Climate change is intensifying drought stress in viticultural regions, threatening grapevine productivity and quality. New genotypes previously bred for disease resistance are an untested resource regarding their water deficit tolerance. Field experiments were conducted over two seasons, applying well-watered and water-stress treatments in two disease-resistant varieties, Fleurtai and Cabernet Volos. Physiological measurements, RNA sequencing, and GC-MS-based metabolite profiling of leaves were integrated, correlating gene expression and metabolite accumulation with stem water potential (ΨS) using a factorial analyses design by FaDSeqSes script. We identified three categories of response: (1) genes and metabolites similarly regulated by water deficit in both genotypes, belonging to several different pathways (such as carbohydrate metabolism, amino acid metabolism, secondary metabolism, and hormone metabolism), among which sugar metabolism was one of the most striking ones, including changes in accumulation of raffinose and galactinol, and induction of genes coding for their synthesis; (2) responses specific to Cabernet Volos, characterized by downregulation of kinase and receptor genes likely to be involved in shutting down biotic defense response; and (3) responses specific to Fleurtai, including accumulation of caffeoylquinate and upregulation of genes involved terpene synthesis and in ABA regulation. We found that each genotype has an individual way to combat water deficits; Cabernet Volos accumulates more osmoprotectant compounds at a constant and higher level, while Fleurtai synthesizes these compounds as needed when stress occurs. This study underscores overall grapevine responses to water deficits, as well as the contribution of the genotype.

Potato (Solanum tuberosum) is highly water and space efficient but susceptible to abiotic stresses such as heat, drought, and flooding, which are severely exacerbated by climate change. Our understanding of crop acclimation to abiotic stress, however, remains limited. Here, we present a comprehensive molecular and physiological high-throughput profiling of potato (Solanum tuberosum, cv. Désirée) under heat, drought, and waterlogging applied as single stresses or in combinations designed to mimic realistic future scenarios. Stress responses were monitored via daily phenotyping and multi-omics analyses of leaf samples comprising proteomics, targeted transcriptomics, metabolomics, and hormonomics at several timepoints during and after stress treatments. Additionally, critical metabolites of tuber samples were analyzed at the end of the stress period. We performed integrative multi-omics data analysis using a bioinformatic pipeline that we established based on machine learning and knowledge networks. Waterlogging produced the most immediate and dramatic effects on potato plants, interestingly activating ABA responses similar to drought stress. In addition, we observed distinct stress signatures at multiple molecular levels in response to heat or drought and to a combination of both. In response to all treatments, we found a downregulation of photosynthesis at different molecular levels, an accumulation of minor amino acids, and diverse stress-induced hormones. Our integrative multi-omics analysis provides global insights into plant stress responses, facilitating improved breeding strategies toward climate-adapted potato varieties.

Potato, the most important non-cereal crop, is highly water and space efficient but susceptible to abiotic stress such as heat, drought, or flooding. Climate change is severely increasing the likelihood of such stresses to occur individually, sequentially, or simultaneously. However, the understanding of acclimation to abiotic stress in crops in general, especially with multiple stresses, is still very limited. Here, we present a comprehensive one month-long molecular and physiological high-throughput profiling of potato ( Solanum tuberosum , cv. Désirée) under both single and multiple abiotic stresses, designed to mimic realistic future scenarios. Acclimation time-responses were monitored via daily phenomic analysis and leaf samples were processed for multi-omics spanning from transcriptomics to proteomics and hormonomics. Additionally, critical metabolites of tuber samples were analysed at the end of the period. To facilitate the multi-omics analyses, the dataset was integrated with prior knowledge, which is indispensable for development of high-throughput pipelines in agricultural research. Waterlogging had the most immediate and dramatic effects, with responses similar to drought stress. In addition, we observed distinct stress signatures at multiple molecular levels in response to heat or drought and to a combination of both. In general, there was a downregulation of photosynthesis at different molecular levels, accumulation of minor amino acids and diverse stress induced hormones. Our integrative multi-omics analysis provides global insights into plant stress responses, facilitating improved breeding strategies. One Sentence Summary Integrated multi-omics analysis of high-throughput phenotyping in potato reveals distinct molecular signatures of acclimation to single and combined abiotic stresses related to climate change. Experimentally acquired data and data required to reproduce the analysis are available from Supp. Table 5 and NIBs’ GitHub repository . Proteomics data are available via ProteomeXchange with identifier PXD052587.

