scholarly journals Blue light-induced gene expression alterations in cultured neurons are the result of phototoxic interactions with neuronal culture media

2019 ◽  
Author(s):  
Corey G. Duke ◽  
Katherine E. Savell ◽  
Robert A. Phillips ◽  
Jeremy J. Day
Keyword(s):  
Gene Expression ◽  
Blue Light ◽  
Cortical Neurons ◽  
Clock Genes ◽  
Culture Media ◽  
Light Exposure ◽  
Neuronal Culture ◽  
Gene Expression Alterations

Blue waveform light is used as an optical actuator in numerous optogenetic technologies employed in neuronal systems. However, the potential side effects of blue waveform light in neurons has not been thoroughly explored, and recent reports suggest that neuronal exposure to blue light can induce transcriptional alterations in vitro and in vivo. Here, we examined the effects of blue waveform light in cultured primary rat cortical neurons. Exposure to blue light (470nm) resulted in upregulation of several immediate early genes (IEGs) traditionally used as markers of neuronal activity, including Fos and Fosb, but did not alter the expression of circadian clock genes Bmal1, Cry1, Cry2, Clock, or Per2. IEG expression was increased following 4 hours of 5% duty cycle light exposure, and IEG induction was not dependent on light pulse width. Elevated levels of blue light exposure induced a loss of cell viability in vitro, suggestive of overt phototoxicity. Changes in gene expression induced by blue waveform light were prevented when neurons were cultured in a photoinert media supplemented with a photostable neuronal supplement instead of commonly utilized neuronal culture media and supplements. Together, these findings suggest that light-induced gene expression alterations observed in vitro stem from a phototoxic interaction between commonly used media and neurons, and offer a solution to prevent this toxicity when using photoactivatable technology in vitro.

scholarly journals Blue Light-Induced Gene Expression Alterations in Cultured Neurons Are the Result of Phototoxic Interactions with Neuronal Culture Media

eNeuro ◽  
2019 ◽  
Vol 7 (1) ◽  
pp. ENEURO.0386-19.2019 ◽  
Author(s):  
Corey G. Duke ◽  
Katherine E. Savell ◽  
Jennifer J. Tuscher ◽  
Robert A. Phillips ◽  
Jeremy J. Day
Keyword(s):  
Gene Expression ◽  
Blue Light ◽  
Culture Media ◽  
Neuronal Culture ◽  
Cultured Neurons ◽  
Gene Expression Alterations

179 EFFECT OF SERUM DURING CULTURE ON DAY 14 ELONGATED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 191 ◽  
Author(s):  
J. Angulo ◽  
G. T. Gentry ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Culture Media ◽  
Calf Serum ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Expression Levels ◽  
The Mean

It has been reported that the addition of serum to embryo culture media alters gene expression and triggers the development of large offspring syndrome. The objectives of this study were to determine gene expression levels in embryos cultured in the absence or presence of 5% calf serum and in vivo-derived (IVD) embryos and to determine the effects of serum on the length of elongated embryos. Abattoir-derived oocytes were obtained from a commercial provider and fertilized at 24 h of maturation with semen from a bull previously used for IVF. At 18 h post-insemination (hpi), embryos were denuded and groups of 15 presumptive zygotes were cultured in 30-μL drops of modified SOF medium with amino acids and 6 mg mL–1 of BSA (mSOFaa). At 72 hpi, cleavage rate was assessed and embryos were randomly allocated into 2 treatments: mSOFaa without and with 5% calf serum. Embryos were then cultured to 168 hpi and blastocyst rates were assessed and recorded. Blastocysts (n = 5 to 10) from each treatment were transferred into synchronized recipients, and Day 14 embryos were recovered 7 days post-transfer. Embryos were photographed, measured, and immediately stored at –80°C in a minimal volume of PBS + 0.1% polyvinyl alcohol. Messenger RNA was isolated using a Dynabeads mRNA Direct Kit™ (Invitrogen, Carlsbad, CA), and reverse transcription was performed using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., CA). Quantitative PCR was performed to determine the transcript abundance for COX6A, IFNT1a, PLAC8, IGF2R, and GAPDH for each sample. The GAPDH was used as a reference gene, and gene expression was calculated as a ratio of expression levels between each gene of interest and GAPDH. Expression levels for each gene were determined from standard curves generated by serial dilutions of PCR amplicons starting with 0.4 pg/reaction. Blastocyst development rates were higher in embryos cultured with serum compared with the nonserum treatment (14.9 and 7.4% respectively; chi-square, P < 0.001). Lengths of elongated embryos from the serum (3395.3 ± 414.7 μm) and nonserum (2784 ± 741.8 μm) culture treatments differed from the IVD (6297.7 ± 677.2 μm) treatment (mean ± SE; ANOVA, P < 0.0052). There were no differences in the mean expression levels for COX6A, IFNT1a, PLAC8, and IGF2R across treatment groups, but in the serum treatment, 3 out 11 overexpressed IFNT1a, 4 out of 11 overexpressed IGF2R, and 2 out of 11 overexpressed PLAC8, defined as being 2 standard deviations above the mean of the IVD treatment for each respective gene. In the in vitro-produced nonserum and IVD treatments, overexpression by this definition was not observed. Although mean expression levels were not affected by culture with serum under these conditions, very high expression of IFNT1a, IGF2R, and PLAC8 was observed in some embryos cultured with serum, but not in embryos cultured without serum or IVD embryos.

