Effects of individual traits vs. trait syndromes on assemblages of various herbivore guilds associated with central European Salix.

Plants employ diverse anti-herbivore defences that can covary to form syndromes consisting of multiple traits. Such syndromes are hypothesized to impact herbivores more than individual defences. We studied 16 species of lowland willows occurring in central Europe and explored if their chemical and physical traits form detectable syndromes. We tested for phylogenetic trends in the syndromes and explored whether three herbivore guilds (i.e., generalist leaf-chewers, specialist leaf-chewers, and gallers) are affected more by the detected syndromes or individual traits. The recovered syndromes showed low phylogenetic signal and were mainly defined by investment in concentration, richness, or uniqueness of structurally related phenolic metabolites. Resource acquisition traits or inducible volatile organic compounds exhibited a limited correlation with the syndromes. Individual traits composing the syndromes showed various correlations to the assemblages of herbivores from the three studied guilds. In turn, we found some support for the hypothesis that defence syndromes are composed of traits that provide defence against various herbivores. However, individual traits rather than trait syndromes explained more variation for all studied herbivore assemblages. The detected negative correlations between various phenolics suggest that investment trade-offs may occur primarily among plant metabolites with shared metabolic pathways that may compete for their precursors. Moreover, several traits characterizing the recovered syndromes play additional roles in willows other than defence from herbivory. Taken together, our findings suggest that the detected syndromes did not solely evolve as an anti-herbivore defence.

Drug Penetration Analysis in 3D Cell Cultures Using Fiducial-Based Semiautomatic Coregistration of MALDI MSI and Immunofluorescence Images.

In this paper, we present an easy-to-follow procedure for the analysis of tissue sections from 3D cell cultures (spheroids) by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and laser scanning confocal microscopy (LSCM). MALDI MSI was chosen to detect the distribution of the drug of interest, while fluorescence immunohistochemistry (IHC) followed by LSCM was used to localize the cells featuring specific markers of viability, proliferation, apoptosis and metastasis. The overlay of the mass spectrometry (MS) and IHC spheroid images, typically without any morphological features, required fiducial-based coregistration. The MALDI MSI protocol was optimized in terms of fiducial composition and antigen epitope preservation to allow MALDI MSI to be performed and directly followed by IHC analysis on exactly the same spheroid section. Once MS and IHC images were coregistered, the quantification of the MS and IHC signals was performed by an algorithm evaluating signal intensities along equidistant layers from the spheroid boundary to its center. This accurate colocalization of MS and IHC signals showed limited penetration of the clinically tested drug perifosine into spheroids during a 24 h period, revealing the fraction of proliferating and promigratory/proinvasive cells present in the perifosine-free areas, decrease of their abundance in the perifosine-positive regions, and distinguishing between apoptosis resulting from hypoxia/nutrient deprivation and drug exposure.

Faster speciation of fig-wasps than their host figs leads to decoupled speciation dynamics: Snapshots across the speciation continuum.

Even though speciation involving multiple interacting partners, such as plants and their pollinators, has attracted much research, most studies focus on isolated phases of the process. This currently precludes an integrated understanding of the mechanisms leading to cospeciation. Here, we examine population genetic structure across six species-pairs of figs and their pollinating wasps along an elevational gradient in New Guinea. Specifically, we test three hypotheses on the genetic structure within the examined species-pairs and find that the hypothesized genetic structures represent different phases of a single continuum, from incipient cospeciation to the full formation of new species. Our results also illuminate the mechanisms governing cospeciation, namely that fig wasps tend to accumulate population genetic differences faster than their figs, which initially decouples the speciation dynamics between the two interacting partners and breaks down their one-to-one matching. This intermediate phase is followed by genetic divergence of both partners, which may eventually restore the one-to-one matching among the fully formed species. Together, these findings integrate current knowledge on the mechanisms operating during different phases of the cospeciation process. They also reveal that the increasingly reported breakdowns in one-to-one matching may be an inherent part of the cospeciation process. Mechanistic understanding of this process is needed to explain how the extraordinary diversity of species, especially in the tropics, has emerged. Knowing which breakdowns in species interactions are a natural phase of cospeciation and which may endanger further generation of diversity seems critical in a constantly changing world.

