Evaluation of suitable qRT-PCR normalization genes for various citrus rootstocks

Background:: In Traditional Chinese Medicine (TCM), the heads and tails of Angelica sinensis (Oliv.) Diels (AS) is used in treating different diseases due to their different pharmaceutical efficacies. The underline mechanisms, however, have not been fully explored. Objective:: Novel mechanisms responsible for the discrepant activities between AS heads and tails were explored by a combined strategy of transcriptomes and metabolomics. Method:: Six pairs of the heads and tails of AS roots were collected in Min County, China. Total RNA and metabolites, which were used for RNA-seq and untargeted metabolomics analysis, were respectively isolated from each AS sample (0.1 g) by Trizol and methanol reagent. Subsequently, differentially expressed genes (DEGs) and discrepant pharmaceutical metabolites were identified for comparing AS heads and tails. Key DEGs and metabolites were quantified by qRT-PCR and targeted metabolomics experiment. Results:: Comprehensive analysis of transcriptomes and metabolomics results suggested that five KEGG pathways with significant differences included 57 DEGs. Especially, fourteen DEGs and six key metabolites were relation to the metabolic regulation of Phenylpropanoid biosynthesis (PB) pathway. Results of qRT-PCR and targeted metabolomics indicated that higher levels of expression of crucial genes in PB pathway, such as PAL, CAD, COMT and peroxidase in the tail of AS were positively correlated with levels of ferulic acid-related metabolites. The average content of ferulic acid in tails (569.58162.39 nmol/g) was higher than those in the heads (168.73  67.30 nmol/g) (P˂0.01); Caffeic acid in tails (3.82  0.88 nmol/g) vs heads (1.37  0.41 nmol/g) (P˂0.01), and Cinnamic acid in tails (0.24  0.09 nmol/g) vs heads (0.14  0.02 nmol/g) (P˂0.05). Conclusion:: Our work demonstrated that overexpressed genes and accumulated metabolites derived from PB pathway might be responsible for the discrepant pharmaceutical efficacies between AS heads and tails.

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MiR-493 Induces Cytotoxic Autophagy in Prostate Cancer Cells through Regulation on PHLPP2

Current Pharmaceutical Biotechnology ◽

10.2174/1389201021666200318120733 ◽

2020 ◽

Vol 21 (14) ◽

pp. 1451-1456 ◽

Cited By ~ 2

Author(s):

Jun Deng ◽

Ming Ma ◽

Wei Jiang ◽

Liangliang Zheng ◽

Suping Cui

Keyword(s):

Prostate Cancer ◽

Protein Expression ◽

Cell Function ◽

Mrna Levels ◽

Prostate Cancer Cells ◽

Potent Inducer ◽

Qrt Pcr ◽

Autophagy Gene ◽

The Relationship ◽

Cancer Inhibition

Background: MiR-493 promotes the proliferation of prostate cancer (PC) cells by targeting PHLPP2. We aimed to explore the relationship between miR-493 and autophagy in PC. Methods: qRT-PCR and western blotting were used to determine the mRNA levels and protein expression of miR-493, PHLPP2, autophagy gene BECN1 and ATG7 in PC cells. The autophagy gene expression was determined after PC cells transfected with miR-493 precursor or PHLPP2 precursor. Corresponding changes of autophagy phenotype and PC cell function were also studied. Results: The mRNA levels and protein expression of miR-493, PHLPP2, BECN1 and ATG7 in PC cells were significantly decreased in PC cells. Overexpression of miR-493 or PHLPP2 markedly upregulated the expression levels of BECN1 and ATG7 in PC cells. Overexpression of miR-493 and PHLPP2 markedly promoted autophagy, and inhibited the invasion and cloning formation of PC cells. Conclusion: MiR-493 is a potent inducer of cytotoxic autophagy that leads to prostate cancer inhibition by regulating on PHLPP2.

