Reference gene selection in bovine caruncular epithelial cells under pregnancy-associated hormones exposure.
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ABSTRACT:
SUBMITTER: Sozoniuk M
PROVIDER: S-EPMC9325760 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Listeria monocytogenes is an important foodborne pathogen in human and veterinary health, causing significant morbidity and mortality including abortion. It has a particular tropism for the gravid uterus, however, the route of infection in reproductive tissues of ruminants (i.e. placentome), is much less clear. In this study, we aimed to investigate a bovine caruncular epithelial cell (BCEC) line as a model for L. monocytogenes infection of the bovine reproductive tract. The BCEC infection model was used to assess the ability of 14 different L. monocytogenes isolates to infect these cells. Lysozyme sensitivity and bacterial survival in 580 ?g lysozyme/ml correlated with attenuated ability to proliferate in BCEC (p = 0.004 and p = 0.02, respectively). Four isolates were significantly attenuated compared to the control strain 10403S. One of these strains (AR008) showed evidence of compromised cell wall leading to increased sensitivity to ß-lactam antibiotics, and another (7644) had compromised cell membrane integrity leading to increased sensitivity to cationic peptides. Whole genome sequencing followed by Multi Locus Sequence Type analysis identified that five invasive isolates had the same sequence type, ST59, despite originating from three different clinical conditions. Virulence gene analysis showed that the attenuated isolate LM4 was lacking two virulence genes (uhpT, virR) known to be involved in intracellular growth and virulence. In conclusion, the BCEC model was able to differentiate between the infective potential of different isolates. Moreover, resistance to lysozyme correlated with the ability to invade and replicate within BCEC, suggesting co-selection for surviving challenging environments as the abomasum.
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Project description:Changes in the expression of various genes, including pregnancy-associated hormone receptors and extracellular matrix proteins, have been suggested to play a significant role in bovine placental development. This study aimed to examine the influence of sex steroids and PGF2α on decorin (DCN) expression in the epithelial cells of bovine caruncle in early−mid pregnancy in cows. The expression patterns of DCN, PTGFR, PGR and ESR1 were analyzed by RT-qPCR and Western blotting in primary caruncular epithelial cell cultures (PCECC) and placental tissue homogenates derived from the 2nd and 4th months of pregnancy. PCECC were found to express DCN, PTGFR, PGR and ESR1. The intensity of PGR staining was higher in cells derived from the 4th month of pregnancy (p < 0.05). The 17β-estradiol, progesterone and PGF2α have not been shown to affect DCN expression. PGF2α decreased PTGFR expression in cells derived from the 4th month of gestation (p < 0.05). In conclusion, the results of the present preliminary study showed that the expression of the PTGFR, ESR1, PGR and DCN in PCECC does not vary throughout early−mid pregnancy. Further studies should be carried out to observe the relationship between hormonal status and cellular adhesion to determine their importance for properly developing placentation and pregnancy in cows.
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Project description:BACKGROUND:Bovine milk contains not only a variety of nutritional ingredients but also microRNAs (miRNAs) that are thought to be secreted by the bovine mammary epithelial cells (BMECs). The objective of this study was to elucidate the production of milk-related miRNAs in BMECs under the influence of lactogenic hormones. RESULTS:According to a microarray result of milk exosomal miRNAs prior to cellular analyses, a total of 257 miRNAs were detected in a Holstein cow milk. Of these, 18 major miRNAs of interest in the milk were selected for an expression analysis in BMEC culture that was treated with or without dexamethasone, insulin, and prolactin (DIP) to induce a lactogenic differentiation. Quantitative polymerase chain reaction (qPCR) results showed that the expressions of miR-21-5p (P?=?0.005), miR-26a (P?=?0.016), and miR-320a (P?=?0.011) were lower in the DIP-treated cells than in the untreated cells. In contrast, the expression of miR-339a (P?=?0.017) in the cell culture medium were lower in the DIP-treated culture than in the untreated culture. Intriguingly, the miR-148a expression in cell culture medium was elevated by DIP treatment of BMEC culture (P?=?0.018). The medium-to-cell expression ratios of miR-103 (P?=?0.025), miR-148a (P?<?0.001), and miR-223 (P?=?0.013) were elevated in the DIP-treated BMECs, suggesting that the lactogenic differentiation-induced secretion of these three miRNAs in BMECs. A bioinformatic analysis showed that the miRNAs down-regulated in the BMECs were associated with the suppression of genes related to transcriptional regulation, protein phosphorylation, and tube development. CONCLUSION:The results suggest that the miRNAs changed by lactogenic hormones are associated with milk protein synthesis, and mammary gland development and maturation. The elevated miR-148a level in DIP-treated BMECs may be associated with its increase in milk during the lactation period of cows.
