Selection and validation of reference genes for gene expression studies in Pseudomonas brassicacearum GS20 using real-time quantitative reverse transcription PCR

Bai, Bianxia; Ren, Jia-Lin; Bai, Fenling; Lin, Hao

doi:10.1371/journal.pone.0227927

Cited by 23 publications

(18 citation statements)

References 40 publications

(35 reference statements)

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“…Identical observations were demonstrated by DeLorenzo and Moon 41 . Unfortunately, according to the literature, there were some discrepancies in the results obtained by BestKeeper, geNorm and NormFinder software 36,39,40 . It should be emphasized that such calculation differences may be a result of these three programs being based on different algorithms.…”

Section: Discussionmentioning

confidence: 99%

“…proved that the 16S rRNA-coding gene was characterized by poor expression stability in Ochrobactrum quorumnocens (Cp value = 10.26) and was one of the most unstable candidate reference genes under 10 different tested culture conditions 39 . The 16S rRNA gene has also been proven unsuitable for the analysis of iron-regulated gene expression in Pseudomonas brassicacearum (it ranked sixth of the eight genes tested) 40 . On the other hand, based on mathematical models (BestKeeper, geNorm and NormFinder software), 16S rRNA was classified as one of the best reference genes to evaluate expression levels in Rhodococcus opacus under different growth conditions 41 .…”

Section: Discussionmentioning

confidence: 99%

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Validation of stable reference genes in Staphylococcus aureus to study gene expression under photodynamic treatment: a case study of SEB virulence factor analysis

Ogonowska

Nakonieczna

2020

Sci Rep

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Staphylococcal enterotoxin B (SEB), encoded by the seb gene, is a virulence factor produced by Staphylococcus aureus that is involved mainly in food poisoning and is known to act as an aggravating factor in patients with atopic dermatitis. Research results in animal infection models support the concept that superantigens, including SEB contribute to sepsis and skin and soft tissue infections. In contrast to antibiotics, antimicrobial photodynamic inactivation (aPDI) is a promising method to combat both bacterial cells and virulence factors. The main aims of this research were to (1) select the most stable reference genes under sublethal aPDI treatments and (2) evaluate the impact of aPDI on seb. Two aPDI combinations were applied under sublethal conditions: rose bengal (RB) and green light (λmax = 515 nm) and new methylene blue (NMB) and red light (λmax = 632 nm). The stability of ten candidate reference genes (16S rRNA, fabD, ftsZ, gmk, gyrB, proC, pyk, rho, rpoB and tpiA) was evaluated upon aPDI using four software packages—BestKeeper, geNorm, NormFinder and RefFinder. Statistical analyses ranked ftsZ and gmk (RB + green light) and ftsZ, proC, and fabD (NMB + red light) as the most stable reference genes upon photodynamic treatment. Our studies showed downregulation of seb under both aPDI conditions, suggesting that aPDI could decrease the level of virulence factors.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Validation of stable reference genes in Staphylococcus aureus to study gene expression under photodynamic treatment: a case study of SEB virulence factor analysis

Ogonowska

Nakonieczna

2020

Sci Rep

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“…The rat primer sequences of Col2a1, Aggrecan, Sox9, and GAPDH used were as follows: Col2a1: 5′-GGAGCAGCAAGAGCAAGGAGAAG-3′ (Forward) and 5′-GGAGCCCTCAGTGGACAGTAGAC-3′ (Reverse), Aggrecan: 5′-GCTACGACGCCATCTGCTACAC-3′ (Forward) and 5′- ATGTCCTCTTCACCACCCACTCC-3′ (Reverse), Sox9: 5′-TGGCAGAGGGTGGCAGACAG-3′ (Forward) and 5′-CGTTGGGCGGCAGGTATTGG-3′ (Reverse), and GAPDH: 5′- ATGGTGAAGGTCGGTGTGAA-3′ (Forward) and 5′-CACCACCCTGTTGCTGTAGC-3′ (Reverse). Relative amounts of mRNA were standardized and calculated as previously described [ 25 , 26 ].…”

Section: Methodsmentioning

confidence: 99%

Positive feedback regulation between USP15 and ERK2 inhibits osteoarthritis progression through TGF-β/SMAD2 signaling