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Overexpression of Pygo2 Increases Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Cardiomyocyte-like Cells

Current Molecular Medicine ◽

10.2174/1566524019666191017150416 ◽

2020 ◽

Vol 20 (4) ◽

pp. 318-324 ◽

Cited By ~ 3

Author(s):

Lei Yang ◽

Shuoji Zhu ◽

Yongqing Li ◽

Jian Zhuang ◽

Jimei Chen ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Umbilical Cord ◽

Heart Function ◽

Critical Role ◽

Human Umbilical Cord ◽

Stem Cell Markers ◽

Quantitative Real Time Pcr ◽

Qrt Pcr

Background: Our previous studies have shown that Pygo (Pygopus) in Drosophila plays a critical role in adult heart function that is likely conserved in mammals. However, its role in the differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) into cardiomyocytes remains unknown. Objective: To investigate the role of pygo2 in the differentiation of hUC-MSCs into cardiomyocytes. Methods: Third passage hUC-MSCs were divided into two groups: a p+ group infected with the GV492-pygo2 virus and a p− group infected with the GV492 virus. After infection and 3 or 21 days of incubation, Quantitative real-time PCR (qRT-PCR) was performed to detect pluripotency markers, including OCT-4 and SOX2. Nkx2.5, Gata-4 and cTnT were detected by immunofluorescence at 7, 14 and 21 days post-infection, respectively. Expression of cardiac-related genes—including Nkx2.5, Gata-4, TNNT2, MEF2c, ISL-1, FOXH1, KDR, αMHC and α-Actin—were analyzed by qRT-PCR following transfection with the virus at one, two and three weeks. Results : After three days of incubation, there were no significant changes in the expression of the pluripotency stem cell markers OCT-4 and SOX2 in the p+ group hUC-MSCs relative to controls (OCT-4: 1.03 ± 0.096 VS 1, P > 0.05, SOX2: 1.071 ± 0.189 VS 1, P > 0.05); however, after 21 days, significant decreases were observed (OCT-4: 0.164 ± 0.098 VS 1, P < 0.01, SOX2: 0.209 ± 0.109 VS 1, P < 0.001). Seven days following incubation, expression of mesoderm specialisation markers, such as Nkx2.5, Gata-4, MEF2c and KDR, were increased; at 14 days following incubation, expression of cardiac genes, such as Nkx2.5, Gata-4, TNNT2, MEF2c, ISL-1, FOXH1, KDR, αMHC and α-Actin, were significantly upregulated in the p+ group relative to the p− group (P < 0.05). Taken together, these findings suggest that overexpression of pygo2 results in more hUCMSCs gradually differentiating into cardiomyocyte-like cells. Conclusion: We are the first to show that overexpression of pygo2 significantly enhances the expression of cardiac-genic genes, including Nkx2.5 and Gata-4, and promotes the differentiation of hUC-MSCs into cardiomyocyte-like cells.

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lncRNAs as Potential Targets in Small Cell Lung Cancer: MYC -dependent Regulation

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200721130700 ◽

2020 ◽

Vol 20 (17) ◽

pp. 2074-2081

Author(s):

Onur Tokgun ◽

Pervin E. Tokgun ◽

Kubilay Inci ◽

Hakan Akca

Keyword(s):

Lung Cancer ◽

Stem Cell ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Therapeutic Strategy ◽

Small Cell ◽

Small Cell Lung ◽

Expression Levels ◽

Qrt Pcr ◽

Shrna Vector

Background: Small Cell Lung Cancer (SCLC) is a highly aggressive malignancy. MYC family oncogenes are amplified and overexpressed in 20% of SCLCs, showing that MYC oncogenes and MYC regulated genes are strong candidates as therapeutic targets for SCLC. c-MYC plays a fundamental role in cancer stem cell properties and malignant transformation. Several targets have been identified by the activation/repression of MYC. Deregulated expression levels of lncRNAs have also been observed in many cancers. Objective: The aim of the present study is to investigate the lncRNA profiles which depend on MYC expression levels in SCLC. Methods: Firstly, we constructed lentiviral vectors for MYC overexpression/inhibition. MYC expression is suppressed by lentiviral shRNA vector in MYC amplified H82 and N417 cells, and overexpressed by lentiviral inducible overexpression vector in MYC non-amplified H345 cells. LncRNA cDNA is transcribed from total RNA samples, and 91 lncRNAs are evaluated by qRT-PCR. Results: We observed that N417, H82 and H345 cells require MYC for their growth. Besides, MYC is not only found to regulate the expressions of genes related to invasion, stem cell properties, apoptosis and cell cycle (p21, Bcl2, cyclinD1, Sox2, Aldh1a1, and N-Cadherin), but also found to regulate lncRNAs. With this respect, expressions of AK23948, ANRIL, E2F4AS, GAS5, MEG3, H19, L1PA16, SFMBT2, ZEB2NAT, HOTAIR, Sox2OT, PVT1, and BC200 were observed to be in parallel with MYC expression, whereas expressions of Malat1, PTENP1, Neat1, UCA1, SNHG3, and SNHG6 were inversely correlated. Conclusion: Targeting MYC-regulated genes as a therapeutic strategy can be important for SCLC therapy. This study indicated the importance of identifying MYC-regulated lncRNAs and that these can be utilized to develop a therapeutic strategy for SCLC.