We developed pISA-tree, a straightforward and flexible data management solution for organisation of life science project-associated research data and metadata. pISA-tree was initiated by end-user requirements thus its strong points are practicality and low maintenance cost. It enables on-the-fly creation of enriched directory tree structure ( p roject/ I nvestigation/ S tudy/ A ssay) based on the ISA model, in a standardised manner via consecutive batch files. Templates-based metadata is generated in parallel at each level enabling guided submission of experiment metadata. pISA-tree is complemented by two R packages, pisar and seekr . pisar facilitates integration of pISA-tree datasets into bioinformatic pipelines and generation of ISA-Tab exports. seekr enables synchronisation with the FAIRDOMHub repository. Applicability of pISA-tree was demonstrated in several national and international multi-partner projects. The system thus supports findable, accessible, interoperable and reusable (FAIR) research and is in accordance with the Open Science initiative. Source code and documentation of pISA-tree are available at https://github.com/NIB-SI/pISA-tree .

Our experiences shape our knowledge and understanding of the world around us. The natural vibrational environment (vibroscape) is hidden to human senses but is nevertheless perceived and exploited by the majority of animals. Here, we show that the vibroscape recorded on plants in a temperate hay meadow is a dynamic low-frequency world, rich in species-specific vibrational signals. The overall vibroscape composition changed throughout the season and also depended on the plant species, as well as on the spatial position of individual plants within the meadow. Within the studied community, vibrationally signaling species sharing this communication channel avoided interference primarily by partitioning vibrational space on a fine temporal scale. The vibroscape is a reliable source of information in the environment and expands our understanding of ecological and evolutionary processes.

SUMMARYWhereas the activation of resistance (R) proteins has been intensively studied, the downstream signaling mechanisms leading to the restriction of the pathogen remain mostly unknown. We studied the immunity network response conditioned by the potato Ny‐1 gene against potato virus Y. We analyzed the processes in the cell death zone and surrounding tissue on the biochemical and gene expression levels in order to reveal the spatiotemporal regulation of the immune response. We show that the transcriptional response in the cell death zone and surrounding tissue is dependent on salicylic acid (SA). For some genes the spatiotemporal regulation is completely lost in the SA‐deficient line, whereas other genes show a different response, indicating multiple connections between hormonal signaling modules. The induction of NADPH oxidase RBOHD expression occurs specifically on the lesion border during the resistance response. In plants with silenced RBOHD, the functionality of the resistance response is perturbed and the spread of the virus is not arrested at the site of infection. RBOHD is required for the spatial accumulation of SA, and conversely RBOHD is under the transcriptional regulation of SA. Using spatially resolved RNA‐seq, we also identified spatial regulation of an UDP‐glucosyltransferase, another component in feedback activation of SA biosynthesis, thus deciphering a novel aspect of resistance signaling.

While activation of resistance (R) proteins has been intensively studied, the mechanisms acting downstream of R protein activation remain elusive. We studied effector-triggered immunity (ETI) conditioned by the potato Ny-1 gene against potato virus Y. Transcriptomic, ultrastructural and biochemical analyses of four consecutive tissue sections in and surrounding the foci of viral infection at three different lesion developmental stages revealed processes that are spatiotemporally regulated. The transcriptional response in the cell death zone and surrounding tissue is dependent on SA. For some of the genes, spatiotemporal regulation is completely lost while for others, the regulation is different in SA-deficient line indicating multiple connections between hormonal signaling modules. The induction of RBOHD NADPH oxidase expression, together with expression of Thioredoxin H gene, occurs specifically in the border region of the lesion during ETI. In plants with silenced RBOHD, ETI is perturbed and virus spread is not arrested at the site of infection. Expression of RBOHD is under the control of SA. On the other hand, RBOHD activity is required for spatial regulation of SA accumulation. We identified an UDP-glucosyltransferase, encoding an enzyme involved in feedback activation of SA biosynthesis, that is derepressed at the border of the lesion in RBOHD silenced plants. Altogether, we revealed a novel aspect of viral ETI, the RBOHD-SA feedback loop required for its spatial regulation.

