scholarly journals mRNA/microRNA Profile at the Metamorphic Stage of Olive Flounder (Paralichthys olivaceus)

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Caixia Xie ◽  
Shanliang Xu ◽  
Linlin Yang ◽  
Zhonghe Ke ◽  
Jubin Xing ◽  
...  
Keyword(s):  
Paralichthys Olivaceus ◽  
Whole Body ◽  
Related Gene ◽  
Rt Pcr ◽  
Metamorphic Stage ◽  
Stem Loop ◽  
Qrt Pcr ◽  
Microrna Profile ◽  
Asymmetric Transformation ◽  
Mirna Library

Flatfish is famous for the asymmetric transformation during metamorphosis. The molecular mechanism behind the asymmetric development has been speculated over a century and is still not well understood. To date, none of the metamorphosis-related genes has been identified in flatfish. As the first step to screen metamorphosis-related gene, we constructed a whole-body cDNA library and a whole-body miRNA library in this study and identified 1051 unique ESTs, 23 unique miRNAs, and 4 snoRNAs in premetamorphosing and prometamorphosingParalichthys olivaceus. 1005 of the ESTs were novel, suggesting that there was a special gene expression profile at metamorphic stage. Four miRNAs (pol-miR-20c,pol-miR-23c,pol-miR-130d, andpol-miR-181e) were novel toP. olivaceus; they were characterized as highly preserved homologies of published miRNAs but with at least one nucleotide differed. Representative 24 mRNAs and 23 miRNAs were quantified during metamorphosis ofP. olivaceusby using quantitative RT PCR or stem-loop qRT PCR. Our results showed that 20 of mRNAs might be associated with early metamorphic events, 10 of mRNAs might be related with later metamorphic events, and 16 of miRNAs might be involved in the regulation of metamorphosis. The data provided in this study would be helpful for further identifying metamorphosis-related gene inP. olivaceus.

scholarly journals Development of an RNA Extraction Protocol for a Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1804
Author(s):  
Daniel Plante ◽  
Julio Alexander Bran Barrera ◽  
Maude Lord ◽  
Irène Iugovaz ◽  
Neda Nasheri
Keyword(s):  
Hepatitis A Virus ◽  
Rna Extraction ◽  
Complex Nature ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Pcr Inhibitors ◽  
Qrt Pcr ◽  
Extraction Protocol ◽  
Viral Particles ◽  
Hands On

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

scholarly journals Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Dipak K. Dube ◽  
Syamalima Dube ◽  
Lynn Abbott ◽  
Ruham Alshiekh-Nasany ◽  
Charles Mitschow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Human Heart ◽  
Amino Acid Sequences ◽  
Rt Pcr ◽  
Adult Heart ◽  
Qrt Pcr ◽  
Long Time ◽  
Alternate Promoters ◽  
Computational Analyses

In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.

scholarly journals Evaluation of the performance of the COVID-19 rapid antigen tests in a tertiary level hospital in Bangladesh.

2021 ◽  
Author(s):  
Mohammad Jahidur Rahman Khan ◽  
Md. Shahadat Hossain ◽  
Samshad Jahan Shumu ◽  
Md. Selim Reza ◽  
Farzana Mim ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Detection Rate ◽  
High Sensitivity ◽  
Rt Pcr ◽  
College Hospital ◽  
Medical College ◽  
Qrt Pcr ◽  
Antigen Tests

Abstract Background: While the COVID-19 pandemic is a worldwide crisis, tests with high sensitivity and specificity are essential for identifying and managing COVID-19 patients. Globally, several rapid antigen tests RATs for COVID-19 have been developed, but their clinical efficacy has not been well established. This study aimed to evaluate the performance of several rapid antigen tests (RATs) to diagnose SARS-CoV-2 infection.Methods: This prospective observational study was conducted at Shaheed Suhrawardy Medical College hospital from February 2021 to April 2021 in Dhaka, Bangladesh. This study included the patients admitted in this hospital at the COVID-19 isolation unit or referred from the triage facility of the outdoor department of this hospital suspected as COVID-19 case. Two nasopharyngeal samples were collected simultaneously. one sample was used on the spot for the RAT. The other was sent to the adjacent Shaheed Suhrawardy Medical College COVID-19 RT-PCR laboratory for real-time reverse transcription-polymerase chain reaction (qRT-PCR). The performance of the RAT was evaluated using the results of qRT-PCR as a reference.Results: A total of 223 patients were included in this study, and the real-time RT-PCR detected SARS-CoV-2 in 84 (37.7%) patients. Of these 84 patients, 9 (10.7%) were asymptomatic. The overall sensitivity and specificity of RATs were 78.6% and 99.3%, respectively. The sensitivity was 81.3% in symptomatic cases and 55.6% in asymptomatic cases. False-negatives were observed in 18 patients, 3 of whom were asymptomatic and had a low viral load (cycle threshold (Ct) > 30). The detection rate of RATs was 100% when the Ct value was up to 24. The detection rate was 42.3% when the Ct was >29. The detection rate of RATs was 92.3% when the onset of symptoms was within three days. The detection rate was 33.3% when the onset of symptoms was >7 days.Conclusions: RATs for COVID-19 used in this study delivered an acceptable performance in patients with high viral load and within the first week of the onset of symptoms. They can be used as a supplementary method to RT-PCR for the diagnosis of COVID-19 patients.

