High release of Candida albicans eDNA as protection for the scaffolding of polymicrobial biofilm formed with Staphylococcus aureus and Streptococcus mutans against the enzymatic activity of DNase I.

This study aimed to determine the protective role of the high release of C. albicans extracellular DNA (eDNA) in a polymicrobial biofilm formed by S. aureus and S. mutans in the course of DNase I treatment. A tube-flow biofilm bioreactor was developed to mimic biofilm formation in the oral cavity. eDNA release was quantified by real-time PCR (qPCR) and confocal microscopy analysis were used to determine the concentration and distribution of eDNA and intracellular DNA (iDNA). The mean amount of eDNA released by each species in the polymicrobial was higher than that in monospecies biofilms (S. aureus: 3.1 × 10-2 ng/µl polymicrobial versus 5.1 × 10-4 ng/µl monospecies; S. mutans: 3 × 10-1 ng/µl polymicrobial versus 2.97 × 10-2 ng/µl monospecies; C. albicans: 8.35 ng/µl polymicrobial versus 4.85 ng/µl monospecies). The large amounts of eDNA released by C. albicans (96%) in polymicrobial biofilms protects the S. aureus and S. mutans cells against the degradation by DNase I and dampens the effect of clindamycin.

COBRA-LIKE4 Modulates Cellulose Synthase Velocity and Facilitates Cellulose Deposition in the Secondary Cell Wall.

Cellulose is a critical component of secondary cell walls and woody tissues of plants. Cellulose synthase (CESA) complexes (CSCs) produce cellulose as they move within the plasma membrane, extruding glucan chains into the cell wall that coalesce and crystallize into cellulose fibrils. Here we examine COBRA-LIKE4 (COBL4), a GPI-anchored protein on the outer leaflet of the plasma membrane that is required for normal cellulose deposition in secondary cell walls. Characterization of the Arabidopsis (Arabidopsis thaliana) cobl4 mutant alleles called irregular xylem6, irx6-2 and irx6-3, showed reduced ⍺-cellulose content and lower crystallinity, supporting a role for COBL4 in maintaining cellulose quantity and quality. In live-cell imaging, mNeon Green-tagged CESA7 moved in the plasma membrane at higher speeds in the irx6-2 background compared to wild type. To test conservation of COBL4 function between herbaceous and woody plants, poplar (Populus trichocarpa) COBL4 homologs PtCOBL4a and PtCOBL4b were transformed into, and rescued, the Arabidopsis irx6 mutants. Using the Arabidopsis secondary cell wall-inducible VND7-GR system to study poplar COBL4 dynamics, YFP-tagged PtCOBL4a localized to the plasma membrane in regions of high cellulose deposition in secondary cell wall bands. As predicted for a lipid-linked protein, COBL4 was more mobile in the plane of the plasma membrane than CESA7 or a control plasma membrane marker. Following programmed cell death, COBL4 anchored to the secondary cell wall bands. These data support a role for COBL4 as a modulator of cellulose organization in the secondary cell wall, influencing cellulose production and CSC velocity at the plasma membrane.

Novel molecular insights into the machinery driving secondary cell wall synthesis and patterning.

The essential role of water-conducting xylem tissue in plant growth and crop yield is well-established. However, the molecular mechanisms underlying xylem formation and its unique functionality, which is acquired post-mortem, remain poorly understood. Recent advancements in genetic tools and model systems have significantly enhanced the ability to microscopically study xylem development, particularly its distinctive cell wall patterning. Early molecular mechanisms enabling pattern formation have been elucidated and validated through computational models. Despite these advancements, numerous questions remain unresolved but are approachable with current methodologies. This mini-review takes in the latest research findings in xylem cell wall synthesis and patterning and highlights prospective directions for future investigations.

Mechano-chemical regulation of complex cell shape formation: Epidermal pavement cells-A case study.

