Rearing medium dictates variability across replicates in untreated and arsenic challenged zebrafish larvae

ABSTRACTReproducibility and consistency are hallmarks of scientific integrity. Biological systems are inherently noisy, posing a challenge to reproducibility. This is particularly relevant to the field of environmental toxicology, where many unaccounted experimental parameters can have a marked influence on the biological response to exposure. Here, we extend the use of zebrafish as a robust toxicological model for studying the effects of inorganic arsenic (iAs) on liver biology. We observed that iAs toxicity in this system is not influenced by important parameters including genetic background, rearing container material or rearing volume but the dose response to iAs is influenced by the rearing medium. We compared mortality as a measure of iAs toxicity to embryos cultured in two standard rearing media: egg water made from dehydrated ocean salts dissolved in water and a defined embryo medium which is a pH adjusted, buffered salt solution. Larvae reared in egg water were more susceptible to iAs compared to those reared in embryo medium. This effect was independent of the pH differences between these solutions. These culture conditions did not cause any difference in the global hepatic transcriptome of control zebrafish. Further, no difference in the expression of genes involved in the unfolded protein response (UPR) in larvae exposed to iAs treatment or in a stress independent system to activate UPR genes by transgenic overexpression of activating transcription factor 6 (nAtf6) in hepatocytes was observed. However, the clutch-to-clutch variation in gene expression was significantly greater in larvae reared in egg water compared to those in embryo medium. These data demonstrate that egg water affects reproducibility across replicates in terms of gene expression and exacerbates iAs mediated toxic response. This highlights the importance of rigorous evaluation of experimental conditions to assure reproducibility.

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Transcription factor ATF4 directs basal and stress-induced gene expression in the unfolded protein response and cholesterol metabolism in the liver

Molecular Biology of the Cell ◽

10.1091/mbc.e16-01-0039 ◽

2016 ◽

Vol 27 (9) ◽

pp. 1536-1551 ◽

Cited By ~ 72

Author(s):

Michael E. Fusakio ◽

Jeffrey A. Willy ◽

Yongping Wang ◽

Emily T. Mirek ◽

Rana J. T. Al Baghdadi ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Transcription Factor ◽

Er Stress ◽

Unfolded Protein Response ◽

Cholesterol Metabolism ◽

Unfolded Protein ◽

Expression Of Genes ◽

The Unfolded Protein Response ◽

Protein Response

Disturbances in protein folding and membrane compositions in the endoplasmic reticulum (ER) elicit the unfolded protein response (UPR). Each of three UPR sensory proteins—PERK (PEK/EIF2AK3), IRE1, and ATF6—is activated by ER stress. PERK phosphorylation of eIF2 represses global protein synthesis, lowering influx of nascent polypeptides into the stressed ER, coincident with preferential translation of ATF4 (CREB2). In cultured cells, ATF4 induces transcriptional expression of genes directed by the PERK arm of the UPR, including genes involved in amino acid metabolism, resistance to oxidative stress, and the proapoptotic transcription factor CHOP (GADD153/DDIT3). In this study, we characterize whole-body and tissue-specific ATF4-knockout mice and show in liver exposed to ER stress that ATF4 is not required for CHOP expression, but instead ATF6 is a primary inducer. RNA-Seq analysis indicates that ATF4 is responsible for a small portion of the PERK-dependent UPR genes and reveals a requirement for expression of ATF4 for expression of genes involved in oxidative stress response basally and cholesterol metabolism both basally and under stress. Consistent with this pattern of gene expression, loss of ATF4 resulted in enhanced oxidative damage, and increased free cholesterol in liver under stress accompanied by lowered cholesterol in sera.

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A new set of reference genes for RT-qPCR assays in the yeast Dekkera bruxellensis

Canadian Journal of Microbiology ◽

10.1139/cjm-2012-0457 ◽

2012 ◽

Vol 58 (12) ◽

pp. 1362-1367 ◽

Cited By ~ 10

Author(s):

Will de Barros Pita ◽

Fernanda Cristina Bezerra Leite ◽

Anna Theresa de Souza Liberal ◽

Luciana Filgueira Pereira ◽

Marcelo Falsarella Carazzolle ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Expression Analysis ◽