et al. 2021

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Background The transforming growth factor-β (TGF-β) signaling pathway plays an essential role in maintaining homeostasis in joints affected by osteoarthritis (OA). However, the specific mechanism of non-SMAD and classical SMAD signaling interactions is still unclear, which needs to be further explored. Methods In ATDC5 cells, USP15 overexpression and knockout were performed using the transfected lentivirus USP15 and Crispr/Cas9. Western blotting and immunofluorescence staining were used to test p-SMAD2 and cartilage phenotype-related molecular markers. In rat OA models, immunohistochemistry, hematoxylin and eosin (HE)/Safranin-O fast green staining, and histology were used to examine the regulatory activity of USP15 in TGF-β/SMAD2 signaling and the cartilage phenotype. Then, ERK2 overexpression and knockout were performed. The expressions of USP15, p-SMAD2, and the cartilage phenotype were evaluated in vitro and in vivo. To address whether USP15 is required for ERK2 and TGF-β/SMAD2 signaling, we performed rescue experiments in vitro and in vivo. Immunoprecipitation and deubiquitination assays were used to examine whether USP15 could bind to ERK2 and affect the deubiquitination of ERK2. Finally, whether USP15 regulates the level of p-ERK1/2 was evaluated by western blotting, immunofluorescence staining, and immunohistochemistry in vitro and in vivo. Results Our results indicated that USP15 stimulated TGF-β/SMAD2 signaling and the cartilage phenotype. Moreover, ERK2 required USP15 to influence TGF-β/SMAD2 signaling for regulating the cartilage phenotype in vivo and in vitro. And USP15 can form a complex with ERK2 to regulate ubiquitination of ERK2. Interestingly, USP15 did not regulate the stability of ERK2 but increased the level of p-ERK1/2 to further enhance the TGF-β/SMAD2 signaling pathway. Conclusions Taken together, our study revealed positive feedback regulation between USP15 and ERK2, which played a critical role in TGF-β/SMAD2 signaling to inhibit OA progression. Therefore, this specific mechanism can guide the clinical treatment of OA.

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“…The mouse primer sequences of the Col2a1, Col10a1, Sox9, Runx2 and GAPDH used were as follows: Col2a1: 5'-TACTGGAGTGACTGGTCCTAAG-3' (Reverse). Relative amounts of mRNA were standardized and calculated as previously described (25,26) .…”

Section: Immunohistochemistry He/safranin-o Fast Green Staining and mentioning

confidence: 99%

Positive Feedback Regulation Between USP15 and ERK2 Promotes Cartilage Repair in Experimental Osteoarthritis

Wang¹,

Zhu²,

Sun³

et al. 2020

Preprint

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Background.The transforming growth factor-β (TGF-β) signaling pathway plays an essential role in maintaining homeostasis in joints affected by osteoarthritis (OA). Determine the specific relationship between non-SMAD and classical SMAD signaling pathways.Methods.First,we found from human cartilage tissue specimens positive expressions of TGF-β1 and USP15between normal andosteoarthritiscartilage tissueby immunohistochemical.Then, overexpressing and knocking out USP15 or ERK2 followed bythe latest gene editing technology Crispr/Cas9 supported the studyinvitro.And we established rat knee OAmodelsthrough anterior cruciate ligament transection in combination with partial medial meniscectomy (ACLT+pMMx)after 8 weeksand conducted the corresponding Adeno-associated virus (AAV) intraarticular injections. Next, experimental data including immunohistochemical staining,HE/Safranin-O fast green staining,histological evidence analysis,relevant Osteoarthritis Research Society International (OARSI) scoresand expressions of molecules in cartilage anabolismindicated that the relationship betweenERK2 and USP15 throughTGF-β signaling pathway in vivo.Finally, we observed the specific mechanismof USP15 on ERK2 throughco-immunoprecipitation,deubiquitination assay, immunofluorescence and nuclear plasma separation.Results.Here, we detected that ERK2 of non-SMAD signaling can maintain the cartilage phenotype by activating the TGF-β/Smad signaling pathway throughincreasing USP15 in vitro and in vivo. Most importantly, USP15 is required during this process and can form a complex with ERK2 to regulate ubiquitination of ERK2.Specifically,USP15, instead of USP15 C269S, deubiquitinates and activates ERK2, followed by translocation of phosphorylated ERK2 from the cytoplasm to the nucleus. Correspondingly, the level of phosphorylated ERK2 was increased by injecting AAV-mediated overexpressing USP15 into the rat OA model. Conclusion.Taken together, our study revealed a positive feedback regulation between USP15 and ERK2, which plays a critical role in the interaction of SMAD and non-SMAD signaling pathways to promote cartilage repair in experimental OA.

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Selection and validation of reference genes for gene expression studies in Pseudomonas brassicacearum GS20 using real-time quantitative reverse transcription PCR

Cited by 23 publications

References 40 publications

Validation of stable reference genes in Staphylococcus aureus to study gene expression under photodynamic treatment: a case study of SEB virulence factor analysis

Validation of stable reference genes in Staphylococcus aureus to study gene expression under photodynamic treatment: a case study of SEB virulence factor analysis

Positive feedback regulation between USP15 and ERK2 inhibits osteoarthritis progression through TGF-β/SMAD2 signaling

Positive Feedback Regulation Between USP15 and ERK2 Promotes Cartilage Repair in Experimental Osteoarthritis

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