Fast and accurate compensation of signal offset for T2 mapping.

OBJECTIVE: T2 maps are more vendor independent than other MRI protocols. Multi-echo spin-echo signal decays to a non-zero offset due to imperfect refocusing pulses and Rician noise, causing T2 overestimation by the vendor's 2-parameter algorithm. The accuracy of the T2 estimate is improved, if the non-zero offset is estimated as a third parameter. Three-parameter Levenberg-Marquardt (LM) T2 estimation takes several minutes to calculate, and it is sensitive to initial values. We aimed for a 3-parameter fitting algorithm that was comparably accurate, yet substantially faster. METHODS: Our approach gains speed by converting the 3-parameter minimisation problem into an empirically unimodal univariate problem, which is quickly minimised using the golden section line search (GS). RESULTS: To enable comparison, we propose a novel noise-masking algorithm. For clinical data, the agreement between the GS and the LM fit is excellent, yet the GS algorithm is two orders of magnitude faster. For synthetic data, the accuracy of the GS algorithm is on par with that of the LM fit, and the GS algorithm is significantly faster. The GS algorithm requires no parametrisation or initialisation by the user. DISCUSSION: The new GS T2 mapping algorithm offers a fast and much more accurate off-the-shelf replacement for the inaccurate 2-parameter fit in the vendor's software.

Quantitative Assessment of Anti-Cancer Drug Efficacy From Coregistered Mass Spectrometry and Fluorescence Microscopy Images of Multicellular Tumor Spheroids.

Spheroids-three-dimensional aggregates of cells grown from a cancer cell line-represent a model of living tissue for chemotherapy investigation. Distribution of chemotherapeutics in spheroid sections was determined using the matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Proliferating or apoptotic cells were immunohistochemically labeled and visualized by laser scanning confocal fluorescence microscopy (LSCM). Drug efficacy was evaluated by comparing coregistered MALDI MSI and LSCM data of drug-treated spheroids with LSCM only data of untreated control spheroids. We developed a fiducial-based workflow for coregistration of low-resolution MALDI MS with high-resolution LSCM images. To allow comparison of drug and cell distribution between the drug-treated and untreated spheroids of different shapes or diameters, we introduced a common diffusion-related coordinate, the distance from the spheroid boundary. In a procedure referred to as "peeling", we correlated average drug distribution at a certain distance with the average reduction in the affected cells between the untreated and the treated spheroids. This novel approach makes it possible to differentiate between peripheral cells that died due to therapy and the innermost cells which died naturally. Two novel algorithms-for MALDI MS image denoising and for weighting of MALDI MSI and LSCM data by the presence of cell nuclei-are also presented.

Divergent mitochondrial respiratory chains in phototrophic relatives of apicomplexan parasites.

Four respiratory complexes and ATP-synthase represent central functional units in mitochondria. In some mitochondria and derived anaerobic organelles, a few or all of these respiratory complexes have been lost during evolution. We show that the respiratory chain of Chromera velia, a phototrophic relative of parasitic apicomplexans, lacks complexes I and III, making it a uniquely reduced aerobic mitochondrion. In Chromera, putative lactate:cytochrome c oxidoreductases are predicted to transfer electrons from lactate to cytochrome c, rendering complex III unnecessary. The mitochondrial genome of Chromera has the smallest known protein-coding capacity of all mitochondria, encoding just cox1 and cox3 on heterogeneous linear molecules. In contrast, another photosynthetic relative of apicomplexans, Vitrella brassicaformis, retains the same set of genes as apicomplexans and dinoflagellates (cox1, cox3, and cob).