All plant cells are encased by walls, which provide structural support and control their morphology. How plant cells regulate the deposition of the wall to generate complex shapes is a topic of ongoing research. Scientists have identified several model systems, the epidermal pavement cells of cotyledons and leaves being an ideal platform to study the formation of complex cell shapes. These cells indeed grow alternating protrusions and indentations resulting in jigsaw puzzle cell shapes. How and why these cells adopt such shapes has shown to be a challenging problem to solve, notably because it involves the integration of molecular and mechanical regulation together with cytoskeletal dynamics and cell wall modifications. In this review, we highlight some recent progress focusing on how these processes may be integrated at the cellular level along with recent quantitative morphometric approaches.

Micro-Cup Architecture for Printing and Coating Asymmetric 2d-Material-Based Solid-State Supercapacitors.

High energy density micro-supercapacitors (MSCs) are in high demand for miniaturized electronics and microsystems. Research efforts today focus on materials development, applied in the planar interdigitated, symmetric electrode architecture. A novel "cup & core" device architecture that allows for printing of asymmetric devices without the need of accurately positioning the second finger electrode here have been introduced. The bottom electrode is either produced by laser ablation of a blade-coated graphene layer or directly screen-printed with graphene inks to create grids with high aspect ratio walls forming an array of "micro-cups". A quasi-solid-state ionic liquid electrolyte is spray-deposited on the walls; the top electrode material -MXene inks- is then spray-coated to fill the cup structure. The architecture combines the advantages of interdigitated electrodes for facilitated ion-diffusion, which is critical for 2D-material-based energy storage systems by providing vertical interfaces with the layer-by-layer processing of the sandwich geometry. Compared to flat reference devices, volumetric capacitance of printed "micro-cups" MSC increased considerably, while the time constant decreased (by 58%). Importantly, the high energy density (3.99 µWh cm-2 ) of the "micro-cups" MSC is also superior to other reported MXene and graphene-based MSCs.

Noninvasive Long-Term Imaging of the Cytoskeleton in Arabidopsis Seedlings.

The preparation of biological samples, especially for live-cell microscopy, remains a major experimental challenge in the lab despite technological advances. In addition, high-resolution microscopy techniques require higher sample quality and uniformity, which is difficult to ensure during manual preparation while maintaining "ideal" growth conditions. In this protocol, we provide a way out by growing Arabidopsis thaliana seedlings directly in an imaging chamber, which eliminates invasive sample preparation directly before imaging. This method hinges on the precise placement of seeds into imaging chambers, which can be grown in conventional climate chambers. We detail three methods to grow hypocotyls, cotyledons, leaves, and roots for high-resolution and long-term imaging of the plant cytoskeleton. Furthermore, we show that the growth and development of seedlings inside the chambers can be externally manipulated by the addition of chemicals.

Microtubule nucleation complex behavior is critical for cortical array homogeneity and xylem wall patterning.

Plant cell walls are versatile materials that can adopt a wide range of mechanical properties through controlled deposition of cellulose fibrils. Wall integrity requires a sufficiently homogeneous fibril distribution to cope effectively with wall stresses. Additionally, specific conditions, such as the negative pressure in water transporting xylem vessels, may require more complex wall patterns, e.g., bands in protoxylem. The orientation and patterning of cellulose fibrils are guided by dynamic cortical microtubules. New microtubules are predominantly nucleated from parent microtubules causing positive feedback on local microtubule density with the potential to yield highly inhomogeneous patterns. Inhomogeneity indeed appears in all current cortical array simulations that include microtubule-based nucleation, suggesting that plant cells must possess an as-yet unknown balancing mechanism to prevent it. Here, in a combined simulation and experimental approach, we show that a limited local recruitment of nucleation complexes to microtubules can counter the positive feedback, whereas local tubulin depletion cannot. We observe that nucleation complexes preferentially appear at the plasma membrane near microtubules. By incorporating our experimental findings in stochastic simulations, we find that the spatial behavior of nucleation complexes delicately balances the positive feedback, such that differences in local microtubule dynamics-as in developing protoxylem-can quickly turn a homogeneous array into a banded one. Our results provide insight into how the plant cytoskeleton has evolved to meet diverse mechanical requirements and greatly increase the predictive power of computational cell biology studies.

