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Twisted growth serves myriad adaptive functions in plants. Unlike animal motions, plant motions require symmetry breaking during growth and typically involve microtubule-related genes. But how macroscopic twisting emerges from molecular-level perturbations remains unclear. Here, we show that microtubule-based symmetry breaking propagates across multiple organizational scales via the epidermis to produce handed root skewing. At the nanoscale, aberrant patterning of cellulose microfibrils is associated with microscale skewed cell expansion, both of which precede the millimeter scale emergence of helical epidermal cell files. The resulting chiral torsion of the epidermis mediates organ level symmetry breaking in the form of whole-root skewing through macroscale interactions between the root and its surrounding environment. We demonstrate the dominant role of the epidermis by complementation of microtubule activity in the epidermis alone, which is sufficient to restore transverse cortical microtubule orientation, wild-type-like morphology in cortical cells, and straight root growth. Nolan et al use Arabidopsis twisted mutants to show that frustrated distortion of the epidermis is the primary organ-level determinant of multilevel asymmetries that underlie skewed root growth.

Background Lung cancer is the second most commonly occurring cancer. The ability to metastasize and spread to distant locations renders the tumor more aggressive. Members of the Rho subfamily of small GTP-binding proteins (GTPases) play a central role in the regulation of the actin cytoskeleton and in cancer cell migration and metastasis. In this study we investigated the role of the RhoA/Cdc42 GAP, StarD13, a previously described tumor suppressor, in malignancy, migration and invasion of the lung cancer cells A549. Methods We knocked down StarD13 expression in A549 lung cancer cells and tested the effect on cell migration and invadopodia formation using time lapse imaging and invasion assays. We also performed rescue experiments to determine the signaling pathways downstream of StarD13 and transfected the cells with FRET biosensors for RhoGTPases to identify the proteins involved in invadopodia formation. Results We observed a decrease in the level of expression of StarD13 in lung tumor tissues compared to normal lung tissues through immunohistochemistry. StarD13 also showed a lower expression in the lung adenocarcinoma cell line A549 compared to normal lung cells, WI38. In addition, the depletion of StarD13 increased cell proliferation and viability in WI38 and A549 cells, suggesting that StarD13 might potentially be a tumor suppressor in lung cancer. The depletion of StarD13, however, inhibited cell motility, conversely demonstrating a positive regulatory role in cell migration. This was potentially due to the constitutive activation of RhoA detected by pull down and FRET assays. Surprisingly, StarD13 suppressed cell invasion by inhibiting Cdc42-mediated invadopodia formation. Indeed, TKS4 staining and invadopodia assay revealed that StarD13 depletion increased Cdc42 activation as well as invadopodia formation and matrix degradation. Normal lung cells depleted of StarD13 also produced invadopodia, otherwise a unique hallmark of invasive cancer cells. Cdc42 knock down mimicked the effects of StarD13, while overexpression of a constitutively active Cdc42 mimicked the effects of its depletion. Finally, immunostaining and FRET analysis revealed the absence of StarD13 in invadopodia as compared to Cdc42, which was activated in invadopodia at the sites of matrix degradation. Conclusion In conclusion, StarD13 plays distinct roles in lung cancer cell migration and invasion through its differential regulation of Rho GTPases. 92FP97T_AMtXGPZ_u9jGL2 Video abstract.

Background Fibroblast activation protein (FAP)-targeting radioligands have gained attention for the ability to image multiple tumor types. Current FAP-targeting radioligands are labeled with 68 Ga and 18 F, but their short half-lives limit distribution range after production and later time-point imaging. This study describes the development Kalios, a novel class of NODAGA-conjugated FAP-targeting radioligands labeled with the cyclotron-produced Copper-61 (t 1/2  = 3.33 h), for greater temporal range for FAP-targeted imaging. Results Four Kalios ligands were synthesized and radiolabeled with [ 61 Cu]CuCl 2 in high yield and radiochemical purity within 5 min at room temperature. All radioligands demonstrated high hydrophilicity and strong affinity for FAP, and were primarily internalized after incubation with FAP-positive cells. PET/CT images obtained at 0–1 h and 4 h post-injection (p.i.) illustrated accumulation of all radioligands in FAP-positive tumors. Biodistribution studies of [ 61 Cu]Cu-Kalios-02 demonstrated stable tumor uptake between 1 and 4 h p.i., with washout from normal tissues at 4 h, resulting in improved tumor-to-background ratios. Conclusions Kalios ligands represent a new class of FAP-targeting 61 Cu-labeled radioligands. The half-life of 61 Cu allowed delayed 4-h imaging with improved tumor-to-background ratios. The improved delayed imaging and greater distribution range of these 61 Cu-labeled FAP-targeting radioligands demonstrates their clear potential for clinical translation, while combination with the therapeutic twin 67 Cu allows for truly paired Kalios theranostics.