Terrestrial isopods are a successful group of terrestrial crustaceans. Their exoskeletal cuticle has been studied in several species. However, it is not known whether the cuticle of these animals becomes thicker or how its structure changes as the animals grow. We aimed to determine the principles of upscaling of the exoskeletal cuticle during growth in the terrestrial isopods Armadillidium vulgare (Latreille, 1804) and Porcellio scaber Latreille, 1804 with scanning electron microscopy. The tergal cuticle becomes thicker with increasing body length. In A. vulgare, which rolls into a ball in defense against predators, the rate of increase of tergite thickness was greater than in P. scaber, which clings to the substrate when threatened. As the cuticles ofboth species become thicker, the proportion of the endocuticle in the cuticle increases. There is a strong correlation between cuticle thickness and the thickness of endo cuticular lamellae. This indicates that in thicker cuticles, chitin-protein fibers in sequential layers change their orientation by a smaller angle. We found no significant differences in morphometric parameters between the cuticles of A. vulgare and P. scaber when controlling for cuticle thickness. This suggests that known differences in cuticle structure and composition between these two species may result from differences in cuticle thickness.

Structural and functional properties of exoskeleton in moulting sea slaters Ligia pallasii from the Eastern Pacific coast were investigated with CT scanning and electron microscopy. Ultrastructure of preecdysial and postecdysial cuticular layers was described in premoult, intramoult and postmoult animals. Cuticle is a flexible extracellular matrix connected to the epidermal cells through pore channels. During premoult epicuticle and exocuticle are formed and during intramoult and postmoult endocuticular lamellae are deposited and the cuticle is progressively constructed by thickening and mineralization. Cuticle permeability, flexibility and waterproofing capacity change accordingly. Elaboration of epicuticular scales connected to an extensive network of nanotubules, establish its anti-adhesive and hydrophobic properties. Labelling with gold conjugated WGA lectins on Tokuyashu thawed cryosections exposes differences in chitin content between exocuticle and endocuticle. Histochemical staining of cuticle shows presence of acidic carbohydrates/glycoconjugates and lipoproteins in epicuticular layer. Chitin microfibrils are formed at the microvillar border of epidermal cells with abundant Golgi apparatus and secretory vesicles. Numerous spherules associated with nanotubules were observed in the ecdysial space in intramoult animals. The mineral component of the cuticle as visualized with CT scanning indicates progressive mineral resorption from the posterior to the anterior half of the body in premoult animals, its translocation from the anterior to posterior part during intramoult and its progressive deposition in the posterior and anterior exoskeleton during postmoult. Cuticle of sea slaters is a unique biocomposite and biodynamic material constantly reconstructed during frequent moults, and adapted to specific physical and biotic conditions of the high intertidal rocky zone.