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Project description:Findings from national surveys suggest that everyone in the United States is exposed to perchlorate. At high doses, perchlorate, thiocyanate, and nitrate inhibit iodide uptake into the thyroid and decrease thyroid hormone production. Small changes in thyroid hormones during pregnancy, including changes within normal reference ranges, have been linked to cognitive function declines in the offspring.We evaluated the potential effects of low environmental exposures to perchlorate on thyroid function.Serum thyroid hormones and anti-thyroid antibodies and urinary perchlorate, thiocyanate, nitrate, and iodide concentrations were measured in 1,880 pregnant women from San Diego County, California, during 2000-2003, a period when much of the area's water supply was contaminated from an industrial plant with perchlorate at levels near the 2007 California regulatory standard of 6 μg/L. Linear regression was used to evaluate associations between urinary perchlorate and serum thyroid hormone concentrations in models adjusted for urinary creatinine and thiocyanate, maternal age and education, ethnicity, and gestational age at serum collection.The median urinary perchlorate concentration was 6.5 μg/L, about two times higher than in the general U.S.Adjusted associations were identified between increasing log10 perchlorate and decreasing total thyroxine (T4) [regression coefficient (β) = -0.70; 95% CI: -1.06, -0.34], decreasing free thyroxine (fT4) (β = -0.053; 95% CI: -0.092, -0.013), and increasing log10 thyroid-stimulating hormone (β = 0.071; 95% CI: 0.008, 0.133).These results suggest that environmental perchlorate exposures may affect thyroid hormone production during pregnancy. This could have implications for public health given widespread perchlorate exposure and the importance of thyroid hormone in fetal neurodevelopment.Steinmaus C, Pearl M, Kharrazi M, Blount BC, Miller MD, Pearce EN, Valentin-Blasini L, DeLorenze G, Hoofnagle AN, Liaw J. 2016. Thyroid hormones and moderate exposure to perchlorate during pregnancy in women in Southern California. Environ Health Perspect 124:861-867; http://dx.doi.org/10.1289/ehp.1409614.
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Project description:Calotropis procera is a perennial Asian shrub with significant adaptation to adverse climate conditions and poor soils. Given its increased salt and drought stress tolerance, C. procera stands out as a powerful candidate to provide alternative genetic resources for biotechnological approaches. The qPCR (real-time quantitative polymerase chain reaction), widely recognized among the most accurate methods for quantifying gene expression, demands suitable reference genes (RGs) to avoid over- or underestimations of the relative expression and incorrect interpretation. This study aimed at evaluating the stability of ten RGs for normalization of gene expression of root and leaf of C. procera under different salt stress conditions and different collection times. The selected RGs were used on expression analysis of three target genes. Three independent experiments were carried out in greenhouse with young plants: i) Leaf100 = leaf samples collected 30 min, 2 h, 8 h and 45 days after NaCl-stress (100 mM NaCl); ii) Root50 and iii) Root200 = root samples collected 30 min, 2 h, 8 h and 1day after NaCl-stress (50 and 200 mM NaCl, respectively). Stability rank among the three algorithms used showed high agreement for the four most stable RGs. The four most stable RGs showed high congruence among all combination of collection time, for each software studied, with minor disagreements. CYP23 was the best RG (rank of top four) for all experimental conditions (Leaf100, Root50, and Root200). Using appropriated RGs, we validated the relative expression level of three differentially expressed target genes (NAC78, CNBL4, and ND1) in Leaf100 and Root200 samples. This study provides the first selection of stable reference genes for C. procera under salinity. Our results emphasize the need for caution when evaluating the stability RGs under different amplitude of variable factors.
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