Abstract Stomatal function in plants is regulated by the nanoscale architecture of the cell wall and turgor pressure, which together control stomatal pore size to facilitate gas exchange and photosynthesis. The mechanical properties of the cell wall and cell geometry are critical determinants of stomatal dynamics. However, the specific biomechanical functions of wall constituents, for example, cellulose and pectins, and their impact on the work required to open or close the stomatal pore are unclear. Here, we use nanoindentation in normal and lateral directions, computational modeling, and microscopic imaging of cells from the model plant Arabidopsis thaliana to investigate the precise influences of wall architecture and turgor pressure on stomatal biomechanics. This approach allows us to quantify and compare the unique anisotropic properties of guard cells with normal composition, lower cellulose content, or alterations in pectin molecular weight. Using these data to calculate the work required to open the stomata reveals that the wild type, with a circumferential-to-longitudinal modulus ratio of 3:1, is the most energy-efficient of those studied. In addition, the tested genotypes displayed similar changes in their pore size despite large differences in wall thickness and biomechanical properties. These findings imply that homeostasis in stomatal function is maintained in the face of varying wall compositions and biomechanics by tuning wall thickness.

Stomata are microscopic pores on the leaf surface, flanked by two guard cells that regulate pore size in response to environmental stimuli such as light, CO₂ levels, temperature, and humidity. The regulation of pore size is essential for controlling water transpiration and enabling CO₂ diffusion into the plant for photosynthesis. Understanding stomatal dynamics is crucial for improving water-use efficiency, elucidating plant adaptation to changing environments, and identifying new strategies for crop improvement.In eudicots such as Arabidopsis thaliana , guard cells are formed through the activity of a series of transcription factors and associated cellular and signaling pathways. This is followed by the growth and maturation of the stomatal complex, a process that remains poorly understood in terms of geometry, cell wall dynamics, and mechanics. In Chapter 2, we used a combination of genetics, cell biology, microscopy, mechanical testing, and finite element modeling to investigate stomatal maturation in Arabidopsis from both geometric and mechanical perspectives. We found that stomatal complexes mature through a series of geometric milestones and that, during maturation, guard cells develop mechanical anisotropy and exhibit reduced turgor pressure.Stomata vary in shape and size across plant species. For instance, while Arabidopsis stomata are flanked by kidney-shaped guard cells, grass stomatal complexes are composed of dumbbell-shaped guard cells flanked by subsidiary cells. These subsidiary cells, which are morphologically distinct from other epidermal cells, are thought to aid in regulating pore size. Several hypotheses have been proposed to explain the interactions between guard and subsidiary cells in grass stomatal complexes; however, these models have yet to be rigorously validated. In Chapter 3, we investigated guard–subsidiary cell interactions in the grass species Brachypodium distachyon using targeted laser ablation, turgor pressure measurements, and two-dimensional computational simulations of stomatal cross-sections. Our findings revealed that, contrary to prevailing models, guard and subsidiary cell turgor pressures change in the same direction during stomatal opening and closing, rather than in an inversely proportional manner. Moreover, we identified the subsidiary cells as the primary drivers of stomatal function in Brachypodium, calling for a revision of current models of grass stomatal mechanics.Guard cell walls play a critical role in stomatal function by providing the stiffness necessary to allow anisotropic cell deformation in response to changes in turgor pressure. The cell wall is composed of carbohydrate polymers such as cellulose, hemicelluloses, and pectins. Grasses possess type II primary cell walls, which are typically low in pectin content. However, previous studies have highlighted the significance of homogalacturonan biosynthesis genes in monocots. In Chapter 4, we investigate stomatal dynamics in the gaut1 mutant of Brachypodium in comparison to the wild type. We find that gaut1 mutants exhibit variations in subsidiary cell size and pore area, highlighting the importance of homogalacturonan in regulating cell expansion. Additionally, we describe the development and testing of new protocols to stain the cell wall and visualize the full depth of grass stomatal complexes using multiple imaging techniques, including confocal microscopy, Airyscan microscopy, multiphoton microscopy, and micro-computed tomography, to support the development of an image segmentation pipeline to automatically measure the volumes and geometries of cells in stomatal complexes.In summary, these studies elucidate the biomechanical characteristics of stomatal maturation in the eudicot model Arabidopsis thaliana and critically assess current hypotheses regarding guard– subsidiary cell interactions and the functional importance of cell wall components in the grass model Brachypodium distachyon . Collectively, our findings advance the understanding of stomatal dynamics in both eudicots and grasses and offer new opportunities for enhancing stomatal traits to improve plant resilience and productivity.