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Abnormal expression of rno_circRNA_014900 and rno_circRNA_005442 induced by ketamine in the rat hippocampus

BMC Psychiatry ◽

10.1186/s12888-019-2374-2 ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 2

Author(s):

Jing Mao ◽

Tianmei Li ◽

Di Fan ◽

Hongli Zhou ◽

Jianguo Feng ◽

...

Keyword(s):

Target Genes ◽

Circular Rna ◽

Fold Change ◽

Rat Hippocampus ◽

Antidepressant Effect ◽

Signal Pathways ◽

Qrt Pcr ◽

Abnormal Expression ◽

Reverse Transcription Pcr ◽

Pathway Analyses

Abstract Background Recent studies have shown that circular RNA (circRNA) is rich in microRNA (miRNA) binding sites. We have previously demonstrated that the antidepressant effect of ketamine is related to the abnormal expression of various miRNAs in the brain. This study determined the expression profile of circRNAs in the hippocampus of rats treated with ketamine. Methods The aberrantly expressed circRNAs in rat hippocampus after ketamine injection were analyzed by microarray chip, and we further validated these circRNAs by quantitative reverse-transcription PCR (qRT-PCR). The target genes of the different circRNAs were predicted using bioinformatic analyses, and the functions and signal pathways of these target genes were investigated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Results Microarray analysis showed that five circRNAs were aberrantly expressed in rat hippocampus after ketamine injection (fold change > 2.0, p < 0.05). The results from the qRT-PCR showed that one of the circRNAs was significantly increased (rno_circRNA_014900; fold change = 2.37; p = 0.03), while one was significantly reduced (rno_circRNA_005442; fold change = 0.37; p = 0.01). We discovered a significant enrichment in several GO terms and pathways associated with depression. Conclusion Our findings showed the abnormal expression of ketamine-induced hippocampal circRNAs in rats.

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Detection of three pandemic causing coronaviruses from non-respiratory samples: systematic review and meta-analysis

Scientific Reports ◽

10.1038/s41598-021-95329-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Chandan Mishra ◽

Suneeta Meena ◽

Jitendra Kumar Meena ◽

Suman Tiwari ◽

Purva Mathur

Keyword(s):