scholarly journals Absolute measurement of androgen receptor mRNA in peripheral blood mononuclear, preputial skin and urethral mucosa cells of control individuals with phimosis using qRT-PCR

2011 ◽  
Vol 55 (8) ◽  
pp. 665-668 ◽  
Author(s):  
Tatiane Sousa e Silva ◽  
Flavio Richetti ◽  
Daniela Patricia Palmeira Santos Cunha ◽  
Antonio Carlos Moreira Amarante ◽  
Jovelino Quintino de Souza Leão ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Peripheral Blood ◽  
External Genitalia ◽  
Absolute Measurement ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Skin Cells ◽  
Qrt Pcr ◽  
Specific Manner ◽  
The Mean

INTRODUCTION: Androgen actions are exerted upon the androgen receptor (AR), and complete genital virilization of normal 46,XY individuals depends on adequate function and expression of the AR gene in a tissue-specific manner. OBJECTIVE: Standardization of normal ARmRNA in androgen-sensitive tissues. MATERIALS AND METHODS: In this study, we determined the quantitative amounts of ARmRNA in peripheral blood mononuclear, urethral mucosa and preputial skin cells of control subjects with phimosis by using RT-PCR. RESULTS: The mean (SD) values of AR expression in blood, urethra and prepuce were: 0.01 (0.01); 0.43 (0.32); 0.31 (0.36), respectively. CONCLUSION: The AR expression is low in blood and equivalent in urethral mucosa and preputial skin, which may be useful in the diagnosis of individuals with abnormal external genitalia.

scholarly journals Feeding of 1-Kestose Induces Glutathione-S-Transferase Expression in Mouse Liver

Foods ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 69 ◽  
Author(s):  
Takumi Tochio ◽  
Yuki Ueno ◽  
Yasuyuki Kitaura ◽  
Mikako Shinohara ◽  
Yoshihiro Kadota ◽  
...  
Keyword(s):  
Gene Expression ◽  
Functional Food ◽  
Mouse Liver ◽  
Antioxidative Activity ◽  
Epididymal Adipose Tissue ◽  
Activity Levels ◽  
Rt Pcr ◽  
Glutathione S Transferase ◽  
Food Ingredients ◽  
Qrt Pcr

Functional food ingredients, including prebiotics, have been increasingly developed for human health. The improvement of the human intestinal environment is one of their main targets. Fructooligosaccarides (FOS) are oligosaccharide fructans that are well studied and commercialized prebiotics. 1-Kestose, one of the components of FOS, is considered to be a key prebiotic component in FOS. However, to our knowledge, no studies have been reported on the physiological efficacy of 1-Kestose regarding its anti-oxidative activity. In the present study, we examined the effects of dietary 1-Kestose on gene expression of antioxidative enzymes in the liver, kidney and epididymal adipose tissue of mice by quantitative RT-PCR (qRT-PCR). We demonstrated that a 1-Kestose-rich diet increased mRNA and enzymatic activity levels of glutathione-S-transferase (GST) in mouse liver. These results suggest the possibility that dietary 1-Kestose as a prebiotic may enhance antioxidative activity in mice.

scholarly journals Lipopolysaccharide exposure induces oxidative damage in Caenorhabditis elegans: protective effects of carnosine

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Jing Ma ◽  
Xiaoyuan Xu ◽  
Ranran Wang ◽  
Haijing Yan ◽  
Huijuan Yao ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Caenorhabditis Elegans ◽  
Signaling Pathway ◽  
P38 Mapk ◽  
Mapk Signaling ◽  
Protective Effects ◽  
Related Gene ◽  
Rt Pcr ◽  
C Elegans ◽  
Apoptosis Related Gene

