The Immunocytochemical Detection of P53 Protein In Cytological Specimens: Technical Considerations

The p53 gene encodes a 393 amino acid nuclear phosphoprotein that appears to act as a cell cycle checkpoint, possibly by transactivating other target genes. Abnormalities of the p53 gene are common in a wide range of human tumours and are associated in many cases with immunologically detectable p53 protein. Detection of p53 immunoreactivity is uncommon in normal cells, but is frequently seen in neoplasia. Here we define the optimum conditions for the detection of p53 immunoreactivity in cytological material, including fixation and storage. Immersion in acetone-methanol for 10 min is optimal, and after air drying, smears or cytospin preparations can be stored at - 70°C for at least 6 months. We describe the range of controls necessary, including the use of positive control cell lines with known mutations of the p53 gene and defined abnormalities of p53 protein. Negative controls should include cell lines (or strains) with no p53 abnormality as well as the conventional negative immunological controls. It is only with these technical caveats and controls that p53 immunoreactivity can be performed reliably on cytological specimens. Le géne p53 code pour une phosphoprotéine nucléaire de 393 acides aminés qui semble jouer en rôle dans la régulation du cycle cellulaire, probablement par transactivation d'autres gènes cibles. Les anomalies du gène p53 sont présentes dans un large éventail de tumeurs humaines et sont associées a la présence d'une protéine p53 détectable immunologiquement. La détection d'une immunoréactivité anti p53 est rare dans les cellules normales alors qu'elle est fréquente dans les tumeurs. Nous avons défini dans ce travail les conditions optimales pour la détection de l'immunoréactivité anti p53 sur matériel cytologique, y compris les conditions de fixation et de conservation. L'immersion dans l'acétone-méthanol pendant 10 minutes est optimale. Aprés séchage à l'air, les frottis ou les préparations par cytocentrifugation peuvent être stockés à—70°C pendant au moins 6 mois. Nous décrivons aussi l'éventail des contrôles nécessaires incluant I'utilisation, comme contrôle positif, de lignées cellulaires avec des mutations connues du gène p53 et des anomalies définies de la protéine p53. Les contrôles négatifs doivent comporter des lignées cellulaires (ou des espèces) sans anomalie de p53 ainsi que les contrôles immunologiques négatifs convrentionnels. C'est seulement lorsque ces précautions techniques et ces contrôles sont respectés que l'immunoréactivité anti p53 peut être étudiée valablement sur les prélèvements cytologiques. Das p53 kodiert ein aus 393 Aminosären bestehendes Phosphoprotein, das offensichtlich den Zellzyklus blockiert, möglicherweise durch Aktivierung anderer Gene. Veränderungen des p53-Gens wurden in zahlreichen menschlischen Tumoren nachgewiesen, sodaß eine positive Reaktion in Neoplasien häufig, in Normalzellen jedoch ungewöhnlich ist. Die optimalen Bedingungen für den p53-Nachweis in cytologischem Material werden hinsichtlich Fixation und Lagerung untersucht. Eine 10minütige Aceton-Methanol-Fixierung mit anschlies-sender Lufttrocknung erlaubt die Lagerung von Ausstrichen und Cytozentrifugen-präparaten bei - 70°C für mindestens 6 Monate. Die erforderlichen Kontrollen einschließlich positiver Zellinien mit bekannten Mutationen des p53-Gens und definierten Anomalien des Proteins werden beschrieben. Negativkontrollen sollten Zellinien ohne p53-Anomalie ebenso umfassen wie die üblichen negativen immunologischen Kontrollen. Nur unter diesen Bedingungen ist ein zuverlässiger Nachweis von p53 mögloch.     

New developments in MRI: System characterization,technical advances and radiotherapy applications

A Special Issue of Physica Medica – European Journal of Medical Physics, focused on some important points of contact between the world of magnetic resonance and that of medical physics, was published during 2021. This Editorial describes and comments on the content of this Focus Issue, which contains articles from leading groups invited by the Guest Editors.     