Systematic Review ◽

Detection Rate ◽

Meta Analysis ◽

Total Sample ◽

Urine Samples ◽

Pathogenic Potential ◽

Clinical Specimens ◽

Qrt Pcr ◽

Corona Virus ◽

Positive Rate

AbstractSARS-CoV-2 has posed an unprecedented challenge to the world. Pandemics have been caused previously by viruses of this family like Middle East Respiratory Corona Virus (MERS CoV), Severe Acute Respiratory Syndrome Corona Virus (SARS CoV). Although these viruses are primarily respiratory viruses, but they have been isolated from non-respiratory samples as well. Presently, the detection rate of SARS‐CoV‐2 RNA from different clinical specimens using Real Time Reverse Transcriptase Polymerized Chain Reaction (qRT‐PCR) after onset of symptoms is not yet well established. Therefore, the aim of this systematic review was to establish the profile of detecting SARS‐CoV‐2, MERS CoV, SARS CoV from different types of clinical specimens other than the respiratory using a standard diagnostic test (qRT‐PCR). A total of 3429 non-respiratory specimens were recorded: SARS CoV (total sample—802), MERS CoV (total sample—155), SARS CoV-2 (total sample—2347). Out of all the samples studied high positive rate was seen for saliva with 96.7% (14/14; 95% CI 87.6–100.0%) for SARS CoV and 57.5% (58/250; 95% CI − 1.2 to 116.2%) for SARS CoV-2, while low detection rate in urine samples for SARS CoV-2 with 2.2% (8/318; 95% CI 0.6–3.7%) and 9.6% (12/61; 95% CI − 0.9 to 20.1%) for SARS CoV but there was relatively higher positivity in urine samples for MERS CoV with detection rate of 32.4% (2/38; 95% CI − 37.3 to 102.1%). In Stool sample positivity was 54.9% (396/779; 95% CI 41.0–68.8%), 45.2% (180/430; 95% CI 28.1–62.3%) and 34.7% (4/38; 95% CI − 29.5 to 98.9%) for SARS CoV-2, MERS CoV, and SARS CoV, respectively. In blood sample the positivity was 33.3% (7/21; 95% CI 13.2–53.5%), 23.7% (42/277; 95% CI 10.5–36.9%) and 2.5% (2/81; 95% CI 0.00–5.8%) for MERS CoV, SARS CoV-2 and SARS CoV respectively. SARS‐CoV‐2 along with previous two pandemic causing viruses from this family, were highly detected stool and saliva. A low positive rate was recorded in blood samples. Viruses were also detected in fluids along with unusual samples like semen and vaginal secretions thus highlighting unique pathogenic potential of SARS‐CoV‐2.

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Comparative transcriptomic and metabolomic analyses of carotenoid biosynthesis reveal the basis of white petal color in Brassica napus

Planta ◽

10.1007/s00425-020-03536-6 ◽

2021 ◽

Vol 253 (1) ◽

Author(s):

Ledong Jia ◽

Junsheng Wang ◽

Rui Wang ◽

Mouzheng Duan ◽

Cailin Qiao ◽

...

Keyword(s):

Brassica Napus ◽

Molecular Mechanisms ◽

Flower Color ◽

Carotenoid Biosynthesis ◽

Differentially Expressed ◽

Transcriptome Data ◽

Oilseed Crops ◽

Qrt Pcr ◽

Carotenoid Metabolism ◽

Rapeseed Cultivar

Abstract Main conclusion The molecular mechanism underlying white petal color in Brassica napus was revealed by transcriptomic and metabolomic analyses. Abstract Rapeseed (Brassica napus L.) is one of the most important oilseed crops worldwide, but the mechanisms underlying flower color in this crop are known less. Here, we performed metabolomic and transcriptomic analyses of the yellow-flowered rapeseed cultivar ‘Zhongshuang 11’ (ZS11) and the white-flowered inbred line ‘White Petal’ (WP). The total carotenoid contents were 1.778-fold and 1.969-fold higher in ZS11 vs. WP petals at stages S2 and S4, respectively. Our findings suggest that white petal color in WP flowers is primarily due to decreased lutein and zeaxanthin contents. Transcriptome analysis revealed 10,116 differentially expressed genes with a fourfold or greater change in expression (P-value less than 0.001) in WP vs. ZS11 petals, including 1,209 genes that were differentially expressed at four different stages and 20 genes in the carotenoid metabolism pathway. BnNCED4b, encoding a protein involved in carotenoid degradation, was expressed at abnormally high levels in WP petals, suggesting it might play a key role in white petal formation. The results of qRT-PCR were consistent with the transcriptome data. The results of this study provide important insights into the molecular mechanisms of the carotenoid metabolic pathway in rapeseed petals, and the candidate genes identified in this study provide a resource for the creation of new B. napus germplasms with different petal colors.

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SARS-CoV-2 Persistent Viral Shedding in the Context of Hydroxychloroquine-Azithromycin Treatment

Viruses ◽

10.3390/v13050890 ◽

2021 ◽

Vol 13 (5) ◽

pp. 890

Author(s):

Michel Drancourt ◽

Sébastien Cortaredona ◽

Cléa Melenotte ◽

Sophie Amrane ◽

Carole Eldin ◽

...