scholarly journals Nonmetastatic Colon Cancer Model C26 Upregulates Glycolysis in Osteocytes in Vitro and Bone in Vivo

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
M. Roarke Tollar ◽  
Matthew Prideaux ◽  
Fabrizio Pin ◽  
Lynda F. Bonewald
Keyword(s):  
Gene Expression ◽  
Culture Media ◽  
Bone Mineralization ◽  
Metabolic Effects ◽  
Whole Body ◽  
Rt Pcr ◽  
Systemic Complications ◽  
Whole Body Metabolism

Background: Developing effective treatments for musculoskeletal complications in cancer patients requires understanding metabolic effects of cancer on bone, and particularly osteocytes, the most abundant bone cell and key regulator of bone remodeling. However, little is known regarding how cancer impacts normal osteocyte energy metabolic pathways, such as glycolysis. Given that changes in metabolism are important regulators of cellular function, it is essential to determine how osteocyte metabolism is disrupted by cancer and how this may impact skeletal and whole-body health. Methods: Mice inoculated with saline (N=5) or C26 cells (N=6) were sacrificed after 2 weeks. Bones were harvested for metabolic profiling by GC-MS, gene expression by RT-PCR and bone morphology by µCT. Differentiated IDG-SW3 osteocyte-like cells were cocultured with C26 cells for 12-24hrs and metabolites and gene expression analyzed by GC-MS and RT-PCR. Results: Trabecular bone mass was significantly decreased in the C26 mice. GC-MS analysis revealed decreased glucose in C26 mice tibiae, but no change in lactate. The bone resorption promoting gene Rankl was upregulated, whereas the inhibitor Opg was unchanged. Bone mineralization regulators Mepe and Phex were decreased. In vitro metabolic studies revealed increased glucose and lactate in IDG-SW3 cell lysate; culture media glucose levels were decreased whereas lactate was increased in the co-cultures with C26 cells. RT-PCR demonstrated increases in the glycolysis promoter Hif1α in addition to glycolysis pathway genes including Glut1, Hk2, Slc16a3 and Pdk1. Rankl was also increased in the IDG-SW3 cells co-cultured with the C26 cells whereas Opg, Phex, and Mepe were downregulated. Conclusion: Glycolysis is upregulated in mouse bone and in vitro IDG-SW3 cells exposed to cancer. Our study provides novel understanding for how cancer affects bone metabolism. Integrating these results with whole body metabolism will aid in the development of novel therapeutic strategies to target musculoskeletal and systemic complications of cancer.

scholarly journals The Circadian Oscillator of the Cerebellum: Triiodothyronine Regulates Clock Gene Expression in Granule Cells in vitro and in the Cerebellum of Neonatal Rats in vivo

2021 ◽  
Vol 12 ◽  
Author(s):  
Tenna Bering ◽  
Henrik Hertz ◽  
Martin Fredensborg Rath
Keyword(s):  
Gene Expression ◽  
Clock Gene ◽  
Granule Cells ◽  
Clock Genes ◽  
Circadian Oscillator ◽  
Neonatal Rats ◽  
Clock Gene Expression ◽  
Mixed Gender

The central circadian clock resides in the suprachiasmatic nucleus (SCN) of the hypothalamus, but an SCN-dependent molecular circadian oscillator is present in the cerebellar cortex. Recent findings suggest that circadian release of corticosterone is capable of driving the circadian oscillator of the rat cerebellum. To determine if additional neuroendocrine signals act to shape cerebellar clock gene expression, we here tested the role of the thyroid hormone triiodothyronine (T3) in regulation of the cerebellar circadian oscillator. In cultured cerebellar granule cells from mixed-gender neonatal rats, T3 treatment affected transcript levels of the clock genes Per2, Arntl, Nr1d1, and Dbp, suggesting that T3 acts directly on granule cells to control the circadian oscillator. We then used two different in vivo protocols to test the role of T3 in adult female rats: Firstly, a single injection of T3 did not influence clock gene expression in the cerebellum. Secondly, we established a surgical rat model combining SCN lesion with a programmable micropump infusing circadian physiological levels of T3; however, rhythmic infusion of T3 did not reestablish differential clock gene expression between day and night in SCN lesioned rats. To test if the effects of T3 observed in vitro were related to the developmental stage, acute injections of T3 were performed in mixed-gender neonatal rats in vivo; this procedure significantly affected cerebellar expression of the clock genes Per1, Per2, Nr1d1, and Dbp. Developmental comparisons showed rhythmic expression of all clock genes analyzed in the cerebellum of adult rats only, whereas T3 responsiveness was limited to neonatal animals. Thus, T3 shapes cerebellar clock gene profiles in early postnatal stages, but it does not represent a systemic circadian regulatory mechanism linking the SCN to the cerebellum throughout life.