Gel-permeation chromatography–enzyme-linked immunosorbent assay method for systematic mass distribution profiling of plant cell wall matrix polysaccharides

Cell walls are dynamic and multi-component materials that play important roles in many areas of plant biology. The composition and interactions of the structural elements give rise to material properties, which are modulated by the activity of wall-related enzymes. Studies of the genes and enzymes that determine wall composition and function have made great progress, but rarely take account of potential compensatory changes in wall polymers that may accompany and accommodate changes in other components, particularly for specific polysaccharides. Here, we present a method that allows the simultaneous examination of the mass distributions and quantities of specific cell wall matrix components, allowing insight into direct and indirect consequences of cell wall manipulations. The method employs gel-permeation chromatography fractionation of cell wall polymers followed by enzyme-linked immunosorbent assay to identify polymer types. We demonstrate the potential of this method using glycan-directed monoclonal antibodies to detect epitopes representing xyloglucans, heteromannans, glucuronoxylans, homogalacturonans (HGs) and methyl-esterified HGs. The method was used to explore compositional diversity in different Arabidopsis organs and to examine the impacts of changing wall composition in a number of previously characterized cell wall mutants. As demonstrated in this article, this methodology allows a much deeper understanding of wall composition, its dynamism and plasticity to be obtained, furthering our knowledge of cell wall biology.     

Citizen science and wildlife biology: Synergies and challenges

Citizen science (CS) has evolved over the past decades as a working method involving interested citizens in scientific research, for example by reporting observations, taking measurements or analysing data. In the past, research on animal behaviour has been benefitting from contributions of citizen scientists mainly in the field of ornithology but the full potential of CS in ecological and behavioural sciences is surely still untapped. Here, we present case studies that successfully applied CS to research projects in wildlife biology and discuss potentials and challenges experienced. Our case studies cover a broad range of opportunities: large‐scale CS projects with interactive online tools on bird song dialects, engagement of stakeholders as citizen scientists to reduce human–wildlife conflicts, involvement of students of primary and secondary schools in CS projects as well as collaboration with the media leading to successful recruitment of citizen scientists. Each case study provides a short overview of the scientific questions and how they were approached to showcase the potentials and challenges of CS in wildlife biology. Based on the experience of the case studies, we highlight how CS may support research in wildlife biology and emphasise the value of fostering communication in CS to improve recruitment of participants and to facilitate learning and mutual trust among different groups of interest (e.g., researchers, stakeholders, students). We further show how specific training for the participants may be needed to obtain reliable data. We consider CS as a suitable tool to enhance research in wildlife biology through the application of open science procedures (i.e., open access to articles and the data on publicly available repositories) to support transparency and sharing experiences.     

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress

The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off‐target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR‐induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5‐fold in somatic tissues and up to 100‐fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double‐stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on‐target mutagenesis in plants using CRISPR/Cas9.     

微生物群体感应抑制剂及其在海洋生态中的应用

微生物具有结构多样性和功能多样性,其生态行为受多种信号因子的调节,其一便是群体感应信号(Quorum sensing,QS)。QS可作为菌群的通讯语言调节多种生物学功能,包括微生物被膜(Biofilm)的形成、毒力因子的表达、抗生素的分泌以及活性物质的生成等。相比之下,群体感应抑制剂(Quorum sensing inhibitor,QSI)的作用与QS相反,它能阻断QS信号的合成或传递、降低细菌致病性、干扰Biofilm的生成、阻断QS级联效应,因而被广泛应用于医药、农业和环境等领域。本文聚焦QSI,对其来源、特性、作用机制的最新进展进行总结,并对其在海洋生态领域上的应用进行综述,以期为QSI物质的开发和海洋生态资源的有效利用提供新思路。     