Total Variation-Based Reduction of Streak Artifacts, Ring Artifacts and Noise in 3D Reconstruction from Optical Projection Tomography.

Optical projection tomography (OPT) is a computed tomography technique at optical frequencies for samples of 0.5-15 mm in size, which fills an important "imaging gap" between confocal microscopy (for smaller samples) and large-sample methods such as fluorescence molecular tomography or micro magnetic resonance imaging. OPT operates in either fluorescence or transmission mode. Two-dimensional (2D) projections are taken over 360° with a fixed rotational increment around the vertical axis. Standard 3D reconstruction from 2D OPT uses the filtered backprojection (FBP) algorithm based on the Radon transform. FBP approximates the inverse Radon transform using a ramp filter that spreads reconstructed pixels to neighbor pixels thus producing streak and other types of artifacts, as well as noise. Artifacts increase the variation of grayscale values in the reconstructed images. We present an algorithm that improves the quality of reconstruction even for a low number of projections by simultaneously minimizing the sum of absolute brightness changes in the reconstructed volume (the total variation) and the error between measured and reconstructed data. We demonstrate the efficiency of the method on real biological data acquired on a dedicated OPT device.

The EPOS multi-disciplinary Data Portal for integrated access to solid Earth science datasets.

The European Plate Observing System (EPOS) is a long-term initiative aimed at integrating research infrastructures for solid Earth science in Europe. EPOS provides a sustainable, multidisciplinary user-oriented platform - the EPOS Data Portal - that facilitates data integration, access, use, and re-use, while adhering to the FAIR principles. The paper describes the key governance, community building, and technical aspects for achieving multidisciplinary data integration through the portal. It also outlines the key portal features for aggregating approximately 250 data sources from more than ten different scientific communities. The main architectural concepts underpinning the portal, namely the rich-metadata, the service-driven data provision, and the usage of semantics, are outlined. The paper discusses the challenges encountered during the creation of the portal, describes the community engagement process, and highlights the benefits to the scientific community and society. Future work includes expanding portal functionalities to include data analysis, processing, and visualization and releasing the portal as an open-source software package.

Leaf volatile and nonvolatile metabolites show different levels of specificity in response to herbivory.

Plants produce diverse chemical defenses with contrasting effects on different insect herbivores. Deploying herbivore-specific responses can help plants increase their defensive efficiency. Here, we explore how variation in induced plant responses correlates with herbivore species, order, feeding guild, and level of specialization. In a greenhouse experiment, we exposed 149 plants of Salix fragilis (Linnaeus, 1753) to 22 herbivore species naturally associated with this host. The insects belonged to four orders (Coleoptera, Lepidoptera, Hemiptera, and Hymenoptera), three feeding guilds (external leaf-chewers, leaf-tying chewers, and sap-sucking), and included both dietary specialists and generalists. Following herbivory, we quantified induced changes in volatiles and nonvolatile leaf metabolites. We performed multivariate analyses to assess the correlation between herbivore order, feeding guild, dietary specialization, chewing damage by herbivores, and induced responses. The volatile composition was best explained by chewing damage and insect order, with Coleoptera and Lepidoptera eliciting significantly different responses. Furthermore, we recorded significant differences in elicited volatiles among some species within the two orders. Variation in nonvolatile leaf metabolites was mainly explained by the presence of insects, as plants exposed to herbivores showed significantly different metabolites from controls. Herbivore order also played a role to some extent, with beetles eliciting different responses than other herbivores. The induction of volatile and nonvolatile leaf metabolites shows different levels of specificity. The specificity in volatiles could potentially serve as an important cue to specialized predators or parasitoids, increasing the efficacy of volatiles as indirect defenses. By contrast, the induction of nonvolatile leaf metabolites was largely unaffected by herbivore identity. Most nonvolatile metabolites were downregulated, possibly indicating that plants redirected their resources from leaves in response to herbivory. Our results demonstrate how diverse responses to herbivores can contribute to the diversity of plant defensive strategies.