Regulation Of Gene Expression ◽

Culture Conditions ◽

Mrna Levels ◽

Dekkera Bruxellensis ◽

Expression Of Genes ◽

Normalization Factors

The yeast Dekkera bruxellensis has been recently regarded as an important microorganism for bioethanol production owing to its ability to convert glucose, sucrose, and cellobiose to ethanol. The aim of this work was to validate a new set of reference genes for gene expression analysis by quantitative real-time PCR in D. bruxellensis and compare the influence of the method of choice for quantification of mRNA levels with the reliability of our data. Three candidate reference genes, DbEFA1, DbEFB1, and DbYNA1, were used in a quantitative analysis of 4 genes of interest, DbYNR1, DbTPS1, DbADH7, and DbUBA4, based on an approach for calculating the normalization factors by means of the geNorm applet. Each reference gene was also individually used for a [Formula: see text] (comparative Cq method) calculation of the relative expression of genes of interest. Our results showed that the 3 reference genes provided enough stability and were complementary to the normalization factors method in different culture conditions. This work was able to confirm the usefulness of a previously reported reference gene, EFA1/TEF1, and increased the set of possible reference genes in D. bruxellensis to 4. Moreover, this can improve the reliability of the analysis of the regulation of gene expression in the industrial yeast D. bruxellensis.

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Establishing Standard Protocols for Bacterial Culture in Biological Research in Canisters (BRIC) Hardware

Gravitational and Space Research ◽

10.2478/gsr-2016-0013 ◽

2020 ◽

Vol 4 (2) ◽

pp. 58-69 ◽

Cited By ~ 4

Author(s):

Patricia Fajardo-Cavazos ◽

Wayne L. Nicholson

Keyword(s):

Gene Expression ◽

Bacterial Culture ◽

Culture Conditions ◽

Media Culture ◽

Ground Control ◽

Biological Research ◽

Confounding Factors ◽

Cross Experiment ◽

Spaceflight Experiments ◽

Control Samples

AbstractThe NASA GeneLab Data System (GLDS) was recently developed to facilitate cross-experiment comparisons in order to understand the response of microorganisms to the human spaceflight environment. However, prior spaceflight experiments have been conducted using a wide variety of different hardware, media, culture conditions, and procedures. Such confounding factors could potentially mask true differences in gene expression between spaceflight and ground control samples. In an attempt to mitigate such confounding factors, we describe here the development of a standardized set of hardware, media, and protocols for liquid cultivation of microbes in Biological Research in Canisters (BRIC) spaceflight hardware, using the model bacteria Bacillus subtilis strain 168 and Staphylococcus aureus strain UAMS-1 as examples.

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Mechanical culture conditions effect gene expression

10.2514/6.2001-5016 ◽

2001 ◽

Author(s):

J. Love ◽

T. Hammond ◽

P. Allen ◽

L. Cubano ◽

T. Baker ◽

...

Keyword(s):

Gene Expression ◽

Culture Conditions ◽

Effect Gene

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Assessment of common housekeeping genes as reference for gene expression studies using RT-qPCR in mouse choroid plexus

Scientific Reports ◽

10.1038/s41598-021-82800-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kim Hoa Ho ◽

Annarita Patrizi

Keyword(s):

Gene Expression ◽

Choroid Plexus ◽

Sensory Deprivation ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Experimental Conditions ◽

Brain Repair ◽

Expression Studies ◽

Testing Algorithms ◽

Gene Expression Studies

AbstractChoroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

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Transcriptome comparisons of in vitro intestinal epithelia grown under static and microfluidic gut-on-chip conditions with in vivo human epithelia

Scientific Reports ◽

10.1038/s41598-021-82853-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kornphimol Kulthong ◽

Guido J. E. J. Hooiveld ◽

Loes Duivenvoorde ◽

Ignacio Miro Estruch ◽

Victor Marin ◽

...

Keyword(s):

Gene Expression ◽

Culture Conditions ◽

Gene Set Enrichment Analysis ◽

Shear Stresses ◽

Static Culture ◽

Dynamic Culture ◽

Intestinal Segments ◽

On Chip

AbstractGut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.

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Impact of Environmental Stressors on Gene Expression in the Embryo of the Italian Wall Lizard

Applied Sciences ◽

10.3390/app11114723 ◽

2021 ◽

Vol 11 (11) ◽

pp. 4723

Author(s):

