the confirmation of PCR tests. Thus, RT-PCR followed by restriction fragment length polymorphism analysis may be a useful approach for screening potato samples on a large scale for the presence of AMV.

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Monitoring Current-Season Spread of Potato virus Y in Potato Fields Using ELISA and Real-Time RT-PCR

Plant Disease ◽

10.1094/pdis-03-12-0283-re ◽

2013 ◽

Vol 97 (5) ◽

pp. 641-644 ◽

Cited By ~ 17

Author(s):

Manphool S. Fageria ◽

Mathuresh Singh ◽

Upeksha Nanayakkara ◽

Yvan Pelletier ◽

Xianzhou Nie ◽

...

Keyword(s):

Real Time ◽

Potato Virus ◽

Potato Virus Y ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Pcr Assay ◽

Current Season ◽

Seed Market ◽

Polymerase Chain ◽

Time Of Harvest

The current-season spread of Potato virus Y (PVY) was investigated in New Brunswick, Canada, in 11 potato fields planted with six different cultivars in 2009 and 2010. In all, 100 plants selected from each field were monitored for current-season PVY infections using enzyme-linked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay. Average PVY incidence in fields increased from 0.6% in 2009 and 2% in 2010 in the leaves to 20.3% in 2009 and 21.9% in 2010 in the tubers at the time of harvest. In individual fields, PVY incidence in tubers reached as high as 37% in 2009 and 39% in 2010 at the time of harvest. Real-time RT-PCR assay detected more samples with PVY from leaves than did ELISA. A higher number of positive samples was also detected with real-time RT-PCR from growing tubers compared with the leaves collected from the same plant at the same sampling time. PVY incidence determined from the growing tubers showed a significant positive correlation with the PVY incidence of tubers after harvest. Preharvest testing provides another option to growers to either top-kill the crop immediately to secure the seed market when the PVY incidence is low or leave the tubers to develop further for table or processing purposes when incidence of PVY is high.

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Prevalence and Molecular Characterization Coat Protein Gene of Tobacco streak virus Causing Peanut Stem Necrosis Disease in Coastal and Rayalaseema Districts of Andhra Pradesh, South-India

Legume Research - An International Journal ◽

10.18805/lr-4515 ◽

2021 ◽

Author(s):

K. Saratbabu ◽

K. Vemana ◽

A.K. Patibanda ◽

B. Sreekanth ◽

V. Srinivasa Rao

Keyword(s):

Coat Protein ◽

Molecular Characterization ◽

Andhra Pradesh ◽

Disease Incidence ◽

Tobacco Streak Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Streak Virus ◽

Rt Pcr ◽

Cp Gene ◽

Stem Necrosis

Background: Peanut stem necrosis disease (PSND) caused by Tobacco streak virus (TSV) is a major constraint for groundnut production in Andhra Pradesh (A.P.). However, studies on prevalence and spread of the disease confined to only few districts of A.P. with this background current study focused on incidence and spread of the disease in entire state of A.P. Further an isolate of TSV occurring in A.P. characterized on the basis of genetic features by comparing with other TSV isolates originated from different hosts and locations from world.Methods: Roving survey was conducted during kharif 2017-18 in groundnut growing districts of Andhra Pradesh (A.P.) for peanut stem necrosis disease incidence. Groundnut plants showing PSND symptoms were collected and tested with direct antigen coating enzyme linked immunosorbent assay (DAC-ELISA). Groundnut samples found positive by ELISA once again tested by reverse transcription polymerase chain reaction (RT-PCR). The representative TSV-GN-INDVP groundnut isolate from Prakasham district was maintained on cowpea seedlings by standard sap inoculation method in glasshouse for further molecular characterization. The Phylogenetic tree for coat protein (CP) gene was constructed using aligned sequences with 1000 bootstrap replicates following neighbor-joining phylogeny.Result: Thirty-eight (52.7%) of seventy-two groundnut samples collected from different locations in A.P were given positive reaction to TSV by DAC-ELISA. For the first time, PSND incidence observed in coastal districts (Krishna, Guntur, Sri Pottisriramulu Nellore, Prakasham) of A.P. Maximum PSND incidence recorded from Bathalapalli (22.2%) and the minimum incidence in Mulakalacheruvu (4.1%). The coat protein (CP) gene of TSV-GN-INDVP groundnut isolate was amplified by RT-PCR and it shared maximum per cent nucleotide identity (97.51-98.62%) with TSV isolates from groundnut and other different crops reported in India. All Indian isolates cluster together irrespective of crop and location based on the phylogenetic analysis.

