scholarly journals Abnormal Expression of Fgf9 during the Development of the Anorectum in Rat Embryos with Anorectal Malformations

2019 ◽  
Vol 2019 ◽  
pp. 1-7
Author(s):  
Huiying Liu ◽  
Hailan Zhang ◽  
Meng Li ◽  
Hongzhong Tian ◽  
Xiaobing Tang ◽  
...  
Keyword(s):  
Immunohistochemical Staining ◽  
Quantitative Polymerase Chain Reaction ◽  
Anorectal Malformations ◽  
Positive Staining ◽  
Western Blots ◽  
Study Objective ◽  
Rat Embryos ◽  
Qrt Pcr ◽  
Cloacal Membrane ◽  
Fusion Site

The study objective was to investigate the role of fibroblast growth factor 9 (Fgf9) in normal and anorectal malformation (ARM) embryos during the development of the anorectum. Fgf9 expression was assayed in both normal rat embryos and embryos with ARM induced by exposure to ethylenethiourea (ETU) on embryonic day 10 (E10). Fgf9 expression was assayed by immunohistochemical staining, Western blotting, and real-time quantitative polymerase chain reaction (qRT-PCR). Immunohistochemical staining revealed spatiotemporal changes in Fgf9 expression between E13 and E16. Fgf9-positive cells predominated in the mesenchyme of the cloaca on E13 and E14 and at the fusion site of the urorectal septum and cloacal membrane, rectal epithelium, and anal membrane on E15. Fgf9-positive cells were obviously decreased after the anal membrane ruptured on E16. Fgf9-positive staining was significantly decreased in embryos with ARM compared with normal embryos from E13 to E15. The results of Western blots and qRT-PCR were consistent, with significantly increased Fgf9 expression in the hindgut and rectum of normal embryos than in embryos with ARM from E13 to E15. However, there was no difference between the two groups on E16. These results suggested that the anorectal embryogenesis might depend on the induction of Fgf9 signal. The expression of Fgf9 was downregulated in ETU-induced ARM embryos, which might be related to the development of ARM.

scholarly journals Expression pattern of Wif1 and β-catenin during development of anorectum in fetal rats with anorectal malformations

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4445 ◽  
Author(s):  
Xiao Bing Tang ◽  
Huan Li ◽  
Jin Zhang ◽  
Wei Lin Wang ◽  
Zheng Wei Yuan ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Expression Pattern ◽  
Immunohistochemical Staining ◽  
Anorectal Malformations ◽  
Rt Pcr ◽  
Rat Embryos ◽  
Fetal Rats ◽  
Normal Rat ◽  
Wnt Inhibitory Factor 1

Purpose This study was performed to investigate the expression pattern of Wnt inhibitory factor 1 (Wif1) and β-catenin during anorectal development in normal and anorectal malformation (ARM) embryos and the possible role of Wif1 and β-catenin in the pathogenesis of ARM. Methods ARM was induced with ethylenethiourea on the 10th gestational day in rat embryos. Cesarean deliveries were performed to harvest the embryos. The expression pattern of Wif1 and β-catenin protein and mRNA was evaluated in normal rat embryos (n = 288) and ARM rat embryos (n = 306) from GD13 to GD16 using immunohistochemical staining, Western blot, and real time RT-PCR. Results Immunohistochemical staining revealed that in normal embryos Wif1 was constantly expressed in the cloaca from GD13 to GD16. On GD13 and GD14, Wif1-immunopositive cells were extensively expressed in the cloaca. On GD15, the expression of Wif1 were mainly detected on the very thin anal membrane. In ARM embryos, the epithelium of the hindgut and urorectal septum demonstrated faint immunostaining for Wif1 from GD14 to GD16. Western blot and real time RT-PCR revealed that Wif1 and β-catenin protein and mRNA expression level was significantly decreased in the ARM groups compared with the normal group on GD14 and GD15 (p < 0.05). Conclusions This study demonstrated that the expression pattern of Wif1 and β-catenin was disrupted in ARM embryos during anorectal morphogenesis, which demonstrated that downregulation of Wif1 and β-catenin at the time of cloacal separation into the primitive rectum and urogenital septum might related to the development of ARM.

scholarly journals Expression pattern of Wif 1 during development of anorectum in fetal rats with anorectal malformations

