Optimizing a reliable ex vivo human blood model to analyze expression of Staphylococcus epidermidis genes

Human blood is often used as an ex vivo model to mimic the environment encountered by pathogens inside the host. A significant variety of experimental conditions has been reported. However, optimization strategies are often not described. This study aimed to evaluate key parameters that are expected to influence Staphylococcus epidermidis gene expression when using human blood ex vivo models. Our data confirmed that blood antimicrobial activity was dependent on initial bacterial concentration. Furthermore, blood degradation over time resulted in lower antimicrobial activity, with a 2% loss of leukocytes viability correlating with a 5-fold loss of antimicrobial activity against S. epidermidis. We further demonstrated that the volume of human blood could be reduced to as little as 0.18 mL without affecting the stability of gene expression of the tested genes. Overall, the data described herein highlight experimental parameters that should be considered when using a human blood ex vivo model for S. epidermidis gene expression analysis.

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Anti-Inflammatory Effects of Novel Standardized Platelet Rich Plasma Releasates on Knee Osteoarthritic Chondrocytes and Cartilage in vitro

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190619111118 ◽

2019 ◽

Vol 20 (11) ◽

pp. 920-933 ◽

Cited By ~ 3

Author(s):

Lucía Gato-Calvo ◽

Tamara Hermida-Gómez ◽

Cristina R. Romero ◽

Elena F. Burguera ◽

Francisco J. Blanco

Keyword(s):

Gene Expression ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Cartilage Explants ◽

Ex Vivo Model ◽

Chondrocyte Viability ◽

Platelet Concentration ◽

The Absolute ◽

Anti Inflammatory

Background: Platelet Rich Plasma (PRP) has recently emerged as a potential treatment for osteoarthritis (OA), but composition heterogeneity hampers comparison among studies, with the result that definite conclusions on its efficacy have not been reached. Objective: 1) To develop a novel methodology to prepare a series of standardized PRP releasates (PRP-Rs) with known absolute platelet concentrations, and 2) To evaluate the influence of this standardization parameter on the anti-inflammatory properties of these PRP-Rs in an in vitro and an ex vivo model of OA. Methods: A series of PRPs was prepared using the absolute platelet concentration as the standardization parameter. Doses of platelets ranged from 0% (platelet poor plasma, PPP) to 1.5·105 platelets/µl. PRPs were then activated with CaCl2 to obtain releasates (PRP-R). Chondrocytes were stimulated with 10% of each PRP-R in serum-free culture medium for 72 h to assess proliferation and viability. Cells were co-stimulated with interleukin (IL)-1β (5 ng/ml) and 10% of each PRP-R for 48 h to determine the effects on gene expression, secretion and intra-cellular content of common markers associated with inflammation, catabolism and oxidative stress in OA. OA cartilage explants were co-stimulated with IL-1β (5 ng/ml) and 10% of either PRP-R with 0.75·105 platelets/µl or PRP-R with 1.5·105 platelets/µl for 21 days to assess matrix inflammatory degradation. Results: Chondrocyte viability was not affected, and proliferation was dose-dependently increased. The gene expression of all pro-inflammatory mediators was significantly and dose-independently reduced, except for that of IL-1β and IL-8. Immunoblotting corroborated this effect for inducible NO synthase (NOS2). Secreted matrix metalloproteinase-13 (MMP-13) was reduced to almost basal levels by the PRP-R from PPP. Increasing platelet dosage led to progressive loss to this anti-catabolic ability. Safranin O and toluidine blue stains supported the beneficial effect of low platelet dosage on cartilage matrix preservation. Conclusion: We have developed a methodology to prepare PRP releasates using the absolute platelet concentration as the standardization parameter. Using this approach, the composition of the resulting PRP derived product is independent of the donor initial basal platelet count, thereby allowing the evaluation of its effects objectively and reproducibly. In our OA models, PRP-Rs showed antiinflammatory, anti-oxidant and anti-catabolic properties. Platelet enrichment could favor chondrocyte proliferation but is not necessary for the above effects and could even be counter-productive.

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Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

Genes ◽

10.3390/genes12070960 ◽

2021 ◽

Vol 12 (7) ◽

pp. 960

Author(s):

Meagan Archer ◽

Jianping Xu

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Quantitative Polymerase Chain Reaction ◽

Specific Method ◽

Experimental Conditions ◽

Reference Gene Selection ◽

Physiological Processes ◽

The Stability

Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.

