Real-time RT-PCR quantitative analysis of plant viruses in stone fruit tissues

Real-time PCR assays aiming at quantifying the level of plant infection by pathogens are becoming more and important. Within microbiology, the application of Real-time PCR has had the biggest impact upon the field of virology. However, Real-time PCR application in fundamental plant virology studies is still lagging behind. The use of relative and absolute quantification is discussed in this study. Also, case studies including Plum pox virus in transgenic plums C5 are presented.Keywords: Plum pox virus, C5, Real-time PCR, quantitatio

Evaluation of transgenic Prunus domestica L., clone C5 resistance to Plum pox virus

Plum pox virus (PPV) is one of the most devastating diseases of Prunus species. Since few sources of resistance to PPV have been identified, transgene-based resistance offers a complementary approach to developing PPV-resistant stone fruit cultivars. C5, a transgenic clone of Prunus domestica L., containing the PPV coat protein (CP) gene, has been described as highly resistant to PPV in greenhouse tests, displaying characteristics typical of post-transcriptional gene silencing (PTGS). Moreover, C5 trees exposed to natural aphid vectors in the field remained uninfected after 4 years while susceptible transgenic and untransformed trees developed severe symptoms within the first year. In our study, a high and permanent infection pressure of PPV was provided by bud grafting of inoculum in the field trial of clone C5 conducted in the Czech Republic, in which PPV-infected and healthy control trees were used. Moreover, trees with combined inoculations by PPV, ACLSV and PDV were also used in the trial. The presence of the viruses throughout the tree tissues, the relative titre of the viruses and symptoms on C5 trees have been monitored over the years. The resistance stability of C5 clones under permanent infection pressure is discussed.Keywords: PPV, C5, resistance, real-time PC

The mouse bile duct tapeworm, Hymenolepis microstoma in free-living small mammals in Slovakia. Occurrence and genetic analysis

The mouse bile duct tapeworm Hymenolepis microstoma, is a potentially zoonotic species with a wide variety of reported definitive hosts of rodent genera. In the present study the occurrence of H. microstoma in free-living small mammals in selected areas of Slovakia and the retrospective analysis of epidemiological data published in Slovakia were performed. Hymenolepis microstoma was detected in two animal species, the common shrew (Sorex araneus) and the European hamster (Cricetus cricetus) of 186 small mammals examined from two ecosystems, urban and natural ecosystem of national park. No mention about the presence of this parasite in Slovakia in the past was found following a bibliographical search. Partial sequences of the nuclear paramyosin gene showed the shrew isolate placed in a subclade together with H. microstoma from Portugal, with high bootstrap value for its differentiation from the sister species Hymenolepis nana. Similarly, the analysis of the nuclear ribosomal ITS region placed the hamster isolate in the cluster composed of H. microstoma from Australia, Spain and Portugal. The Slovak isolate was the most distinctive sample among available H. microstoma, differing in 1.4 – 1.9% of nucleotides from the remaining isolates. The difference (seven of 17 nucleotide positions) was partially due to indel polymorphisms associated with two and five nucleotides. To our knowledge, these are the first reports of H. microstoma in Central Europe and also the first record of infection in the common shrew. A recently indicated zoonotic potential of H. microstoma along with a possibility of its direct transmission between animals and/or humans without the need of intermediate hosts pose a public health concern in contaminated areas of Slovakia. The use of molecular techniques may substantially facilitate more thorough understanding of the epidemiological situation of H. microstoma and related tapeworms in various ecosystems of the country

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

Dynamický model ložiska kaolinu

This paper focuses on research within the project TE02000029 Competence Centre for Effective and Ecological Mining of Mineral Resources, granted by The Technology Agency of the Czech Republic, and, more specifically, on the research within its work package WP4 - Spatial modelling of mineral deposits. The focus of this work package is digital modelling of selected nonenergetic raw materials, which belong to the critical commodities, as defined by the European Union. For modelling these deposits, suitable mathematical procedures, based on study and reevaluation of archived data, are needed. One of the selected deposits is a kaolin deposit near the village Jimlíkov near the city Karlovy Vary. In this paper, we show a step-by-step procedure for creation, visualization and evaluation of a 3D model of the deposit. This methodology, along with our recently developed software allows a user to create a variant of this dynamic model for the same or similar types of deposits, enables rapid updating of these models when adding or changing the input data on the basis of new mining exploration or when changing modelling parameters such as using multiple variations interpolation parameters. Our methodology leads to a more advanced deposit evaluation, including adaptive estimates of the reserves based on the usability requirements we choose. In January 2017 our software was tried out in the company Sedlecký kaolin a. s. in Božičany and our dynamic model is fully applied in practice.Článek popisuje část řešení projektu TE02000029 - Centrum kompetence efektivní a ekologické těžby nerostných surovin (CEEMIR) financovaného Technologickou agenturou ČR, přesněji výsledky řešení Work Package WP4 - Prostorové modelování ložisek nerostných surovin. Hlavním cílem WP4 je digitální modelování vybraných neenergetických surovin, které se řadí mezi kritické komodity EU. Pro modelování ložisek se využívají vhodné matematické postupy na základě studia a přehodnocení dat z archivních materiálů. Jedním z vybraných ložisek je ložisko kaolínu v okolí obce Jimlíkov na Karlovarsku. V článku jsou popsány jednotlivé kroky metodického postupu tvorby, vizualizace a zhodnocení 3D modelu tohoto ložiska. Tato metodika spolu s nově vyvinutým programovým vybavením umožňuje vytvářet variantní dynamické modely ložiska tohoto a obdobných typů, umožňuje rychlé aktualizace těchto modelů při doplnění nebo změně vstupních dat na základě prováděného těžebního průzkumu (případně i parametrů modelování - například použití více variant parametrů interpolace). Uplatnění popsaného metodického postupu vede ke komplexnímu zhodnocení ložiska včetně variantních odhadů zásob podle zadaných podmínek využitelnosti. V lednu 2017 proběhlo zaškolení a instalace software u Sedleckého kaolinu a. s. v Božičanech a dynamický model je tak plně využíván v praxi