2017 ◽  
Author(s):  
Xiao Bing Tang ◽  
Huan Li ◽  
Jin Zhang ◽  
Wei Lin Wang ◽  
Zheng Wei Yuan ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Expression Pattern ◽  
Immunohistochemical Staining ◽  
Anorectal Malformations ◽  
Rt Pcr ◽  
Rat Embryos ◽  
Fetal Rats ◽  
Normal Rat ◽  
Wnt Inhibitory Factor 1

Purpose: This study was performed to investigate the expression pattern of Wnt inhibitory factor 1 (Wif1) during anorectal development in normal and anorectal malformation (ARM) embryos and the possible role of Wif1 in the pathogenesis of ARM. Methods: ARM was induced with ethylenethiourea on the 10th gestational day in rat embryos. Cesarean deliveries were performed to harvest the embryos. The expression pattern of Wif1 protein and mRNA was evaluated in normal rat embryos (n=288) and ARM rat embryos (n=306) from GD13 to GD16 using immunohistochemical staining, Western blot, and real time RT-PCR. Results: Immunohistochemical staining revealed that in normal embryos Wif1 was constantly expressed in the cloaca from GD13 to GD16. On GD13 and GD14, Wif1-immunopositive cells were extensively expressed in the cloaca. On GD15, the expression of Wif1 were mainly detected on the very thin anal membrane. In ARM embryos, the epithelium of the hindgut and urorectal septum demonstrated faint immunostaining for Wif1 from GD14 to GD16. Western blot and real time RT-PCR revealed that Wif1 protein and mRNA expression level was significantly decreased in the ARM groups compared with the normal group on GD14 and GD15 (p<0.05).Conclusions: This study demonstrated that the expression pattern of Wif1 was disrupted in ARM embryos during anorectal morphogenesis, which demonstrated that downregulation of Wif1 at the time of cloacal separation into the primitive rectum and urogenital septum might related to the development of ARM.

scholarly journals Expression pattern of Wif 1 during development of anorectum in fetal rats with anorectal malformations

2017 ◽  
Author(s):  
Xiao Bing Tang ◽  
Huan Li ◽  
Jin Zhang ◽  
Wei Lin Wang ◽  
Zheng Wei Yuan ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Expression Pattern ◽  
Immunohistochemical Staining ◽  
Anorectal Malformations ◽  
Rt Pcr ◽  
Rat Embryos ◽  
Fetal Rats ◽  
Normal Rat ◽  
Wnt Inhibitory Factor 1

Purpose: This study was performed to investigate the expression pattern of Wnt inhibitory factor 1 (Wif1) during anorectal development in normal and anorectal malformation (ARM) embryos and the possible role of Wif1 in the pathogenesis of ARM. Methods: ARM was induced with ethylenethiourea on the 10th gestational day in rat embryos. Cesarean deliveries were performed to harvest the embryos. The expression pattern of Wif1 protein and mRNA was evaluated in normal rat embryos (n=288) and ARM rat embryos (n=306) from GD13 to GD16 using immunohistochemical staining, Western blot, and real time RT-PCR. Results: Immunohistochemical staining revealed that in normal embryos Wif1 was constantly expressed in the cloaca from GD13 to GD16. On GD13 and GD14, Wif1-immunopositive cells were extensively expressed in the cloaca. On GD15, the expression of Wif1 were mainly detected on the very thin anal membrane. In ARM embryos, the epithelium of the hindgut and urorectal septum demonstrated faint immunostaining for Wif1 from GD14 to GD16. Western blot and real time RT-PCR revealed that Wif1 protein and mRNA expression level was significantly decreased in the ARM groups compared with the normal group on GD14 and GD15 (p<0.05).Conclusions: This study demonstrated that the expression pattern of Wif1 was disrupted in ARM embryos during anorectal morphogenesis, which demonstrated that downregulation of Wif1 at the time of cloacal separation into the primitive rectum and urogenital septum might related to the development of ARM.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Small Non-Coding RNAome of Ageing Chondrocytes

2020 ◽  
Vol 21 (16) ◽  
pp. 5675
Author(s):  
Panagiotis Balaskas ◽  
Jonathan A. Green ◽  
Tariq M. Haqqi ◽  
Philip Dyer ◽  
Yalda A. Kharaz ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Differential Expression Analysis ◽  
Cartilage Tissue ◽  
Pcr Analysis ◽  
Small Rna Sequencing ◽  
High Grade ◽  
Human Cartilage ◽  
Fragment Analysis ◽  
Qrt Pcr ◽  
Age Related

