Pea root responses under naproxen stress: changes in the formation of structural barriers in the primary root in context with changes of auxin and abscisic acid levels.

Pharmaceuticals belong to pseudo-persistent pollutants because of constant entry into the environment and hazardous potential for non-target organisms, including plants, in which they can influence biochemical and physiological processes. Detailed analysis of results obtained by microscopic observations using fluorescent dyes (berberine hemisulphate, Fluorol Yellow 088), detection of phytohormone levels (radioimmunoassay, enzyme-linked immune sorbent assay) and thermogravimetric analysis of lignin content proved that the drug naproxen (NPX) can stimulate the formation of root structural barriers. In the primary root of plants treated with 0.5, 1, and 10 mg/L NPX, earlier Casparian strip formation and development of the whole endodermis circle closer to its apex were found after five days of cultivation (by 9-20% as compared to control) and after ten days from 0.1 mg/L NPX (by 8-63%). Suberin lamellae (SL) were deposited in endodermal cells significantly closer to the apex under 10 mg/L NPX by up to 75%. Structural barrier formation under NPX treatment can be influenced indirectly by auxin-supported cell division and differentiation caused by its eight-times higher level under 10 mg/L NPX and directly by stimulated SL deposition induced by abscisic acid (higher from 0.5 mg/L NPX), as proved by the higher proportion of cells with SL in the primary root base (by 8-44%). The earlier modification of endodermis in plant roots can help to limit the drug transfer and maintain the homeostasis of the plant.

Symmetry breaking in spreading RAT2 fibroblasts requires the MAPK/ERK pathway scaffold RACK1 that integrates FAK, p190A-RhoGAP and ERK2 signaling.

The spreading of adhering cells is a morphogenetic process during which cells break spherical or radial symmetry and adopt migratory polarity with spatially segregated protruding cell front and non-protruding cell rear. The organization and regulation of these symmetry-breaking events, which are both complex and stochastic, are not fully understood. Here we show that in radially spreading cells, symmetry breaking commences with the development of discrete non-protruding regions characterized by large but sparse focal adhesions and long peripheral actin bundles. Establishment of this non-protruding static region specifies the distally oriented protruding cell front and thus determines the polarity axis and the direction of cell migration. The development of non-protruding regions requires ERK2 and the ERK pathway scaffold protein RACK1. RACK1 promotes adhesion-mediated activation of ERK2 that in turn inhibits p190A-RhoGAP signaling by reducing the peripheral localization of p190A-RhoGAP. We propose that sustained ERK signaling at the prospective cell rear induces p190A-RhoGAP depletion from the cell periphery resulting in peripheral actin bundles and cell rear formation. Since cell adhesion activates both ERK and p190A-RhoGAP signaling this constitutes a spatially confined incoherent feed-forward signaling circuit.

Depletion of calpain2 accelerates epithelial barrier establishment and reduces growth factor-induced cell scattering.

Calpain2 is a conventional member of the non-lysosomal calpain protease family that has been shown to affect the dynamics of focal and cell-cell adhesions by proteolyzing the components of adhesion complexes. Here, we inactivated calpain2 using CRISPR/Cas9 in epithelial MDCK cells. We show that depletion of calpain2 has multiple effects on cell morphology and function. Calpain2-depleted cells develop epithelial shape, however, they cover a smaller area, and cell clusters are more compact. Inactivation of calpain2 enhanced restoration of transepithelial electrical resistance after calcium switch, decreased cell migration, and delayed cell scattering induced by HGF/SF. In addition, calpain2 depletion prevented morphological changes induced by ERK2 overexpression. Interestingly, proteolysis of several calpain2 targets, including E-cadherin, ß-catenin, talin, FAK, and paxillin, was not discernibly affected by calpain2 depletion. Taken together, these data suggest that calpain2 regulates the stability of cell-cell and cell-substratum adhesions indirectly without affecting the proteolysis of these adhesion complexes.

Disaggregation of canopy photosynthesis among tree species in a mixed broadleaf forest.

Carbon dioxide sequestration from the atmosphere is commonly assessed using the eddy covariance method. Its net flux signal can be decomposed into gross primary production and ecosystem respiration components, but these have seldom been tested against independent methods. In addition, eddy covariance lacks the ability to partition carbon sequestration among individual trees or species within mixed forests. Therefore, we compared gross primary production from eddy covariance versus an independent method based on sap flow and water-use efficiency, as measured by the tissue heat balance method and δ13C of phloem contents, respectively. The latter measurements were conducted on individual trees throughout a growing season in a mixed broadleaf forest dominated by three tree species, namely English oak, narrow-leaved ash and common hornbeam (Quercus robur L., Fraxinus angustifolia Vahl, and Carpinus betulus L., respectively). In this context, we applied an alternative ecophysiological method aimed at verifying the accuracy of a state-of-the-art eddy covariance system while also offering a solution to the partitioning problem. We observed strong agreement in the ecosystem gross primary production estimates (R2 = 0.56; P < 0.0001), with correlation being especially high and nearly on the 1:1 line in the period before the end of July (R2 = 0.85; P < 0.0001). After this period, the estimates of gross primary production began to diverge. Possible reasons for the divergence are discussed, focusing especially on phenology and the limitation of the isotopic data. English oak showed the highest per-tree daily photosynthetic rates among tree species, but the smaller, more abundant common hornbeam contributed most to the stand-level summation, especially early in the spring. These findings provide a rigorous test of the methods and the species-level photosynthesis offers avenues for enhancing forest management aimed at carbon sequestration.