The biotechnology sector in a latecomer country: The case of Poland.

This paper offers a detailed firm-level analysis of innovation strategies, exporting activities and employment and sales dynamics of Polish biotechnology companies. The study is based on the unique dataset provided by three runs of the Polish edition of the Community Innovation Survey (CIS). Poland is a latecomer in the biotechnology industry, as are all the Central and Eastern European Countries (CEECs). In addition, the Polish biotechnology sector faced relatively unfavorable starting conditions: no partners in the pharmaceutical industry and, unlike some other CEECs, little government support. Nevertheless, biotechnology companies have developed in several Polish industries. It is shown that these companies implement innovation strategies that are typical for the biotechnology sector; however, they do not cooperate much with their clients. The innovation expenditure of biotechnology companies is distinctively higher than that of other companies. In addition, government and EU grants play a crucial role in funding the R&D activities of Polish biotechnology companies. This reflects the change in the innovation policy in Poland, but also raises the question of the sustainability of these efforts. Biotechnology companies are strongly export-oriented but, contrary to expectations, their dynamics are average: when controlling for firm characteristics and export performance, biotechnology companies neither outperform nor underperform other companies in terms of sales or employment growth.

How big a drop in agricultural exports to the United Kingdom after Brexit? Simulations for sensitive products of four Visegrad countries.

After the European Union (EU) was left by the United Kingdom (UK), a free trade area was established between these economies. Although bilateral trade in all goods is tariff-free, regulatory requirements make exports more costly and burdensome. We used a partial equilibrium model to analyze the implications of Brexit for agricultural exports from Visegrad countries (Czechia, Hungary, Poland and Slovakia). We assess trade creation and trade diversion effects resulting from an increase in non-tariff measures and border costs for 4-digit agricultural products identified as sensitive in each of the Visegrad countries. The simulations reveal that exports of sensitive products from Visegrad countries to the UK could decrease by up to 20%. While the macroeconomic importance of this change is not significant, for the producers of the sensitive goods such export losses are substantial.

Predicting distributions of Wolbachia strains through host ecological contact-Who's manipulating whom?

Reproductive isolation in response to divergent selection is often mediated via third-party interactions. Under these conditions, speciation is inextricably linked to ecological context. We present a novel framework for understanding arthropod speciation as mediated by Wolbachia, a microbial endosymbiont capable of causing host cytoplasmic incompatibility (CI). We predict that sympatric host sister-species harbor paraphyletic Wolbachia strains that provide CI, while well-defined congeners in ecological contact and recently diverged noninteracting congeners are uninfected due to Wolbachia redundancy. We argue that Wolbachia provides an adaptive advantage when coupled with reduced hybrid fitness, facilitating assortative mating between co-occurring divergent phenotypes-the contact contingency hypothesis. To test this, we applied a predictive algorithm to empirical pollinating fig wasp data, achieving up to 91.60% accuracy. We further postulate that observed temporal decay of Wolbachia incidence results from adaptive host purging-adaptive decay hypothesis-but implementation failed to predict systematic patterns. We then account for post-zygotic offspring mortality during CI mating, modeling fitness clines across developmental resources-the fecundity trade-off hypothesis. This model regularly favored CI despite fecundity losses. We demonstrate that a rules-based algorithm accurately predicts Wolbachia infection status. This has implications among other systems where closely related sympatric species encounter adaptive disadvantage through hybridization.

Nonrigid registration of CLSM images of physical sections with discontinuous deformations.