Abstract Background Glioblastoma is a highly aggressive central nervous system neoplasm characterized by extensive infiltration of malignant cells into brain parenchyma, thus preventing complete tumor eradication. Cysteine cathepsins B, S, L and K are involved in cancer progression and are overexpressed in glioblastoma. We report here for the first time that cathepsin X mRNA and protein are also abundantly present in malignant glioma. Materials and methods Gene expression of cathepsins K and X was analyzed using publically-available tran-scriptomic datasets and correlated with glioma grade and glioblastoma subtype. Kaplan-Maier survival analysis was performed to evaluate the predictive value of cathepsin K and X mRNA expression. Cathepsin protein expression was localized and semi-quantified in tumor tissues by immunohistochemistry. Results Highest gene expression of cathepsins K and X was found in glioblastoma, in particular in the mesenchymal subtype. Overall, high mRNA expression of cathepsin X, but not that of cathepsin K, correlated with poor patients’ survival. Cathepsin K and X proteins were abundantly and heterogeneously expressed in glioblastoma tissue. Immuno-labeling of cathepsins K and X was observed in areas of CD133-positive glioblastoma stem cells, localized around arterioles in their niches that also expressed SDF-1α and CD68. mRNA levels of both cathepsins K and X correlated with mRNA levels of markers of glioblastoma stem cells and their niches. Conclusions The presence of both cathepsins in glioblastoma stem cell niche regions indicates their possible role in regulation of glioblastoma stem cell homing in their niches. The clinical relevance of this data needs to be elaborated in further prospective studies.

BACKGROUND:Progress in high-throughput molecular methods accompanied by more complex experimental designs demands novel data visualisation solutions. To specifically answer the question which parts of the specifical biological system are responding in particular perturbation, integrative approach in which experimental data are superimposed on a prior knowledge network is shown to be advantageous.RESULTS:We have developed DiNAR, Differential Network Analysis in R, a user-friendly application with dynamic visualisation that integrates multiple condition high-throughput data and extensive biological prior knowledge. Implemented differential network approach and embedded network analysis allow users to analyse condition-specific responses in the context of topology of interest (e.g. immune signalling network) and extract knowledge concerning patterns of signalling dynamics (i.e. rewiring in network structure between two or more biological conditions). We validated the usability of software on the Arabidopsis thaliana and Solanum tuberosum datasets, but it is set to handle any biological instances.CONCLUSIONS:DiNAR facilitates detection of network-rewiring events, gene prioritisation for future experimental design and allows capturing dynamics of complex biological system. The fully cross-platform Shiny App is hosted and freely available at https://nib-si.shinyapps.io/DiNAR. The most recent version of the source code is available at https://github.com/NIB-SI/DiNAR/ with a DOI 10.5281/zenodo.1230523 of the archived version in Zenodo.

Bois noir (BN) is the most widespread European grapevine yellows disease caused by ‘Candidatus Phytoplasma solani’. Although our knowledge of the mechanisms of interactions of this pathogenic bacteria with host is largely unknown, the plant-pathogen system of BN is commonly used as a model system for studying grapevine yellows diseases. We applied here a conceptual model of general plant pathology – a disease triangle for describing interactions among the host plant, the pathogen and the environment. We generated a proof-of-concept statistical model for disease triangle using original experimental data and different statistical and data mining approaches for a selected system of ‘Ca. P. solani’ infection of cv. ‘Chardonnay’ grapevine plants. We monitored individual plants from a single vineyard over a period of six years. Phytoplasma content, the expression of 21 selected grapevine genes and environmental conditions were recorded and related to disease severity. Our model predicts that in described conditions BN is a function of the expression of grapevine gene VvDMR6, summer rainfall and abundance of ‘Ca. P. solani’. The greatest impact among elements of the disease triangle is attributed to the pathogen, and is independent of the pathogen titer. We showed that this first de facto representation of the disease triangle is useful for showing disease dynamics over several years and could be applied to other plant-pathogen systems. The overall results of this study will contribute to understanding of ‘Ca. P. solani’ biology and its interactions with grapevine host.

In vitro cell-based models are important tools for assessing efficacies of new leads in early phases of drug development. Human osteoarthritic chondrocytes (OACs), obtained from biomedical waste material, represent a valuable, relatively accessible cellular source that could be used for this purpose. By employing reverse transcription-polymerase chain reaction (qRT-PCR) we compared gene expression profiles of key anabolic, catabolic and inflammatory genes of freshly isolated vs. monolayer cultured OACs (passages P0-P2) and non-stimulated vs. tumor necrosis factor alpha (TNF-α) stimulated P2 OACs. After expansion of OACs in monolayer cultures, the expression of almost all analyzed genes significantly decreased. The subsequent addition of TNF-α to OACs at P2 significantly increased expressions of all catabolic and inflammatory genes, leaving the anabolic profile almost unchanged. TNF-α-treated OACs were later utilized for efficacy testing of anti-TNF-α drugs infliximab and etanercept and both significantly reduced the expressions of all catabolic and inflammatory genes tested.