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Validation of Internal Control Genes for Quantitative Real-Time PCR Gene Expression Analysis in Morchella

Molecules ◽

10.3390/molecules23092331 ◽

2018 ◽

Vol 23 (9) ◽

pp. 2331 ◽

Cited By ~ 10

Author(s):

Qianqian Zhang ◽

Wei Liu ◽

Yingli Cai ◽

A-Feng Lan ◽

Yinbing Bian

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Expression Analysis ◽

Internal Control ◽

Reference Genes ◽

Gene Expression Analysis ◽

Stable Gene ◽

Experimental Conditions ◽

The Stability ◽

Vacuolar Protein

The reliability of qRT-PCR results depend on the stability of reference genes used for normalization, suggesting the necessity of identification of reference genes before gene expression analysis. Morels are edible mushrooms well-known across the world and highly prized by many culinary kitchens. Here, several candidate genes were selected and designed according to the Morchella importuna transcriptome data. The stability of the candidate genes was evaluated with geNorm and NormFinder under three different experimental conditions, and several genes with excellent stability were selected. The extensive adaptability of the selected genes was tested in ten Morchella species. Results from the three experimental conditions revealed that ACT1 and INTF7 were the most prominent genes in Morchella, CYC3 was the most stable gene in different development stages, INTF4/AEF3 were the top-ranked genes across carbon sources, while INTF3/CYC3 pair showed the robust stability for temperature stress treatment. We suggest using ACT1, AEF3, CYC3, INTF3, INTF4 and INTF7 as reference genes for gene expression analysis studies for any of the 10 Morchella strains tested in this study. The stability and practicality of the gene, vacuolar protein sorting (INTF3), vacuolar ATP synthase (INTF4) and14-3-3 protein (INTF7) involving the basic biological processes were validated for the first time as the candidate reference genes for quantitative PCR. Furthermore, the stability of the reference genes was found to vary under the three different experimental conditions, indicating the importance of identifying specific reference genes for particular conditions.

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Evaluation of endogenous control gene(s) for gene expression studies in human blood exposed to 60 Co γ-rays ex vivo

Journal of Radiation Research ◽

10.1093/jrr/rru074 ◽

2014 ◽

Vol 56 (1) ◽

pp. 177-185 ◽

Cited By ~ 9

Author(s):

S. Thangminlal Vaiphei ◽

Joshua Keppen ◽

Saibadaiahun Nongrum ◽

R.C. Chaubey ◽

L. Kma ◽

...

Keyword(s):

Gene Expression ◽

Human Blood ◽

Ex Vivo ◽

Endogenous Control ◽

Control Gene ◽

Endogenous Control Gene ◽

Expression Studies ◽

Γ Rays ◽

Gene Expression Studies

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Unravelling the role of Mesenchymal Stem/Stromal Cells Secretome in intervertebral disc degeneration in a pro-inflammatory/degenerative ex vivo model

10.21203/rs.2.22688/v1 ◽

2020 ◽

Author(s):

JR Ferreira ◽

GQ Teixeira ◽

E Neto ◽

C Ribeiro-Machado ◽

AM Silva ◽

...

Keyword(s):

Gene Expression ◽

Intervertebral Disc ◽

Stromal Cells ◽

Ex Vivo ◽

Low Oxygen ◽

Ex Vivo Model ◽

A Cell ◽

New Perspective ◽

Regulated Gene Expression ◽

Ivd Degeneration

Abstract Background: Mesenchymal stem/stromal cells (MSCs) have been increasingly used in clinical trials for intervertebral disc (IVD) degeneration. Here, we aimed to evaluate the potential of a cell-free approach to degenerated IVD, testing if MSCs secretome can stimulate a regenerative response by modulating the IVD inflammatory cascade. Methods: Human bone marrow-derived MSCs were pre-conditioned with IL-1β (10 ng/mL) and low oxygen (6% O2). The secretome of MSCs (MSCsec) was collected after 48h. Bovine IVD tissue explants cultured in pro-inflammatory/degenerative conditions (needle puncture + IL-1β) were treated with MSCsec or co-cultured with MSCs. Results: MSCsec obtained upon IL-1β-pre-conditioning, as well as MSCs co-culture, down-regulated gene expression of pro-inflammatory cytokines, bIL-6 and bIL-8 after 48h, in IVD. IVD matrix degrading enzymes, bMMP1 and bMMP3, were downregulated and upregulated, respectively, in the presence of MSCsec, but not MSCs. After 14 days, MSCsec-treated IVDs revealed increased aggrecan content at the protein level, contrarily to MSCs/IVD co-cultures. Interestingly, IL-1β-preconditioning only, but not IL-1β-IVD, increased gene expression of hADAMTS5 and hTIMP-1in MSCs. Additionally, conditioned medium from MSCsec-treated IVDs did not promote angiogenesis or neurogenesis. In MSCsec-treated IVD, an increase in MCP-3 and GCP-2 was observed, while SDF-1α, TNF-α, IGF-1, Eotaxin 3, FGF-9, MIP-1δ, IFN-γ, IL-5, TNF-β, IL-4, TGF-β1, IL-16, IGFBP-3 and IGFBP-4 were decreased, compared with MSCs/IVD co-cultures. Conclusions: MSCsec obtained upon IL1β-preconditioning, present an immunomodulatory role in degenerated IVD, as well as MSCs. Nevertheless, MSCsec but not MSCs, potentiate aggrecan deposition in IVD in pro-inflammatory/degenerative conditions. This finding can open new perspective on the use of MSCsec as a cell-based/cell-free approach to LBP.