Ageing is a leading risk factor predisposing cartilage to osteoarthritis. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs (sncRNAs). RNA from young and old chondrocytes from macroscopically normal equine metacarpophalangeal joints was extracted and subjected to small RNA sequencing (RNA-seq). Differential expression analysis was performed in R using package DESeq2. For transfer RNA (tRNA) fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5. Selected microRNA (miRNAs or miRs) and small nucleolar RNA (snoRNA) findings were validated using real-time quantitative Polymerase Chain Reaction (qRT-PCR) in an extended cohort of equine chondrocytes. tRNA fragments were further investigated in low- and high-grade OA human cartilage tissue. In total, 83 sncRNAs were differentially expressed between young and old equine chondrocytes, including miRNAs, snoRNAs, small nuclear RNAs (snRNAs), and tRNAs. qRT-PCR analysis confirmed findings. tRNA fragment analysis revealed that tRNA halves (tiRNAs), tiRNA-5035-GluCTC and tiRNA-5031-GluCTC-1 were reduced in both high grade OA human cartilage and old equine chondrocytes. For the first time, we have measured the effect of ageing on the expression of sncRNAs in equine chondrocytes. Changes were detected in a number of different sncRNA species. This study supports a role for sncRNAs in ageing cartilage and their potential involvement in age-related cartilage diseases.

Pepsinogen C Expression in Tumors of Extragastric Origin

2000 ◽  
Vol 15 (2) ◽  
pp. 165-170 ◽  
Author(s):  
A.M. Merino ◽  
J. Vázquez ◽  
J.C. Rodríguez ◽  
R. Fernández ◽  
I. Quintela ◽  
...  
Keyword(s):  
Basal Cell ◽  
Immunohistochemical Staining ◽  
Proteolytic Enzyme ◽  
Human Tumors ◽  
Positive Staining ◽  
Human Tissues ◽  
Breast Carcinomas ◽  
Pepsinogen C ◽  
Human Carcinomas

We have examined by immunohistochemistry the ability of human carcinomas of various origin to produce pepsinogen C, an aspartyl proteinase mainly involved in the digestion of proteins in the stomach and recently found to be associated with breast carcinomas. Of the 268 tumors analyzed 80 (29.8%) showed positive staining for pepsinogen C. These positive tumors included 12 gastric (38.7% of the 31 examined cases), nine pancreatic (42.8%), two renal (20%), 12 prostatic (40%), three bladder (27.3%), 14 endometrial (29.7%) and 18 ovarian (40%) carcinomas. We also detected 10 melanomas (50%) that were positive for pepsinogen C. By contrast, immunohistochemical staining for the proteinase was not detected in colorectal, cervical, lung and basal cell skin carcinomas. These results demonstrate that pepsinogen C, a proteolytic enzyme of highly restricted expression in human tissues, can also be expressed by a wide variety of human carcinomas. In addition, and similar to pepsinogen C expression in breast carcinomas, the production of this enzyme by different human tumors might be related to putative hormonal alterations associated with the development and progression of these tumors.

Detection of fos protein during osteogenesis by monoclonal antibodies

1988 ◽  
Vol 8 (5) ◽  
pp. 2251-2256
Author(s):  
P De Togni ◽  
H Niman ◽  
V Raymond ◽  
P Sawchenko ◽  
I M Verma
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Indirect Immunofluorescence ◽  
Protein Complex ◽  
Synthetic Peptide ◽  
Immunohistochemical Staining ◽  
Nuclear Staining ◽  
Rat Embryos ◽  
Fos Protein ◽  
Modified Forms

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.

scholarly journals The Protective Effect of Cordycepin On Alcohol-Induced Osteonecrosis of the Femoral Head

2017 ◽  
Vol 42 (6) ◽  
pp. 2391-2403 ◽  
Author(s):  
Yi-Xuan Chen ◽  
Dao-Yu Zhu ◽  
Zheng-Liang Xu ◽  
Jun-Hui Yin ◽  
Xiao-Wei Yu ◽  
...  
Keyword(s):  
Femoral Head ◽  
Protective Effect ◽  
Immunohistochemical Staining ◽  
Critical Role ◽  
Micro Ct ◽  
Bone Mesenchymal Stem Cells ◽  
Western Blots ◽  
Alizarin Red ◽  
Alp Activity