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Comparison of ELISA and RT-PCR assays for the detection of Tomato spotted wilt virus in peanut

Peanut Science ◽

10.3146/ps08-024.1 ◽

2009 ◽

Vol 36 (2) ◽

pp. 133-137 ◽

Cited By ~ 1

Author(s):

P. M. Dang ◽

D. L. Rowland ◽

W. H. Faircloth

Keyword(s):

Tomato Spotted Wilt Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Infection Rates ◽

Rt Pcr ◽

Chi Square ◽

Tomato Spotted Wilt ◽

Significant Difference ◽

Pcr Assays ◽

Chi Square Analysis ◽

Wilt Virus

Abstract Diagnosis of Tomato spotted wilt virus (TSWV) in peanut can be accomplished by enzyme-linked immunosorbent assay (ELISA) or reverse transcription polymerase chain reaction (RT-PCR) but there has been no report of a direct comparison of the success of the two assays in evaluating infection rates of field-grown peanut. We collected peanut root samples from field-grown plants, 76 in 2006 and 48 in 2007, and tested these samples by both ELISA and RT-PCR assays for the presence of TSWV. Out of 124 samples, 50 (40.3%) and 57 (46.0%) were positive for TSWV by ELISA and RT-PCR respectively. In 13.7% of these samples, ELISA and RT-PCR differed in their results. However, Chi square analysis showed no significant difference between the results for these two assays. This result supports the conclusion that ELISA and RT-PCR are comparable for detecting TSWV infection rates in field-grown peanuts.

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Epidemiological Survey and Screening Strategy for Dengue Virus in Blood Donors from Yunnan Province

10.21203/rs.3.rs-50346/v1 ◽

2020 ◽

Author(s):

LING LI ◽

YING LI ◽

Shaofang Lu ◽

Jing Dong ◽

Haixia Xu ◽

...

Keyword(s):

Dengue Virus ◽

Prevalence Rate ◽

Screening Strategy ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Pcr Assay ◽

Igm Antibodies ◽

Blood Center ◽

Nonstructural Protein 1 ◽

Elisa Testing

Abstract BACKGROUND Dengue virus (DENV) can be transmitted through blood transfusion. DENV was not screened regularly in Xishuangbanna Blood Center. This study was conducted in Xishuangbanna Blood Center with an attempt to develop DENV screening strategies in one of China’s high-incidence areas.METHODS Blood samples were collected randomly between June 2019 and August 2019. These samples were first screened for dengue IgG and IgM antibodies using enzyme-linked immunosorbent assay (ELISA). All reactive samples and some randomly-chosen non-reactive samples were used to detect DENV RNAs using real time polymerase-chain-reaction (RT-PCR) assay. After RT-PCR assay, these samples were further tested for soluble nonstructural protein 1 (NS1) using colloidal gold method. The demographic data of DENV positive donors were collected.RESULTS A total of 2,254 donor samples were collected and tested for dengue IgG and IgM antibodies by ELISA between June 2019 and August 2019. ELISA testing revealed that 598 donor samples were anti-IgG and/or anti-IgM reactive, with a serological prevalence rate of 26.53%. Among all the donor samples, 26 were RT-PCR positive and/or NS1 positive. Moreover, there were significant differences in the prevalence rate of DENV in terms of occupation (P=0.001), education(P<0.001) and ethnicity (P=0.026). CONCLUSION The prevalence of DENV in Xishuangbanna Blood Center was higher than most other blood centers that have implemented DENV donor screening. Our study provides the first-hand data about the prevalence of DENV and allows development of a screening strategy for clinical use.

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