Tethering of cellulose synthase to microtubules dampens mechano-induced cytoskeletal organization in Arabidopsis pavement cells.

Mechanical forces control development in plants and animals, acting as cues in pattern formation and as the driving force of morphogenesis. In mammalian cells, molecular assemblies residing at the interface of the cell membrane and the extracellular matrix play an important role in perceiving and transmitting external mechanical signals to trigger physiological responses. Similar processes occur in plants, but there is little understanding of the molecular mechanisms and their genetic basis. Here, we show that the number and movement directions of cellulose synthase complexes (CSCs) at the plasma membrane vary during initial stages of development in the cotyledon epidermis of Arabidopsis, closely mirroring the microtubule organization. Uncoupling microtubules and CSCs resulted in enhanced microtubule co-alignment as caused by mechanical stimuli driven either by cell shape or by tissue-scale physical perturbations. Furthermore, micromechanical perturbation resulted in depletion of CSCs from the plasma membrane, suggesting a possible link between cellulose synthase removal from the plasma membrane and microtubule response to mechanical stimuli. Taken together, our results suggest that the interaction of cellulose synthase with cortical microtubules forms a physical continuum between the cell wall, plasma membrane and the cytoskeleton that modulates the mechano-response of the cytoskeleton.

Secondary cell wall patterning-connecting the dots, pits and helices.

All plant cells are encased in primary cell walls that determine plant morphology, but also protect the cells against the environment. Certain cells also produce a secondary wall that supports mechanically demanding processes, such as maintaining plant body stature and water transport inside plants. Both these walls are primarily composed of polysaccharides that are arranged in certain patterns to support cell functions. A key requisite for patterned cell walls is the arrangement of cortical microtubules that may direct the delivery of wall polymers and/or cell wall producing enzymes to certain plasma membrane locations. Microtubules also steer the synthesis of cellulose-the load-bearing structure in cell walls-at the plasma membrane. The organization and behaviour of the microtubule array are thus of fundamental importance to cell wall patterns. These aspects are controlled by the coordinated effort of small GTPases that probably coordinate a Turing's reaction-diffusion mechanism to drive microtubule patterns. Here, we give an overview on how wall patterns form in the water-transporting xylem vessels of plants. We discuss systems that have been used to dissect mechanisms that underpin the xylem wall patterns, emphasizing the VND6 and VND7 inducible systems, and outline challenges that lay ahead in this field.

A Universal Approach for Room-Temperature Printing and Coating of 2D Materials.

Processing 2D materials into printable or coatable inks for the fabrication of functional devices has proven to be quite difficult. Additives are often used in large concentrations to address the processing challenges, but they drastically degrade the electronic properties of the materials. To remove the additives a high-temperature post-deposition treatment can be used, but this complicates the fabrication process and limits the choice of materials (i.e., no heat-sensitive materials). In this work, by exploiting the unique properties of 2D materials, a universal strategy for the formulation of additive-free inks is developed, in which the roles of the additives are taken over by van der Waals (vdW) interactions. In this new class of inks, which is termed "vdW inks", solvents are dispersed within the interconnected network of 2D materials, minimizing the dispersibility-related limitations on solvent selection. Furthermore, flow behavior of the inks and mechanical properties of the resultant films are mainly controlled by the interflake vdW attractions. The structure of the vdW inks, their rheological properties, and film-formation behavior are discussed in detail. Large-scale production and formulation of the vdW inks for major high-throughput printing and coating methods, as well as their application for room-temperature fabrication of functional films/devices are demonstrated.