Impacts of elevated CO2 levels and temperature on photosynthesis and stomatal closure along an altitudinal gradient are counteracted by the rising atmospheric vapor pressure deficit.

The efficiency of water use in plants, a critical ecophysiological parameter closely related to water and carbon cycles, is essential for understanding the interactions between plants and their environment. This study investigates the effects of ongoing climate change and increasing atmospheric CO2 concentration on intrinsic (stomata-based; iWUE) and evaporative (transpiration-based; eWUE) water use efficiency in oak trees along a naturally small altitudinal gradient (130-630 m a.s.l.) of Vihorlat Mountains (eastern Slovakia, Central Europe). To assess changes in iWUE and eWUE values over the past 60 years (1961-2020), stable carbon isotope ratios in latewood cellulose (δ13Ccell) of annually resolved tree rings were analyzed. Such an approach was sensitive enough to distinguish tree responses to growth environments at different altitudes. Our findings revealed a rising trend in iWUE, particularly in oak trees at low and middle altitudes. However, this increase was negligible at high altitudes. Warmer and drier conditions at lower altitudes likely led to significant stomatal closure and enhanced efficiency in photosynthetic CO2 uptake due to rising CO2 concentration. Conversely, the increasing intracellular-to-ambient CO2 ratio (Ci/Ca) at higher altitudes indicated lower efficiency in photosynthetic CO2 uptake. In contrast to iWUE, eWUE showed no increasing trends over the last 60 years. This suggests that the positive impacts of elevated CO2 concentrations and temperature on photosynthesis and stomatal closure are counteracted by the rising atmospheric vapor pressure deficit (VPD). These differences underscore the importance of the correct interpretation of stomata-based and transpiration-based WUEs and highlight the necessity of atmospheric VPD correction when applying tree-ring δ13C-derived WUE at ecosystem and global levels.

Grain carbon isotopes indicate the ability of wheat plants to maintain enhanced intrinsic water-use efficiency even after short-term exposure to high temperatures and drought.

Minimizing the impact of heat and drought on crop yields requires varieties with effective protective mechanisms. We tested the hypothesis that even a short-term high temperature amplifies the negative effects of reduced water availability on leaf gas-exchange, but can induce long-lasting improvement in plant water-use efficiency after the stress period. Accordingly, three common varieties of winter wheat (Triticum aestivum) were grown under field conditions. During the stem extension, the plants were exposed to distinct temperatures (daily maximum 26 vs. 38 °C), water availabilities (75% of field water capacity vs. permanent wilting point), and their combination for 14 days. All treatments reduced light-saturated rates of CO2 assimilation and transpiration, particularly when heat and drought were combined. Drought enhanced water-use efficiency (WUE) in all varieties (31.4-36.4%), but not at high temperatures (decrease by 17-52%). Intrinsic WUE (iWUE), determined from the stable carbon isotope composition of grains, was enhanced by 7.9-37% in all treatments and varieties; however, not all changes were significant. The combination of heat and drought tended to increase total protein content in grains but reduced spike productivity. Noticeably, the strongest decline in spike productivity was observed in Elan - the variety displaying the smallest enhancement of iWUE, while it was negligible in Pannonia which shows the most pronounced improvement of iWUE. We conclude that even several hot and dry days can improve iWUE for the rest of the vegetation season. This improvement, however, does not necessarily lead to increased crop productivity possibly due to physiological trade-offs.

ERK2 signaling regulates cell-cell adhesion of epithelial cells and enhances growth factor-induced cell scattering.