Abstract Background The present study was designed to investigate the protective effects and mechanisms of carnosine on lipopolysaccharide (LPS)-induced injury in Caenorhabditis elegans. Methods C. elegans individuals were stimulated for 24 h with LPS (100 μg/mL), with or without carnosine (0.1, 1, 10 mM). The survival rates and behaviors were determined. The activities of superoxide dismutase (SOD), glutathione reductase (GR), and catalase (CAT) and levels of malondialdehyde (MDA) and glutathione (GSH) were determined using the respective kits. Reverse transcription polymerase chain reaction (RT-PCR) was performed to validate the differential expression of sod-1, sod-2, sod-3, daf-16, ced-3, ced-9, sek-1, and pmk-1. Western blotting was used to determine the levels of SEK1, p38 mitogen-activated protein kinase (MAPK), cleaved caspase3, and Bcl-2. C. elegans sek-1 (km2) mutants and pmk-1 (km25) mutants were used to elucidate the role of the p38 MAPK signaling pathway. Results Carnosine improved the survival of LPS-treated C. elegans and rescued behavioral phenotypes. It also restrained oxidative stress by decreasing MDA levels and increasing SOD, GR, CAT, and GSH levels. RT-PCR results showed that carnosine treatment of wild-type C. elegans up-regulated the mRNA expression of the antioxidant-related genes sod-1, sod-2, sod-3, and daf-16. The expression of the anti-apoptosis-related gene ced-9 and apoptosis-related gene ced-3 was reversed by carnosine. In addition, carnosine treatment significantly decreased cleaved caspase3 levels and increased Bcl-2 levels in LPS-treated C. elegans. Apoptosis in the loss-of-function strains of the p38 MAPK signaling pathway was suppressed under LPS stress; however, the apoptotic effects of LPS were blocked in the sek-1 and pmk-1 mutants. The expression levels of sek-1 and pmk-1 mRNAs were up-regulated by LPS and reversed by carnosine. Finally, the expression of p-p38MAPK and SEK1 was significantly increased by LPS, which was reversed by carnosine. Conclusion Carnosine treatment protected against LPS injury by decreasing oxidative stress and inhibiting apoptosis through the p38 MAPK pathway.

Hydroxychloroquine Blocks Autophagy and Promotes Apoptosis of the Prostate after Castration in Rats

2020 ◽  
Vol 104 (11-12) ◽  
pp. 968-974
Author(s):  
Sheng-Tao Ling ◽  
Chun-Lei Deng ◽  
Li Huang ◽  
Qi-Sheng Yao ◽  
Cui Liu ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Morphological Changes ◽  
Control Group ◽  
Sham Operation ◽  
Related Gene ◽  
Rt Pcr ◽  
Bax Protein ◽  
Sd Rats ◽  
Immunohistochemical Analyses ◽  
Scanning Electron

Autophagy is an important pro-survival mechanism and closely related to apoptosis. The aim of this study was to investigate whether hydroxychloroquine (HCQ) blocks autophagy and promotes apoptosis of the prostate after castration. <b><i>Methods:</i></b> Thirty-six male SD rats were randomly divided into 3 groups (<i>n</i> = 12): control group (sham operation), castration group, and HCQ group (castrated and treated with HCQ). On day 7, all mice were executed and prostates were isolated. The morphological changes of prostates were observed by light microscope, and the ultrastructure changes were observed under scanning electron microscope (SEM). The protein expression of Beclin-l, P62, caspase-3, Bcl-2, and Bax was assessed by immunohistochemical analyses. The mRNA expression of microtubule-associated protein light chain 3 (LC3) and autophagy-related gene 5 (Atg5) was detected by RT-PCR. <b><i>Results:</i></b> Prostates of castration group shrank remarkably and prostates of HCQ group shrank more remarkably than castration group. Cytolysosomes were visible in the prostates of the castration group under SEM. Immunohistochemistry showed that the protein of Beclin-1 increased in the castration group compared to the control group, while decreased in the HCQ group compared to the castration group. While P62 protein moderately dyed in the control group and weakly dyed in the castration group, it strongly dyed in the HCQ group. Caspase-3 and Bax protein were weakly dyed in the control group but moderately dyed in the castration group and strongly dyed in the HCQ group. The expressions of apoptosis suppressor Bcl-2 were reduced in the castration group and further reduced in the HCQ group compared to the castration group. RT-PCR revealed that the mRNA of LC3 and Atg5 in the castration group increased compared to the control group, while decreased after treated with HCQ. <b><i>Conclusion:</i></b> Autophagy increased after castrated in prostates, while decreased after treated with HCQ; all these indicated that HCQ blocked autophagy and then promoted prostate apoptosis of castrated mice.

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