OBJECTIVES:To evaluate the diagnostic and prognostic potential of preoperative serum CA-125 and HE4 levels in patients with endometrial cancer. METHODS:Prospective case-control study of 133 women who underwent surgical treatment at the University Medical Centre Ljubljana (64 patients with endometrial cancer, 69 control patients with prolapsed uterus or myoma). Serum CA-125 and HE4 levels were determined using electrochemiluminescent assays. RESULTS:Serum CA-125 and HE4 levels were significantly higher in patients with endometrial cancer, compared to the controls (p=2.67×10-4, 1.36×10-7, respectively). A diagnostic model that combines serum CA-125 and HE4 levels and body mass index separated patients with endometrial cancer from controls, with AUC of 0.804, sensitivity of 66.7%, and specificity of 84.6%. Serum HE4 levels showed good prognostic potential and stratified the patients according to presence/absence of deep myometrial invasion (p=0.001) or lymphovascular invasion (p=0.003), with AUCs of 0.78 and 0.81, respectively. In low-risk patients with grade 1 and 2 endometrioid cancer for whom lymphadenectomy can be avoided, HE4 allowed stratification according to deep myometrial invasion (p=3.39×10-4), with AUC of 0.84. Although median HE4 levels were higher in patients with lymphovascular invasion, this difference did not reach significance (p=0.06). CONCLUSIONS:A model based on preoperative serum CA-125 and HE4 levels and body mass index has good diagnostic accuracy for separation of patients with endometrial cancer and control patients. In patients with endometrial cancer, serum HE4 levels allow prediction of deep myometrial and lymphovascular invasion.

Quantitative PCR(qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR(dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility(expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision(coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.

Basic idea of science is reproducibility of phenomena and experiments. Reproducibility of data analyses and reports is becoming more and more important. It requires structured organization of data, augmented with enought metadata for future re-use of data. Our aim is to provide a system to store data in a way that can be used for small and moderate size projects and fulfill minimal requirements of ISA-tab and FAIR paradigm. Standard directory trees are applicable to research data storage. The main condition is that information is organized in files and we are not interested in the system that enables access to individual line/record or column/variable in a tabular data structure in the file. The tree structure is generated on the fly by use of batch files (on Windows platform) that generate necessary folders and meta-data template files. We implemented the system of standard directory trees for support of the research in our research unit. Most often, our research projects can be hierarchically structured into, what can be called, Investigation which is composed of several Studies. Each individual study can have one or more Assays. To reflect this hierarchy, we named such directory tree the pISA-tree. To make new levels we provide three batch files: makeInvestigation, makeStudy, and makeAssay. Special attention is given to Description files that contain meta-information about research, protocols, samples, features et cetera. They are in line with the standards accepted for particular assays (e.g. MIQE, MIAMI, MIRIAM,...) which allow exchange of data with other data management services. In particular, we have in mind FAIRdom platform and some digital notebooks (e.g. SciNote). pISA-tree structure rely on directories that are readily available on any computer platform and familiar to use by the researchers. Since the translation of meta-data into ISA-tab standard format is not too complex, pISA-tree is a step towards the FAIR paradigm.

Understanding statistical concepts is important for proper use of statistics. The idea of using simulations and dynamic graphics to foster understanding of statistical concepts is not new. In recent years, R became the lingua franca for statistical data analysis. While R graphical devices are not meant for display of animated graphics, my aim is to use base R graphics for display of animated graphical sequences. To enable dynamic graphics in R, I developed a package animatoR, which supports smooth transitions of graphical elements and simplifies preparation of animated displays. I will show some animations that can be useful for statistics teaching and present basic features of the animatoR package. The animatoR package is freely available at https://github.com/ablejec/animatoR.