Spike‐dip transformation of Setaria viridis

Traditional method of Agrobacterium‐mediated transformation through the generation of tissue culture had limited success for Setaria viridis, an emerging C4 monocot model. Here we present an efficient in planta method for Agrobacterium‐mediated genetic transformation of S. viridis using spike dip. Pre‐anthesis developing spikes were dipped into a solution of Agrobacterium tumefaciens strain AGL1 harboring the β‐glucuronidase (GUS) reporter gene driven by the cauliflower mosaic virus 35S (CaMV35S) promoter to standardize and optimize conditions for transient as well as stable transformations. A transformation efficiency of 0.8 ± 0.1% was obtained after dipping of 5‐day‐old S3 spikes for 20 min in Agrobacterium cultures containing S. viridis spike‐dip medium supplemented with 0.025% Silwet L‐77 and 200 μm acetosyringone. Reproducibility of this method was demonstrated by generating stable transgenic lines expressing β‐glucuronidase plus (GUSplus), green fluorescent protein (GFP) and Discosoma sp. red fluorescent protein (DsRed) reporter genes driven by either CaMV35S or intron‐interrupted maize ubiquitin (Ubi) promoters from three S. viridis genotypes. Expression of these reporter genes in transient assays as well as in T1 stable transformed plants was monitored using histochemical, fluorometric GUS activity and fluorescence microscopy. Molecular analysis of transgenic lines revealed stable integration of transgenes into the genome, and inherited transgenes expressed in the subsequent generations. This approach provides opportunities for the high‐throughput transformation and potentially facilitates translational research in a monocot model plant.     

The impact of mechanical compression on cortical microtubules in Arabidopsis: a quantitative pipeline

Exogenous mechanical perturbations on living tissues are commonly used to investigate whether cell effectors can respond to mechanical cues. However, in most of these experiments, the applied mechanical stress and/or the biological response are described only qualitatively. We developed a quantitative pipeline based on microindentation and image analysis to investigate the impact of a controlled and prolonged compression on microtubule behaviour in the Arabidopsis shoot apical meristem, using microtubule fluorescent marker lines. We found that a compressive stress, in the order of magnitude of turgor pressure, induced apparent microtubule bundling. Importantly, that response could be reversed several hours after the release of compression. Next, we tested the contribution of microtubule severing to compression‐induced bundling: microtubule bundling seemed less pronounced in the katanin mutant, in which microtubule severing is dramatically reduced. Conversely, some microtubule bundles could still be observed 16 h after the release of compression in the spiral2 mutant, in which severing rate is instead increased. To quantify the impact of mechanical stress on anisotropy and orientation of microtubule arrays, we used the nematic tensor based FibrilTool ImageJ/Fiji plugin. To assess the degree of apparent bundling of the network, we developed several methods, some of which were borrowed from geostatistics. The final microtubule bundling response could notably be related to tissue growth velocity that was recorded by the indenter during compression. Because both input and output are quantified, this pipeline is an initial step towards correlating more precisely the cytoskeleton response to mechanical stress in living tissues.     

An efficient method for measuring copy number variation applied to improvement of nematode resistance in soybean

Copy number variation (CNV) is implicated in important traits in multiple crop plants, but can be challenging to genotype using conventional methods. The Rhg1 locus of soybean, which confers resistance to soybean cyst nematode (SCN), is a CNV of multiple 31.2‐kb genomic units each containing four genes. Reliable, high‐throughput methods to quantify Rhg1 and other CNVs for selective breeding were developed. The CNV genotyping assay described here uses a homeologous gene copy within the paleopolyploid soybean genome to provide the internal control for a single‐tube TaqMan copy number assay. Using this assay, CNV in breeding populations can be tracked with high precision. We also show that extensive CNV exists within Fayette, a released, inbred SCN‐resistant soybean cultivar with a high copy number at Rhg1 derived from a single donor parent. Copy number at Rhg1 is therefore unstable within a released variety over a relatively small number of generations. Using this assay to select for individuals with altered copy number, plants were obtained with both increased copy number and increased SCN resistance relative to control plants. Thus, CNV genotyping technologies can be used as a new type of marker‐assisted selection to select for desirable traits in breeding populations, and to control for undesirable variation within cultivars.