KATANIN and CLASP function at different spatial scales to mediate microtubule response to mechanical stress in Arabidopsis cotyledons.

Mechanical stress influences cell- and tissue-scale processes across all kingdoms. It remains challenging to delineate how mechanical stress, originating at these different length scales, impacts cell and tissue form. We combine growth tracking of cells, quantitative image analysis, as well as molecular and mechanical perturbations to address this problem in pavement cells of Arabidopsis thaliana cotyledon tissue. We show that microtubule organization based on chemical signals and cell-shape-derived mechanical stress varies during early stages of pavement cell development and is mediated by the evolutionary conserved proteins, KATANIN and CLASP. However, we find that these proteins regulate microtubule organization in response to tissue-scale mechanical stress to different extents in the cotyledon epidermis. Our results further demonstrate that regulation of cotyledon form is uncoupled from the mechanical-stress-dependent control of pavement cell shape that relies on microtubule organization governed by subcellular mechanical stress.

Long-term single-cell imaging and simulations of microtubules reveal principles behind wall patterning during proto-xylem development.

Plants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. Here, we establish long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to understand microtubule re-organization. We find that the re-organization requires local microtubule de-stabilization in band-interspersing gaps. Using microtubule simulations, we recapitulate the process in silico and predict that spatio-temporal control of microtubule nucleation is critical for pattern formation, which we confirm in vivo. By combining simulations and live-cell imaging we further explain how the xylem wall-deficient and microtubule-severing KATANIN contributes to microtubule and wall patterning. Hence, by combining quantitative microscopy and modelling we devise a framework to understand how microtubule re-organization supports wall patterning.

Microtubule-Mediated Wall Anisotropy Contributes to Leaf Blade Flattening.

Plant organs can adopt a wide range of shapes, resulting from highly directional cell growth and divisions. We focus here on leaves and leaf-like organs in Arabidopsis and tomato, characterized by the formation of thin, flat laminae. Combining experimental approaches with 3D mechanical modeling, we provide evidence that leaf shape depends on cortical microtubule mediated cellulose deposition along the main predicted stress orientations, in particular, along the adaxial-abaxial axis in internal cell walls. This behavior can be explained by a mechanical feedback and has the potential to sustain and even amplify a preexisting degree of flatness, which in turn depends on genes involved in the control of organ polarity and leaf margin formation.

Turning Trash into Treasure: Additive Free MXene Sediment Inks for Screen-Printed Micro-Supercapacitors.

Printed functional conductive inks have triggered scalable production of smart electronics such as energy-storage devices, antennas, wearable electronics, etc. Of particular interest are highly conductive-additive-free inks devoid of costly postdeposition treatments to eliminate sacrificial components. Due to the high filler concentration required, formulation of such waste-free inks has proven quite challenging. Here, additive-free, 2D titanium carbide MXene aqueous inks with appropriate rheological properties for scalable screen printing are demonstrated. Importantly, the inks consist essentially of the sediments of unetched precursor and multilayered MXene, which are usually discarded after delamination. Screen-printed structures are presented on paper with high resolution and spatial uniformity, including micro-supercapacitors, conductive tracks, integrated circuit paths, and others. It is revealed that the delaminated nanosheets among the layered particles function as efficient conductive binders, maintaining the mechanical integrity and thus the metallic conductive network. The areal capacitance (158 mF cm-2 ) and energy density (1.64 µWh cm-2 ) of the printed micro-supercapacitors are much superior to other devices based on MXene or graphene. The ink formulation strategy of "turning trash into treasure" for screen printing highlights the potential of waste-free MXene sediment printing for scalable and sustainable production of next-generation wearable smart electronics.

Quantification of Cytoskeletal Dynamics in Time-Lapse Recordings.

