Reference Gene Selection for Quantitative Real-time PCR Normalization in Quercus suber

The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber), have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1α, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq) were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old) or collected in different dates (active growth period versus dormancy). The three statistical methods (geNorm, NormFinder, and CV method) used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut

<p>Abstract</p> <p>Background</p> <p>Wild peanut species (<it>Arachis </it>spp.) are a rich source of new alleles for peanut improvement. Plant transcriptome analysis under specific experimental conditions helps the understanding of cellular processes related, for instance, to development, stress response, and crop yield. The validation of these studies has been generally accomplished by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) which requires normalization of mRNA levels among samples. This can be achieved by comparing the expression ratio between a gene of interest and a reference gene which is constitutively expressed. Nowadays there is a lack of appropriate reference genes for both wild and cultivated <it>Arachis</it>. The identification of such genes would allow a consistent analysis of qRT-PCR data and speed up candidate gene validation in peanut.</p> <p>Results</p> <p>A set of ten reference genes were analyzed in four <it>Arachis </it>species (<it>A. magna</it>; <it>A. duranensis</it>; <it>A. stenosperma </it>and <it>A. hypogaea</it>) subjected to biotic (root-knot nematode and leaf spot fungus) and abiotic (drought) stresses, in two distinct plant organs (roots and leaves). By the use of three programs (GeNorm, NormFinder and BestKeeper) and taking into account the entire dataset, five of these ten genes, <it>ACT1 </it>(actin depolymerizing factor-like protein), <it>UBI1 </it>(polyubiquitin), <it>GAPDH </it>(glyceraldehyde-3-phosphate dehydrogenase), <it>60S </it>(60S ribosomal protein L10) and <it>UBI2 </it>(ubiquitin/ribosomal protein S27a) emerged as top reference genes, with their stability varying in eight subsets. The former three genes were the most stable across all species, organs and treatments studied.</p> <p>Conclusions</p> <p>This first in-depth study of reference genes validation in wild <it>Arachis </it>species will allow the use of specific combinations of secure and stable reference genes in qRT-PCR assays. The use of these appropriate references characterized here should improve the accuracy and reliability of gene expression analysis in both wild and cultivated Arachis and contribute for the better understanding of gene expression in, for instance, stress tolerance/resistance mechanisms in plants.</p

Dictionaries and their users

It is only recently that dictionary users have become a central consideration in the design of dictionaries, and this focus has both stimulated and benefited from research into dictionary use. The present contribution reviews the major issues in dictionary design from the user perspective, taking stock of the relevant findings from user research, insofar as such research can assist lexicographers in producing improved lexical tools

Distribution of phthalic acid esters in agricultural plants and soil

The study observed the occurrence of di-n-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) in the soil and agricultural crops (Triticum aestivum, Brassica napus, Zea mays) and their distribution to the individual parts. For the experiment were selected 4 locations in central Moravia. At two locations (L1, L2) winter wheat (Triticum aestivum) was grown, at the third location (L3) winter oilseed rape (Brassica napus), and at the fourth location (L4) flint corn (Zea mays). The soil samples (n = 72) and whole plant samples (n = 78) were collected during the vegetation. The aboveground and underground parts of plants, ears, siliques and seeds were analyzed separately.The values of DBP in soil at all areas ranged from 0.28 to 1.59 mg.kg−1 of dry matter and DEHP &lt; 0.03 to 0.73 mg.kg−1 of dry matter. The soil at the L4 site, which was the most fertilized with organic fertilizers, especially manure, was detected significantly (P &lt; 0.05) to show the highest values of both the PAEs (phthalic acid esters) observed (DBPL4 1.59 ± 0.07 mg.kg−1 of dry matter, DEHPL4 0.73 ± 0.18 mg.kg−1 of dry matter).Average concentrations measured in underground parts of monitored crops ranged from 1.68 to 14.26 mg.kg−1 of dry matter for DBP, and 0.12 to 10.34 mg.kg−1 of dry matter for DEHP. Values detected in aboveground parts were 0.03 to 8.84 mg.kg−1 of dry matter for DBP, and 0.25 to 4.59 mg.kg−1 of dry matter for DEHP. Average values of DBP in final products ranged from 0.05 to 0.83 mg.kg−1 of dry matter, and &lt; 0.06 to 0.98 mg.kg−1 of dry matter for DEHP

Assessment of COVID-19 Fear in Five European Countries before Mass Vaccination and Key Predictors among Nurses and Nursing Students

Background: Levels of fear have increased since the COVID-19 pandemic outbreak. The absence of a safe and effective vaccine for mass-vaccination deteriorates this situation, which has a significant impact on mental health. This study aimed to assess the feelings of fear among nurses and nursing students in five European countries. Methods: A multicenter cross-sectional study was conducted in five European countries (Greece, Albania, Cyprus, Spain, and Kosovo) before the start of mass vaccination in Europe. Data collection was conducted in December 2020–January 2021 using an online questionnaire for nursing students and professional nurses. Fear of COVID-19 Scale (FCV-19S) was used for measuring levels of fear. IBM SPSS version 21.0 was used for statistical analysis. Results: The study population included 1135 nurses and 1920 nursing students from Kosovo (n = 1085), Spain (n = 663), Greece (n = 534), Albania (n = 529), and Cyprus (n = 244). According to multivariable analysis, females (OR = 2.53, 95% CI = 1.89–3.15), married (OR = 0.86, 95% CI = 0.24–1.48), nurses (OR = 0.87, 95% CI = 0.28–1.45) and those with a chronic disease (OR = 0.86, 95% CI = 0.11–1.62) were more fearful of COVID-19. Conclusions: It is important to decrease fear in the population of nurses who are at the frontlines of the pandemic. The provision of appropriate education and training activities for nurses and students to manage their stress levels is of high importance. Future studies should focus on levels of fear after the administration of several safe and effective vaccines worldwide. © 2022 by the authors. Licensee MDPI, Basel, Switzerland