391 A COMPARATIVE GENE EXPRESSION ANALYSIS BETWEEN SHEEP EARLY EMBRYONIC DEVELOPMENT AND PUTATIVE EMBRYONIC STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 352
Author(s):  
B. M. Murray ◽  
S. Schmoelzl ◽  
N. M. Andronicos ◽  
J. R. Hill ◽  
P. J. Verma ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
In Vitro Culture ◽  
Culture Media ◽  
Embryonic Stem ◽  
Oct4 Expression ◽  
Pluripotency Markers

The optimization of culture media to support the isolation of embryonic stem cells relies on methods to monitor whether the pluripotent state of the cultured cells has been maintained. We developed a panel of gene expression assays that allowed us to correlate molecular measures of pluripotency or lineage differentiation with a developmental time course. By conducting quantitative PCR analysis of sheep embryos over Day 6.5 to 24 and sheep inner cell mass (ICM) cells cultured over 25 days, we tested whether culture media designed to inhibit differentiation are able to maintain sheep ICM cells in a pluripotent state. Briefly, embryos were collected from Merino ewes (n = 50, 3 years) at Day 6.5, 12, 16, 20, and 24 post-AI. Embryos were collected from the dissected uterine tracts of slaughtered ewes, excluding Day 6.5 blastocysts, which were surgically recovered from superovulated ewes. For the in vitro culture, Day 6.5 ICM cells were isolated by immunosurgery and cultured on mitomycin-C-treated mouse embryonic fibroblasts in an inhibitor-based medium (3i, based on Ying Q-L et al. 2008 Nature 453, 519-523). Real-time PCR assays for pluripotency (OCT4, SOX2, NANOG) and differentiation (ectodermal: FGF5, PAX6; endodermal: GATA4, GATA6, Somatostatin; mesodermal: BMP4, Connexin40) of sheep candidate genes were conducted on cDNA prepared from these samples and normalized against the reference genes RPL19 and RPS26. In in vivo embryos, pluripotency markers OCT4, SOX2, and NANOG all decreased between Day 6.5 and Day 20, although OCT4 expression spiked around Day 16. More interestingly, pluripotency expression decreased during in vitro culture, with NANOG expression completely lost by passage 2 at Day 11 and OCT4 expression at an equivalent Day 24 embryo basal level by Day 14. The endodermal markers GATA6 and GATA4 decreased between Day 6.5 and Day 12, respectively, although in vitro GATA4 was only expressed once at Day 7. In vivo FGF5 and both PAX6 and Somatostatin displayed a delayed onset, increasing expression from Day 16 and 20, respectively, whereas the ectodermal markers were already expressed by Day 7 in vitro. Both mesodermal markers Connexin40 and BMP4 presented minor fold changes in both data sets. In conclusion, this study has verified the primer sets and described a sheep in vivo embryo gene expression profile comprising both pluripotent and differentiation candidates. Furthermore, the decrease of pluripotency markers together with the appearance of differentiation markers during in vitro culture of ICM cells suggest that culturing ICM cells in 3i media is not sufficient to maintain a sheep-specific pluripotent population of cells. Therefore, future studies will be aimed at manipulating the current in vitro system to focus on maintaining pluripotent genes such as NANOG and OCT4 in culture.

scholarly journals Tetracycline-Regulatable System To Tightly Control Gene Expression in the Pathogenic Fungus Candida albicans

2000 ◽  
Vol 68 (12) ◽  
pp. 6712-6719 ◽  
Author(s):  
Hironobu Nakayama ◽  
Toshiyuki Mio ◽  
Shigehisa Nagahashi ◽  
Michiko Kokado ◽  
Mikio Arisawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Culture Media ◽  
Pathogenic Fungus ◽  
Elongation Factor ◽  
Growth Defect ◽  
Control Gene ◽  
Control Gene Expression

ABSTRACT Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in C. albicans by the tetracycline-regulatable (TR) promoters. When the sea pansy Renilla reniformisluciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement ofN-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.

Pharmacokinetics explain in vivo/in vitro discrepancies of carcinogen-induced gene expression alterations in rat liver and cultivated hepatocytes

2012 ◽  
Vol 87 (2) ◽  
pp. 337-345 ◽  
Author(s):  
Markus Schug ◽  
Regina Stöber ◽  
Tanja Heise ◽  
Hans Mielke ◽  
Ursula Gundert-Remy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rat Liver ◽  
Cultivated Hepatocytes ◽  
Gene Expression Alterations

In Vitro - In Vivo Correlation of Gene Expression Alterations Induced by Liver Carcinogens

2012 ◽  
Vol 19 (11) ◽  
pp. 1721-1730 ◽  
Author(s):  
T. Heise ◽  
M. Schug ◽  
D. Storm ◽  
H. Ellinger-Ziegelbauer ◽  
H. J. Ahr ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Alterations

Effect of X-irradiation on gene expression of rat liver chemokines: in vivo and in vitro studies

2008 ◽  
Vol 46 (01) ◽  
Author(s):  
F Moriconi ◽  
H Christiansen ◽  
H Christiansen ◽  
N Sheikh ◽  
J Dudas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rat Liver ◽  
In Vitro Studies ◽  
X Irradiation
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