When biological specimens are cut into physical sections for three-dimensional (3D) imaging by confocal laser scanning microscopy, the slices may get distorted or ruptured. For subsequent 3D reconstruction, images from different physical sections need to be spatially aligned by optimization of a function composed of a data fidelity term evaluating similarity between the reference and target images, and a regularization term enforcing transformation smoothness. A regularization term evaluating the total variation (TV), which enables the registration algorithm to account for discontinuities in slice deformation (ruptures), while enforcing smoothness on continuously deformed regions, was proposed previously. The function with TV regularization was optimized using a graph-cut (GC) based iterative solution. However, GC may generate visible registration artifacts, which impair the 3D reconstruction. We present an alternative, multilabel TV optimization algorithm, which in the examined samples prevents the artifacts produced by GC. The algorithm is slower than GC but can be sped up several times when implemented in a multiprocessor computing environment. For image pairs with uneven brightness distribution, we introduce a reformulation of the TV-based registration, in which intensity-based data terms are replaced by comparison of salient features in the reference and target images quantified by local image entropies.

Fatty Acid Biosynthesis in Chromerids.

Fatty acids are essential components of biological membranes, important for the maintenance of cellular structures, especially in organisms with complex life cycles like protozoan parasites. Apicomplexans are obligate parasites responsible for various deadly diseases of humans and livestock. We analyzed the fatty acids produced by the closest phototrophic relatives of parasitic apicomplexans, the chromerids Chromera velia and Vitrella brassicaformis, and investigated the genes coding for enzymes involved in fatty acids biosynthesis in chromerids, in comparison to their parasitic relatives. Based on evidence from genomic and metabolomic data, we propose a model of fatty acid synthesis in chromerids: the plastid-localized FAS-II pathway is responsible for the de novo synthesis of fatty acids reaching the maximum length of 18 carbon units. Short saturated fatty acids (C14:0-C18:0) originate from the plastid are then elongated and desaturated in the cytosol and the endoplasmic reticulum. We identified giant FAS I-like multi-modular enzymes in both chromerids, which seem to be involved in polyketide synthesis and fatty acid elongation. This full-scale description of the biosynthesis of fatty acids and their derivatives provides important insights into the reductive evolutionary transition of a phototropic algal ancestor to obligate parasites.

Lactic Acidosis Interferes With Toxicity of Perifosine to Colorectal Cancer Spheroids: Multimodal Imaging Analysis.

Colorectal cancer (CRC) is a disease with constantly increasing incidence and high mortality. The treatment efficacy could be curtailed by drug resistance resulting from poor drug penetration into tumor tissue and the tumor-specific microenvironment, such as hypoxia and acidosis. Furthermore, CRC tumors can be exposed to different pH depending on the position in the intestinal tract. CRC tumors often share upregulation of the Akt signaling pathway. In this study, we investigated the role of external pH in control of cytotoxicity of perifosine, the Akt signaling pathway inhibitor, to CRC cells using 2D and 3D tumor models. In 3D settings, we employed an innovative strategy for simultaneous detection of spatial drug distribution and biological markers of proliferation/apoptosis using a combination of mass spectrometry imaging and immunohistochemistry. In 3D conditions, low and heterogeneous penetration of perifosine into the inner parts of the spheroids was observed. The depth of penetration depended on the treatment duration but not on the external pH. However, pH alteration in the tumor microenvironment affected the distribution of proliferation- and apoptosis-specific markers in the perifosine-treated spheroid. Accurate co-registration of perifosine distribution and biological response in the same spheroid section revealed dynamic changes in apoptotic and proliferative markers occurring not only in the perifosine-exposed cells, but also in the perifosine-free regions. Cytotoxicity of perifosine to both 2D and 3D cultures decreased in an acidic environment below pH 6.7. External pH affects cytotoxicity of the other Akt inhibitor, MK-2206, in a similar way. Our innovative approach for accurate determination of drug efficiency in 3D tumor tissue revealed that cytotoxicity of Akt inhibitors to CRC cells is strongly dependent on pH of the tumor microenvironment. Therefore, the effect of pH should be considered during the design and pre-clinical/clinical testing of the Akt-targeted cancer therapy.

Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites.

The eukaryotic phylum Apicomplexa encompasses thousands of obligate intracellular parasites of humans and animals with immense socio-economic and health impacts. We sequenced nuclear genomes of Chromera velia and Vitrella brassicaformis, free-living non-parasitic photosynthetic algae closely related to apicomplexans. Proteins from key metabolic pathways and from the endomembrane trafficking systems associated with a free-living lifestyle have been progressively and non-randomly lost during adaptation to parasitism. The free-living ancestor contained a broad repertoire of genes many of which were repurposed for parasitic processes, such as extracellular proteins, components of a motility apparatus, and DNA- and RNA-binding protein families. Based on transcriptome analyses across 36 environmental conditions, Chromera orthologs of apicomplexan invasion-related motility genes were co-regulated with genes encoding the flagellar apparatus, supporting the functional contribution of flagella to the evolution of invasion machinery. This study provides insights into how obligate parasites with diverse life strategies arose from a once free-living phototrophic marine alga.

A piecewise monotone subgradient algorithm for accurate L¹-TV based registration of physical slices with discontinuities in microscopy.

Image registration tasks are often formulated in terms of minimization of a functional consisting of a data fidelity term penalizing the mismatch between the reference and the target image, and a term enforcing smoothness of shift between neighboring pairs of pixels (a min-sum problem). Most methods for deformable image registration use some form of interpolation between matching control points. The interpolation makes it impossible to account for isolated discontinuities in the deformation field that may appear, e.g., when a physical slice of a microscopy specimen is ruptured by the cutting tool. For registration of neighboring physical slices of microscopy specimens with discontinuities, Janácek proposed an L¹-distance data fidelity term and a total variation (TV) smoothness term, and used a graph-cut (GC) based iterative steepest descent algorithm for minimization. The L¹-TV functional is nonconvex; hence a steepest descent algorithm is not guaranteed to converge to the global minimum. Schlesinger presented transformation of max-sum problems to minimization of a dual quantity called problem power, which is--contrary to the original max-sum functional--convex. Based on Schlesinger's solution to max-sum problems we developed an algorithm for L¹-TV minimization by iterative multi-label steepest descent minimization of the convex dual problem. For Schlesinger's subgradient algorithm we proposed a novel step control heuristics that considerably enhances both speed and accuracy compared with standard step size strategies for subgradient methods. It is shown experimentally that our subgradient scheme achieves consistently better image registration than GC in terms of lower values both of the composite L¹-TV functional, and of its components, i.e., the L¹ distance of the images and the transformation smoothness TV, and yields visually acceptable results even in cases where the GC based algorithm fails. The new algorithm allows easy parallelization and can thus be sped up by running on multi-core graphic processing units.

Compensation of inhomogeneous fluorescence signal distribution in 2D images acquired by confocal microscopy.

In images acquired by confocal laser scanning microscopy (CLSM), regions corresponding to the same concentration of fluorophores in the specimen should be mapped to the same grayscale levels. However, in practice, due to multiple distortion effects, CLSM images of even homogeneous specimen regions suffer from irregular brightness variations, e.g., darkening of image edges and lightening of the center. The effects are yet more pronounced in images of real biological specimens. A spatially varying grayscale map complicates image postprocessing, e.g., in alignment of overlapping regions of two images and in 3D reconstructions, since measures of similarity usually assume a spatially independent grayscale map. We present a fast correction method based on estimating a spatially variable illumination gain, and multiplying acquired CLSM images by the inverse of the estimated gain. The method does not require any special calibration of reference images since the gain estimate is extracted from the CLSM image being corrected itself. The proposed approach exploits two types of morphological filters: the median filter and the upper Lipschitz cover. The presented correction method, tested on images of both artificial (homogeneous fluorescent layer) and real biological specimens, namely sections of a rat embryo and a rat brain, proved to be very fast and yielded a significant visual improvement.