The ERK signaling pathway, consisting of core protein kinases Raf, MEK and effector kinases ERK1/2, regulates various biological outcomes such as cell proliferation, differentiation, apoptosis, or cell migration. Signal transduction through the ERK signaling pathway is tightly controlled at all levels of the pathway. However, it is not well understood whether ERK pathway signaling can be modulated by the abundance of ERK pathway core kinases. In this study, we investigated the effects of low-level overexpression of the ERK2 isoform on the phenotype and scattering of cuboidal MDCK epithelial cells growing in discrete multicellular clusters. We show that ERK2 overexpression reduced the vertical size of lateral membranes that contain cell-cell adhesion complexes. Consequently, ERK2 overexpressing cells were unable to develop cuboidal shape, remained flat with increased spread area and intercellular adhesive contacts were present only on the basal side. Interestingly, ERK2 overexpression was not sufficient to increase phosphorylation of multiple downstream targets including transcription factors and induce global changes in gene expression, namely to increase the expression of pro-migratory transcription factor Fra1. However, ERK2 overexpression enhanced HGF/SF-induced cell scattering as these cells scattered more rapidly and to a greater extent than parental cells. Our results suggest that an increase in ERK2 expression primarily reduces cell-cell cohesion and that weakened intercellular adhesion synergizes with upstream signaling in the conversion of the multicellular epithelium into single migrating cells. This mechanism may be clinically relevant as the analysis of clinical data revealed that in one type of cancer, pancreatic adenocarcinoma, ERK2 overexpression correlates with a worse prognosis.

Overview of Erasmus+ NETCHEM project: ICT networking for overcoming technical and social barriers in instrumental analytical chemistry education.

The paper briefly presents goals, activities, challenges, and outcomes of the NETCHEM project ( http://www.netchem.ac.rs/ ) that was co-funded by the Erasmus+ Program of European Union (573885-EPP-1-2016-1-RS-EPPKA2- CBHE-JP). The project has been started in October 2016 and with extension lasted until April 2020. Western Balkan region has been targeted by upgrading capacities for education and research in environmental and food analysis in cooperation with partners from France, the UK, and Czech Republic. NETCHEM platform providing Web Accessed Remote Instrumental Analytical Laboratories (WARIAL) network, Database service and Open education system was created in order to improve the cooperation, educational, and research capacities of Higher Education Institutions involved, but also targeting whether audience not only from academic domain but from industry as well. The NETCHEM platform is free for access to public; thus, the external users to NETCHEM consortium can not only see its content but also actively participate, enter Database and WARIAL network, and upload their own educational/research material.

Fusarium Mycotoxins Stability during the Malting and Brewing Processes.

Mycotoxins are widely studied by many research groups in all aspects, but the stability of these compounds needs further research for clarification. The objective of this study is to evaluate deoxynivalenol and zearalenone stability during all steps of the malting and brewing processes. The levels of these compounds decreased significantly during the production process (barley to beer). During the malting process, the DON levels decreased significantly in the steeping, germination, and malting steps (62%, 51.5%, and 68%, respectively). Considering ZEN, when the levels were compared between barley and the last step of the process, a significant decrease was observed. Most of the mycotoxins produced were transferred to the rootlets and spent grains, which is advantageous considering the final product. Furthermore, the mycotoxin dietary intake estimation was included in this study. The results proved that if the concentrations of target mycotoxins in raw material are under the limits established by the regulations, the levels decrease during the malting and brewing processes and make the beer secure for consumers. The quality of the five commodities involved in the beer process plays a decisive role in the creation of a safe final product.

The assembly and function of perinuclear actin cap in migrating cells.

Stress fibers are actin bundles encompassing actin filaments, actin-crosslinking, and actin-associated proteins that represent the major contractile system in the cell. Different types of stress fibers assemble in adherent cells, and they are central to diverse cellular processes including establishment of the cell shape, morphogenesis, cell polarization, and migration. Stress fibers display specific cellular organization and localization, with ventral fibers present at the basal side, and dorsal fibers and transverse actin arcs rising at the cell front from the ventral to the dorsal side and toward the nucleus. Perinuclear actin cap fibers are a specific subtype of stress fibers that rise from the leading edge above the nucleus and terminate at the cell rear forming a dome-like structure. Perinuclear actin cap fibers are fixed at three points: both ends are anchored in focal adhesions, while the central part is physically attached to the nucleus and nuclear lamina through the linker of nucleoskeleton and cytoskeleton (LINC) complex. Here, we discuss recent work that provides new insights into the mechanism of assembly and the function of these actin stress fibers that directly link extracellular matrix and focal adhesions with the nuclear envelope.

Synergistic effect of ligating and ionic functions, prearranged on a calix[4]arene.

The covalent attachment of two CMPO-functions and two anionic Cosan groups to the narrow rim of tert-butylcalix[4]arene leads to a dramatic increase of the extraction efficiency for the cone isomer; Am(3+) is removed from 5 x 10(-8) M solution to more than 99% by a single extraction step with a 3 x 10(-6) M solution of the calixarene.

UPLC-MS/MS method for determination of selected pyrrolizidine alkaloids in feed.