Rosaria Scudiero ◽

Chiara Maria Motta ◽

Palma Simoniello

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Membrane Trafficking ◽

Translational Regulation ◽

Cellular Protection ◽

Terrestrial Vertebrate ◽

Expression Of Genes ◽

Stress Changes ◽

Probable Consequence ◽

The Impact

The cleidoic eggs of oviparous reptiles are protected from the external environment by membranes and a parchment shell permeable to water and dissolved molecules. As a consequence, not only physical but also chemical insults can reach the developing embryos, interfering with gene expression. This review provides information on the impact of the exposure to cadmium contamination or thermal stress on gene expression during the development of Italian wall lizards of the genus Podarcis. The results obtained by transcriptomic analysis, although not exhaustive, allowed to identify some stress-reactive genes and, consequently, the molecular pathways in which these genes are involved. Cadmium-responsive genes encode proteins involved in cellular protection, metabolism and proliferation, membrane trafficking, protein interactions, neuronal transmission and plasticity, immune response, and transcription regulatory factors. Cold stress changes the expression of genes involved in transcriptional/translational regulation and chromatin remodeling and inhibits the transcription of a histone methyltransferase with the probable consequence of modifying the epigenetic control of DNA. These findings provide transcriptome-level evidence of how terrestrial vertebrate embryos cope with stress, giving a key to use in population survival and environmental change studies. A better understanding of the genes contributing to stress tolerance in vertebrates would facilitate methodologies and applications aimed at improving resistance to unfavourable environments.

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Transversal gene expression panel to evaluate intestinal health in broiler chickens in different challenging conditions

Scientific Reports ◽

10.1038/s41598-021-85872-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

L. Criado-Mesas ◽

N. Abdelli ◽

A. Noce ◽

M. Farré ◽

J. F. Pérez ◽

...

Keyword(s):

Gene Expression ◽

Immune Response ◽

Barrier Function ◽

Nutrient Transport ◽

Gene Expression Pattern ◽

Vaccine Dose ◽

Special Focus ◽

Intestinal Health ◽

Barrier Failure ◽

Expression Of Genes

AbstractThere is a high interest on gut health in poultry with special focus on consequences of the intestinal diseases, such as coccidiosis and C. perfringens-induced necrotic enteritis (NE). We developed a custom gene expression panel, which could provide a snapshot of gene expression variation under challenging conditions. Ileum gene expression studies were performed through high throughput reverse transcription quantitative real-time polymerase chain reaction. A deep review on the bibliography was done and genes related to intestinal health were selected for barrier function, immune response, oxidation, digestive hormones, nutrient transport, and metabolism. The panel was firstly tested by using a nutritional/Clostridium perfringens model of intestinal barrier failure (induced using commercial reused litter and wheat-based diets without exogenous supplementation of enzymes) and the consistency of results was evaluated by another experiment under a coccidiosis challenge (orally gavaged with a commercial coccidiosis vaccine, 90× vaccine dose). Growth traits and intestinal morphological analysis were performed to check the gut barrier failure occurrence. Results of ileum gene expression showed a higher expression in genes involved in barrier function and nutrient transport in chickens raised in healthy conditions, while genes involved in immune response presented higher expression in C.perfringens-challenged birds. On the other hand, the Eimeria challenge also altered the expression of genes related to barrier function and metabolism, and increased the expression of genes related to immune response and oxidative stress. The panel developed in the current study gives us an overview of genes and pathways involved in broiler response to pathogen challenge. It also allows us to deep into the study of differences in gene expression pattern and magnitude of responses under either a coccidial vaccine or a NE.

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Selection of reference genes for gene expression analysis in Liriodendron hybrids’ somatic embryogenesis and germinative tissues

Scientific Reports ◽

10.1038/s41598-021-84518-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tingting Li ◽

Weigao Yuan ◽

Shuai Qiu ◽

Jisen Shi

Keyword(s):

Gene Expression ◽

Somatic Embryogenesis ◽

Reference Genes ◽

Gene Selection ◽

Elongation Factor ◽

Reverse Transcription Pcr ◽

Expression Of Genes ◽

Suitable Reference ◽

Functional Analyses ◽

Elongation Factor 1

AbstractThe differential expression of genes is crucial for plant somatic embryogenesis (SE), and the accurate quantification of gene expression levels relies on choosing appropriate reference genes. To select the most suitable reference genes for SE studies, 10 commonly used reference genes were examined in synchronized somatic embryogenic and subsequent germinative cultures of Liriodendron hybrids by using quantitative real-time reverse transcription PCR. Four popular normalization algorithms: geNorm, NormFinder, Bestkeeper and Delta-Ct were used to select and validate the suitable reference genes. The results showed that elongation factor 1-gamma, histone H1 linker protein, glyceraldehyde-3-phosphate dehydrogenase and α-tubulin were suitable for SE tissues, while elongation factor 1-gamma and actin were best for the germinative organ tissues. Our work will benefit future studies of gene expression and functional analyses of SE in Liriodendron hybrids. It is also serves as a guide of reference gene selection in early embryonic gene expression analyses for other woody plant species.

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