Background: Alcohol abuse is known to be a leading risk factor for atraumatic osteonecrosis of the femoral head (ONFH), in which the suppression of osteogenesis plays a critical role. Cordycepin benefits bone metabolism; however, there has been no study to determine its effect on osteonecrosis. Methods: Human bone mesenchymal stem cells (hBMSCs) were identified by multi-lineage differentiation. Alkaline phosphatase (ALP) activity, RT-PCR, western blots, immunofluorescent assay and Alizarin red staining of BMSCs were evaluated. A rat model of alcohol-induced ONFH was established to investigate the protective role of cordycepin against ethanol. Hematoxylin & eosin (H&E) staining and micro-computerized tomography (micro-CT) were performed to observe ONFH. Apoptosis was assessed by TdT-mediated dUTP nick end labeling (TUNEL). Immunohistochemical staining was carried out to detect OCN and COL1. Results: Ethanol significantly suppressed ALP activity, decreased gene expression of OCN and BMP2, lowered levels of RUNX2 protein, and reduced immunofluorescence staining of OCN and COL1 and calcium formation of hBMSCs. However, these inhibitory effects were attenuated by cordycepin co-treatment at concentrations of 1 and 10 µg/mL Moreover, it was revealed that the osteo-protective effect of cordycepin was associated with modulation of the Wnt/β-catenin pathway. In vivo, by micro-CT, TUNEL and immunohistochemical staining of OCN and COL1, we found that cordycepin administration prevented alcohol-induced ONFH. Conclusion: Cordycepin treatment to enhance osteogenesis may be considered a potential therapeutic approach to prevent the development of alcohol-induced ONFH.

scholarly journals Anlotinib Inhibits the Growth of Breast Cancer Cells by Promoting Autophagy and Apoptosis via the Akt/GSK-3α Signalling Pathway

2020 ◽  
Author(s):  
shuyi chen ◽  
Ping Zhu ◽  
Xue Wang ◽  
Youping Jin ◽  
Xiuling Zhi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Underlying Mechanism ◽  
Cell Counting ◽  
Tunel Staining ◽  
Western Blots ◽  
Qrt Pcr ◽  
Mcf 7

Abstract Background: Anlotinib, a multi-target tyrosine kinase inhibitor, has already been indicated to have significant anticancer effects on lung cancer, colon cancer and ovarian cancer in a phase II clinical trial, but its effect on breast cancer (BC) has not been adequately investigated. Methods: The proliferation activity of BC cell lines MCF-7 and MDA-MB-231 with the treatment of anlotinib was tested by Cell Counting Kit-8 (CCK-8) assay and immunocytochemistry (ICC) staining. We investigated the alteration of cell cycle and apoptosis and autophagy level and the underlying mechanism in the cell lines by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), Western blots, ICC and TUNEL staining and flow cytometry. Further, AT-3 cells were subcutaneously injected into C57BL/6 mice, followed by anlotinib intragastrically. The extracted tumours were assessed by qRT-PCR, Western blots and immunohistochemistry.Results: We found that anlotinib suppressed the cell viability and proliferation of MCF-7 and MDA-MB-231 cell lines and tumour growth in BC xenografts in mice, likely due to abnormal cell cycle arrest and induction of autophagy and apoptosis. Then, we further examined the underlying mechanism of anlotinib, and the results indicated that anlotinib induced apoptosis by promoting autophagy in MCF-7 and MDA-MB-231 cells by regulating the Akt/GSK-3α pathway. The analysis of data from patients with BC collected in TCGA revealed that increased VEGFA expression was related to BC.Conclusions: Our study demonstrated that anlotinib inhibited the growth of BC cells via promoting apoptosis through autophagy mediated by Akt/GSK-3α signalling and may be an effective new drug for BC treatment.

scholarly journals Detection of fos protein during osteogenesis by monoclonal antibodies.

1988 ◽  
Vol 8 (5) ◽  
pp. 2251-2256 ◽  
Author(s):  
P De Togni ◽  
H Niman ◽  
V Raymond ◽  
P Sawchenko ◽  
I M Verma
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Indirect Immunofluorescence ◽  
Protein Complex ◽  
Synthetic Peptide ◽  
Immunohistochemical Staining ◽  
Nuclear Staining ◽  
Rat Embryos ◽  
Fos Protein ◽  
Modified Forms

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.

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