Implementace modelu ložiska kaolinu v prostředí Visual Studio 2015

This paper focuses on research within the project TE02000029 Competence Centre for Effective and Ecological Mining of Mineral Resources, granted by The Technology Agency of the Czech Republic, and, more specifically, on the research within its work package WP4 - Spatial modelling of mineral deposits. The focus of this work package is digital modelling of selected nonenergetic raw materials, which belong to the critical commodities, as defined by the European Union. For modelling these deposits, suitable mathematical procedures, based on study and reevaluation of archived data, are needed. One of the selected deposits is a kaolin deposit near the village Jimlíkov near the city Karlovy Vary. The article describes the implementation of the methodology used for processing deposits of kaolin in Visual Studio 2015 by means of objects of Surfer and Voxler created by Golden Software. This software solution is installed at the company Sedlecky kaolin a.s. and thus is fully exploited in practice.Článek popisuje část řešení projektu TE02000029 - Centrum kompetence efektivní a ekologické těžby nerostných surovin (CEEMIR) financovaného Technologickou agenturou ČR, přesněji výsledky řešení Work Package WP4 - Prostorové modelování ložisek nerostných surovin. Hlavním cílem WP4 je digitální modelování vybraných neenergetických surovin, které se řadí mezi kritické komodity EU. Pro modelování ložisek se využívají vhodné matematické postupy na základě studia a přehodnocení dat z archivních materiálů. Jedním z vybraných ložisek je ložisko kaolínu v okolí obce Jimlíkov na Karlovarsku. V článku je popis implementace metodiky zpracování ložiska kaolínu v prostředí Visual Studia 2015 s využitím objektů Surfer a Voxler firmy Golden Software. Vytvořené softwarové řešení je instalováno u firmy Sedlecký kaolín a.s. a je tak plně využíváno v praxi

Osetrovatelske intervence v domaci peci

Article in Czech, abstract in both Czech and English. English title: Nursing interventions in home carePříspěvek prezentuje dílčí výsledky pokračujícího projektu IGA MZ ČR, zabývajícího se tvorbou české verze vybran ých částí klasifikace NIC v domácí péči. Klasifikace NIC je mezinárodní klasifikací ošetřovatelských intervencí a aktivit sestry, která zpřehledňuje a dokumentuje autonomní činnosti sester. Ošetřovatelské intervence a aktivity (NIC) byly zvoleny k posouzení expertní skupinou v závislosti na předchozí ošetřovatelské diagnostice 200 klientů agentury domácí péče v Ostravě. Zařazeny byly intervence a aktivity pro ošetřovatelské diagnózy s četností nad 26 % (9 diagnóz). Těmto diagnózám odpovídalo 263 intervencí spolu s 6 356 příslušnými aktivitami sestry. Tento soubor vybraných intervencí NIC byl posouzen akademickými ošetřovatelskými odborníky a expertní skupinou sester s ohledem na platnou českou legislativu a aktuální stav kompetencí sester. Výsledkem je banka 110 ošetřovatelských intervencí dle NIC a 2 150 příslušných aktivit, které byly zaneseny do elektronické dokumentace pro domácí péči. = This paper presents the partial results of an ongoing project dealing with the creation of a Czech version of selected parts of the NIC classification in home care. NIC classification is an international classification of interventions and activities that nurses perform, which streamlines and documents the autonomous nurses' activities. An expert group chose nursing interventions and activities (NIC) that were based on the previous diagnosis of 200 clients from a home care agency in Ostrava for assessment. They included interventions and activities for nursing diagnoses with a frequency above 26 % (9 diagnoses). These diagnoses corresponded to 263 interventions with 6 356 of relevant nurses' activities. This set of selected NIC interventions was assessed by academic nursing specialists and an expert group of nurses with regard to the valid Czech legislation and the current state of competencies of nurses. As a result, there is a database of 110 nursing interventions according to the NIC and 2150 activities, which were entered into the electronic documentation for home care.Jarošová, D., Sikorová, L., Marečková, J., Pajorová, H