Keyword(s):

Intensive Care Unit ◽

Intensive Care ◽

Elderly Patients ◽

Rare Event ◽

Viral Shedding ◽

Qrt Pcr ◽

Viral Culture ◽

D Dimer

SARS-CoV-2 nasopharyngeal shedding contributes to the spread of the COVID-19 epidemic. Among 3271 COVID-19 patients treated at the Hospital University Institute Méditerranée Infection, Marseille, France from 3 March to 27 April 2020, tested at least twice by qRT-PCR, the median SARS-CoV-2 nasopharyngeal shedding duration was 6 days (range 2–54 days). Compared with short shedders (qRT-PCR positivity < 10 days), 34 (1.04%) persistent shedders (qRT-PCR positivity ≥ 17 days; mean ± SD: 23.3 ± 3.8 days) were significantly older, with associated comorbidities, exhibiting lymphopenia, eosinopenia, increased D-dimer and increased troponin (p < 0.05), and were hospitalized in intensive care unit in 17.7% vs. 1.1% of cases (p < 0.0001). Viral culture was positive in six persistent shedders after day 10, including in one patient after day 17, and no viral co-pathogen was detected in 33 tested patients. Persistent shedders received azithromycin plus hydroxychloroquine ≥ 3 days in 26/34 (76.5%) patients, a figure significantly lower than in short shedders (86.6%) (p = 0.042). Accordingly, mortality was 14.7% vs. 0.5% (p < 0.0001). Persistent shedding was significantly associated with persistent dyspnea and anosmia/ageusia (p < 0.05). In the context of COVID-19 treatment, including treatment with azithromycin plus hydroxychloroquine, the persistence of SARS-CoV-2 nasopharyngeal shedding was a rare event, most frequently encountered in elderly patients with comorbidities and lacking azithromycin plus hydroxychloroquine treatment.

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POS0393 FIBRIN DEPOSITION IS AN ACTIVE TRIGGER OF CARTILAGE DEGENERATION IN RHEUMATOID ARTHRITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2521 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 426.1-426

Author(s):

T. Hügle ◽

S. Nasi ◽

D. Ehirchiou ◽

P. Omoumi ◽

A. So ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Cartilage Damage ◽

Cartilage Degeneration ◽

Cartilage Explants ◽

Fibrin Deposition ◽

Qrt Pcr ◽

Catabolic Genes ◽

Key Factor ◽

Calcific Deposits

Background:Fibrin(ogen) maintains inflammation in various disorders but has never been linked to cartilage damage in rheumatoid arthritis (RA) or other forms of inflammatory arthritis.Objectives:To investigate the role of fibrin deposition on cartilage integrity in arthritis.Methods:Fibrin deposition on knee cartilage was analyzed by immunohistochemistry in RA patients and in murine adjuvant-induced arthritis (AIA). In chondrocytes, fibrinogen expression (Fgα, Fgβ, Fgγ) and procoagulant activity were evaluated by qRT-PCR and turbidimetry respectively. Fibrin-induced catabolic genes were assessed by qRT-PCR in chondrocytes. Fibrin-mediated chondro-synovial adhesion (CSA) with subsequent cartilage tears was studied in co-cultures of human RA cartilage with autologous synoviocytes, in the AIA model, and by MRI. The link between fibrin and calcification was examined in human RA cartilage stained for calcific deposits and in vitro in fibrinogen-stimulated chondrocytes.Results:Fibrin deposition on cartilage correlated with the severity of cartilage damage in human RA explants and in AIA wildtype (WT) mice, while fibrinogen deficient (Fg-/-) mice were protected. Accordingly, fibrin upregulated catabolic enzymes (Adamts5 and Mmp13) in chondrocytes. Secondly, CSA was present in fibrin-rich and damaged cartilage in AIA WT but not in Fg-/- mice. In line, autologous human synoviocytes, cultured on RA cartilage explants, adhered exclusively to fibrin-positive degraded areas. Gadolinium-enhanced MRI of human joints showed contrast-enhancement along cartilage surface in RA patients but not in controls. Finally, fibrin co-localized with calcification in human RA cartilage and triggered chondrocyte mineralization inducing pro-calcification genes (Anx5, Pit1, Pc1) and cytokine (IL-6). Although at a much lesser extent, we observed similar fibrin-mediated mechanisms in osteoarthritis (OA).Conclusion:Fibrin deposition directly impacts on cartilage integrity via induction of catabolism, mechanical stress, and calcification. Potentially, fibrin is a key factor of cartilage damage occurring in RA as a secondary consequence of inflammation.Disclosure of Interests:None declared

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