Alkaloids known as secondary metabolites are grouped by typical structural characteristics into large families such as pyrrolizidine alkaloids (PAs) comprising more than 350 individual heterocyclic compounds. The PAs present a serious health risk to human and livestock; hence there is a need for methods that allow these dangerous plant toxins to be determined. In this study, a fast, reliable and sensitive approach is proposed to identify and quantify PAs in feed samples. PAs including monocrotaline, senkirkine, senecionine, seneciphylline and retrorsine were determined by ultra-performance liquid chromatography coupled with tandem mass spectrometry. Sample preparation was based on a modified QuEChERS approach. The mean recovery, precision, matrix effects and limits of quantification were assessed for three matrices within the method validation. The presented method was used to inspect 41 various feed samples, where the presence of PAs was expected. Roughages and feed for rabbits contained the highest levels of PAs, in general.

ERK and RSK regulate distinct steps of a cellular program that induces transition from multicellular epithelium to single cell phenotype.

The ERK (extracellular signal-regulated kinases) cascade has an evolutionarily conserved three tier architecture consisting of protein kinases Raf, MEK (MAPK/ERK kinase) and ERK. Following activation, ERK phosphorylates various cellular elements leading to diverse cellular responses. Downstream of ERK the family of p90 ribosomal S6 kinases (RSKs) has been proven to be an important conveyor of ERK signaling, however, little is known if ERK and RSK coordinate their functions to generate a specific biological response. Here we show that in epithelial cells conditional activation of the ERK pathway causes phenotypic conversion of epithelial cells to autonomously migrating cells. This process involves two sequential steps characterized by loss of apical-basal polarity followed by cell scattering. The activation of ERK, but not RSK, is sufficient for the execution of the first step and it requires calpain mediated remodeling of actin cytoskeleton. Conversely, RSK regulates the successive stage characterized by cell-cell contact weakening and increased cellular migration. Thus, ERK and RSK regulate different cellular subprograms and coordinated execution of these subprograms in time generates a relevant biological response. Our data also suggest that the mechanism by which the ERK pathway controls a cellular response may be distributed between ERK and RSK, rather than being elicited by a single effector kinase.

Comparison of negative ion electrospray mass spectra measured by seven tandem mass analyzers towards library formation.

A library of negative ion electrospray ionization mass spectra and tandem mass spectra (MS/MS) of sulfonated dyes has been developed for fast identification purposes. The uniform protocol has been elaborated and applied to the measurements of more than 50 anionic dyes. Three collision energies are selected in our protocol which ensures that at least one of them provides a suitable ratio of product ions to the precursor ion. The robustness is investigated with altered values of tuning parameters (e.g. the pressure of the nebulizing gas, the temperature and the flow rate of drying gas, and the mobile phase composition). The results of the inter-laboratory comparison of product ion mass spectra recorded on seven different tandem mass spectrometers (three ion traps, two triple quadrupoles and two hybrid quadrupole time of flight instruments) are presented for four representative anionic dyes--azo dye Acid Red 118, anthraquinone dye Acid Violet 43, triphenylmethane dye Acid Blue 1 and Al(III) metal-complex azo dye. The fragmentation patterns are almost identical for all tandem mass analyzers, only the ratios of product ions differ somewhat which confirms the possibility of spectra transfer among different mass analyzers with the goal of library formation.

Comparison of two different solvents employed for pressurised fluid extraction of stevioside from Stevia rebaudiana: methanol versus water.

Pressurised fluid extraction using water or methanol was employed for the extraction of stevioside from Stevia rebaudiana Bertoni. The extraction method was optimised in terms of temperature and duration of the static or the dynamic step. Extracts were analysed by liquid chromatography followed by UV and mass-spectrometric (MS) detections. Thermal degradation of stevioside was the same in both solvents within the range 70-160 degrees C. Methanol showed better extraction ability for isolation of stevioside from Stevia rebaudiana leaves than water within the range 110-160 degrees C. However, water represents the green alternative to methanol. The limit of detection of stevioside in the extract analysed was 30 ng for UV detection and 2 ng for MS detection.

Synthesis, characterization, and UV-vis linear absorption of centrosymmetric pi-systems incorporating closo-dodecaborate clusters.

Single- and multibranched centrosymmetric derivatives incorporating B12 clusters [B12H11-N(H)=C(H)-C6H4-C6H4-C(H)=(H)N-B12H11]2- (3) and [1,3,5-(4-(B12H11-N(H)=C(H))-C6H4)-C6H3]3- (5) have been synthesized. Both derivatives were characterized by multinuclear NMR and ESI-MS analyses. To the best of our knowledge, compound 5 is the first example of a multicage derivative bearing three B12 units. Compounds 3 and 5 are only slightly yellowish colored. The UV-vis absorption curves of 3 and 5 show intense absorption bands at 360 and 314 nm, respectively. This result permits us to confirm the strong donor effect of the B12 cluster. The hypsochrome effect observed for compound 5 compared to that of compound 3 confirms the interest in multibranched derivatives for the preparation of two-photon absorption materials active in the visible range.