Fibroblast activation protein (FAP)-targeting radioligands have gained attention for the ability to image multiple tumor types. Current FAP-targeting radioligands are labeled with Ga and F, but their short half-lives limit distribution range after production and later time-point imaging. This study describes the development Kalios, a novel class of NODAGA-conjugated FAP-targeting radioligands labeled with the cyclotron-produced Copper-61 (t  = 3.33 h), for greater temporal range for FAP-targeted imaging. Four Kalios ligands were synthesized and radiolabeled with [ Cu]CuCl in high yield and radiochemical purity within 5 min at room temperature. All radioligands demonstrated high hydrophilicity and strong affinity for FAP, and were primarily internalized after incubation with FAP-positive cells. PET/CT images obtained at 0-1 h and 4 h post-injection (p.i.) illustrated accumulation of all radioligands in FAP-positive tumors. Biodistribution studies of [ Cu]Cu-Kalios-02 demonstrated stable tumor uptake between 1 and 4 h p.i., with washout from normal tissues at 4 h, resulting in improved tumor-to-background ratios. Kalios ligands represent a new class of FAP-targeting Cu-labeled radioligands. The half-life of Cu allowed delayed 4-h imaging with improved tumor-to-background ratios. The improved delayed imaging and greater distribution range of these Cu-labeled FAP-targeting radioligands demonstrates their clear potential for clinical translation, while combination with the therapeutic twin Cu allows for truly paired Kalios theranostics.

Fibroblast activation protein (FAP)-targeting radioligands have gained attention for the ability to image multiple tumor types. Current FAP-targeting radioligands are labeled with 68Ga and 18F, but their short half-lives limit distribution range after production and later time-point imaging. This study describes the development Kalios, a novel class of NODAGA-conjugated FAP-targeting radioligands labeled with the cyclotron-produced Copper-61 (t1/2 = 3.33 h), for greater temporal range for FAP-targeted imaging.BACKGROUNDFibroblast activation protein (FAP)-targeting radioligands have gained attention for the ability to image multiple tumor types. Current FAP-targeting radioligands are labeled with 68Ga and 18F, but their short half-lives limit distribution range after production and later time-point imaging. This study describes the development Kalios, a novel class of NODAGA-conjugated FAP-targeting radioligands labeled with the cyclotron-produced Copper-61 (t1/2 = 3.33 h), for greater temporal range for FAP-targeted imaging.Four Kalios ligands were synthesized and radiolabeled with [61Cu]CuCl2 in high yield and radiochemical purity within 5 min at room temperature. All radioligands demonstrated high hydrophilicity and strong affinity for FAP, and were primarily internalized after incubation with FAP-positive cells. PET/CT images obtained at 0-1 h and 4 h post-injection (p.i.) illustrated accumulation of all radioligands in FAP-positive tumors. Biodistribution studies of [61Cu]Cu-Kalios-02 demonstrated stable tumor uptake between 1 and 4 h p.i., with washout from normal tissues at 4 h, resulting in improved tumor-to-background ratios.RESULTSFour Kalios ligands were synthesized and radiolabeled with [61Cu]CuCl2 in high yield and radiochemical purity within 5 min at room temperature. All radioligands demonstrated high hydrophilicity and strong affinity for FAP, and were primarily internalized after incubation with FAP-positive cells. PET/CT images obtained at 0-1 h and 4 h post-injection (p.i.) illustrated accumulation of all radioligands in FAP-positive tumors. Biodistribution studies of [61Cu]Cu-Kalios-02 demonstrated stable tumor uptake between 1 and 4 h p.i., with washout from normal tissues at 4 h, resulting in improved tumor-to-background ratios.Kalios ligands represent a new class of FAP-targeting 61Cu-labeled radioligands. The half-life of 61Cu allowed delayed 4-h imaging with improved tumor-to-background ratios. The improved delayed imaging and greater distribution range of these 61Cu-labeled FAP-targeting radioligands demonstrates their clear potential for clinical translation, while combination with the therapeutic twin 67Cu allows for truly paired Kalios theranostics.CONCLUSIONSKalios ligands represent a new class of FAP-targeting 61Cu-labeled radioligands. The half-life of 61Cu allowed delayed 4-h imaging with improved tumor-to-background ratios. The improved delayed imaging and greater distribution range of these 61Cu-labeled FAP-targeting radioligands demonstrates their clear potential for clinical translation, while combination with the therapeutic twin 67Cu allows for truly paired Kalios theranostics.