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Identification and Validation of Reference Genes for Gene Expression Analysis in Different Development Stages of Amylostereum areolatum

Frontiers in Microbiology ◽

10.3389/fmicb.2021.827241 ◽

2022 ◽

Vol 12 ◽

Author(s):

Ningning Fu ◽

Jiaxing Li ◽

Ming Wang ◽

Lili Ren ◽

Shixiang Zong ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Growth And Development ◽

Target Genes ◽

Quantitative Polymerase Chain Reaction ◽

Growth Stages ◽

Experimental Conditions ◽

Development Stages ◽

Symbiotic Fungus ◽

The Stability

A strict relationship exists between the Sirex noctilio and the Amylostereum areolatum, which is carried and spread by its partner. The growth and development of this symbiotic fungus is key to complete the life history of the Sirex woodwasp. Real-time quantitative polymerase chain reaction (RT-qPCR) is used to measure gene expression in samples of A. areolatum at different growth stages and explore the key genes and pathways involved in the growth and development of this symbiotic fungus. To obtain accurate RT-qPCR data, target genes need to be normalized by reference genes that are stably expressed under specific experimental conditions. In our study, the stability of 10 candidate reference genes in symbiotic fungal samples at different growth and development stages was evaluated using geNorm, NormFinder, BestKeeper, delta Ct methods, and RefFinder. Meanwhile, laccase1 was used to validate the stability of the selected reference gene. Under the experimental conditions of this study, p450, CYP, and γ-TUB were identified as suitable reference genes. This work is the first to systematically evaluate the reference genes for RT-qPCR results normalization during the growth of this symbiotic fungus, which lays a foundation for further gene expression experiments and understanding the symbiotic relationship and mechanism between S. noctilio and A. areolatum.

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Interleukin-6 actions in the hypothalamus protects against obesity and is involved in the regulation of neurogenesis

Journal of Neuroinflammation ◽

10.1186/s12974-021-02242-8 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Vanessa C. Bobbo ◽

Daiane F. Engel ◽

Carlos Poblete Jara ◽

Natalia F. Mendes ◽

Roberta Haddad-Tovolli ◽

...

Keyword(s):

Gene Expression ◽

Interleukin 6 ◽

Ex Vivo ◽

Cell Types ◽

Cell Labeling ◽

Specific Cell ◽

Related Gene ◽

Sequencing Data ◽

Ex Vivo Model ◽

Diet Induced Obesity

Abstract Background Interleukin-6 (IL6) produced in the context of exercise acts in the hypothalamus reducing obesity-associated inflammation and restoring the control of food intake and energy expenditure. In the hippocampus, some of the beneficial actions of IL6 are attributed to its neurogenesis-inducing properties. However, in the hypothalamus, the putative neurogenic actions of IL6 have never been explored, and its potential to balance energy intake can be an approach to prevent or attenuate obesity. Methods Wild-type (WT) and IL6 knockout (KO) mice were employed to study the capacity of IL6 to induce neurogenesis. We used cell labeling with Bromodeoxyuridine (BrdU), immunofluorescence, and real-time PCR to determine the expression of markers of neurogenesis and neurotransmitters. We prepared hypothalamic neuroprogenitor cells from KO that were treated with IL6 in order to provide an ex vivo model to further characterizing the neurogenic actions of IL6 through differentiation assays. In addition, we analyzed single-cell RNA sequencing data and determined the expression of IL6 and IL6 receptor in specific cell types of the murine hypothalamus. Results IL6 expression in the hypothalamus is low and restricted to microglia and tanycytes, whereas IL6 receptor is expressed in microglia, ependymocytes, endothelial cells, and astrocytes. Exogenous IL6 reduces diet-induced obesity. In outbred mice, obesity-resistance is accompanied by increased expression of IL6 in the hypothalamus. IL6 induces neurogenesis-related gene expression in the hypothalamus and in neuroprogenitor cells, both from WT as well as from KO mice. Conclusion IL6 induces neurogenesis-related gene expression in the hypothalamus of WT mice. In KO mice, the neurogenic actions of IL6 are preserved; however, the appearance of new fully differentiated proopiomelanocortin (POMC) and neuropeptide Y (NPY) neurons is either delayed or disturbed.

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