Thymosin Beta 4 (TB4), a multi-functional 5 kDa peptide found at up to 500 µM concentration in virtually every mammalian cell, is the main G-actin sequestering protein. Extracellular concentrations are generally in the low nM range. Although no receptor for TB4 is known, higher extracellular levels of TB4 have been linked to enhanced cell migration and epithelial-mesenchymal transition. As these cellular effects involve substantial changes in the cytoskeleton, we measured the effect of TB4 on cytoskeletal mechanics, contractility, and motility. SW480 cells showed a distinct biphasic dose response with increasing concentrations of extracellular TB4. Cells stimulated with 0.2 µM TB4 showed the highest level of stiffness and contractility, displayed a pronounced elongated, mesenchymal morphology, migrated with higher directional persistence on flat 2D surfaces, and became highly invasive in a 3D collagen invasion assay. All parameters return to baseline levels at concentrations larger than 1 µM. Differential scanning calorimetry (DSC) and fluorescence correlation spectroscopy (FCS) measurements using fluorescently labelled TB4 and synthetic lipid membranes demonstrated that TB4 binds to the cell membrane (Kd=72 µM for a 1:5 DMPS/DMPC lipid ratio). The binding of TB4 to the cell membrane presents a novel mechanism linking small extracellular TB4 concentrations to pronounced cell mechanical responses.

Stomata are pores at the leaf surface that enable gas exchange and transpiration. The signaling pathways that regulate the differentiation of stomatal guard cells and the mechanisms of stomatal pore formation have been characterized in Arabidopsis thaliana. However, the process by which stomatal complexes develop after pore formation into fully mature complexes is poorly understood. We tracked the morphogenesis of young stomatal complexes over time to establish characteristic geometric milestones along the path of stomatal maturation. Using 3D-nanoindentation coupled with finite element modeling of young and mature stomata, we found that despite having thicker cell walls than young guard cells, mature guard cells are more energy efficient with respect to stomatal opening, potentially attributable to the increased mechanical anisotropy of their cell walls and smaller changes in turgor pressure between the closed and open states. Comparing geometric changes in young and mature guard cells of wild-type and cellulose-deficient plants revealed that although cellulose is required for normal stomatal maturation, mechanical anisotropy appears to be achieved by the collective influence of cellulose and additional wall components. Together, these data elucidate the dynamic geometric and biomechanical mechanisms underlying the development process of stomatal maturation.Competing Interest StatementThe authors have declared no competing interest.

Insufficient physical activity is considered a strong risk factor associated with non-communicable diseases. This study aimed to assess the impact of COVID-19 on physical (in)activity behavior in 10 Arab countries before and during the lockdown. A cross-sectional study using a validated online survey was launched originally in 38 different countries. The Eastern Mediterranean regional data related to the 10 Arabic countries that participated in the survey were selected for analysis in this study. A total of 12,433 participants were included in this analysis. The mean age of the participants was 30.3 (SD, 11.7) years. Descriptive and regression analyses were conducted to examine the associations between physical activity levels and the participants’ sociodemographic characteristics, watching TV, screen time, and computer usage. Physical activity levels decreased significantly during the lockdown. Participants’ country of origin, gender, and education were associated with physical activity before and during the lockdown (p < 0.050). Older age, watching TV, and using computers had a negative effect on physical activity before and during the lockdown (p < 0.050). Strategies to improve physical activity and minimize sedentary behavior should be implemented, as well as to reduce unhealthy levels of inactive time, especially during times of crisis. Further research on the influence of a lack of physical activity on overall health status, as well as on the COVID-19 disease effect is recommended.