The cytoskeleton is key to many essential processes in a plant cell, e.g., growth, division, and defense. Contrary to what "skeleton" implies, the cytoskeleton is highly dynamic, and is able to re-organize itself continuously. The advent of live-cell microscopy and the development of genetically encoded fluorophores enabled detailed observation of the organization and dynamics of the cytoskeleton. Despite the biological importance of the cytoskeletal dynamics, quantitative analyses remain laborious endeavors that only a handful of research teams regularly conduct. With this protocol, we provide a standardized step-by-step guide to analyze the dynamics of microtubules. We provide example data and code for post-processing in Fiji that enables researchers to modify and adapt the routine to their needs. More such tools are needed to quantitatively assess the cytoskeleton and thus to better understand cell biology. © 2019 by John Wiley & Sons, Inc.

Slow sand filtration for biofouling reduction in seawater desalination by reverse osmosis.

Control of the organic substrate pool that determines the microbial growth potential (MGP) of feedwater in seawater reverse osmosis (SWRO) is a challenge unresolved in conventional or advanced membrane pretreatment. Slow sand filtration (SSF) combines filtration with biodegradation, but its capability of reducing MGP, proteins and carbohydrates on seawater feeds is not known. Two SSF, one constructed with new media (newSSF) and one from a previous filtration run (oldSSF), reduced MGP as measured in a growth assay with the marine organism Pseudoalteromonas songiae by one order of magnitude after maturation periods of 76 and 61 days, respectively. The reduction of the amount of biopolymers deposited on the surfaces of SWRO membranes in laminar fluid flow cells was significant with filtrates from biologically non-acclimated SSF (proteins: 60% (oldSSF) and -66% (new SSF), carbohydrates: 75% (oldSSF) and -70% (newSSF)) and an even greater reduction was observed after filter maturation (proteins: 81% (oldSSF) and -76% (new SSF), carbohydrates: 88% (oldSSF) and -88% (newSSF). Turbidity was less than 0.3 nephelometric turbidity units (NTU) and silt density index (SDI) < 4 immediately after startup and during the 181 days operating period regardless of the oscillations of the raw sea water quality. Filtration and biological activity were restricted to the top 30 cm of the media column, with no significant further contribution of the deeper media layers to filtrate quality.

The companion of cellulose synthase 1 confers salt tolerance through a Tau-like mechanism in plants.

Microtubules are filamentous structures necessary for cell division, motility and morphology, with dynamics critically regulated by microtubule-associated proteins (MAPs). Here we outline the molecular mechanism by which the MAP, COMPANION OF CELLULOSE SYNTHASE1 (CC1), controls microtubule bundling and dynamics to sustain plant growth under salt stress. CC1 contains an intrinsically disordered N-terminus that links microtubules at evenly distributed points through four conserved hydrophobic regions. By NMR and live cell analyses we reveal that two neighboring residues in the first hydrophobic binding motif are crucial for the microtubule interaction. The microtubule-binding mechanism of CC1 is reminiscent to that of the prominent neuropathology-related protein Tau, indicating evolutionary convergence of MAP functions across animal and plant cells.

Controlling the surface structure of electrospun fibers: Effect on endothelial cells and blood coagulation.

The influence of nano- or micron-sized structures on polymer films as well as the impact of fiber diameter of electrospun membranes on endothelial cell (EC) and blood response has been studied for vascular tissue engineering applications. However, the influence of surface structures on micron-sized fibers on endothelial cells and blood interaction is currently not known. In this work, electrospun membranes with distinct fiber surface structures were designed to study their influence on the endothelial cell viability and thrombogenicity. The thermodynamically derived Hansen-solubility-parameters model accurately predicted the formation of solvent dependent fiber surface structured poly(caprolactone) membranes. The electrospun membranes composed of microfibers (MF) or structured MF were of similar fiber diameter, macroscopic roughness, wettability, and elastic modulus. In vitro evaluation with ECs demonstrated that cell proliferation and morphology were not affected by the fiber surface structure. Similarly, investigating the blood response to the fiber meshes showed comparable fibrin network formation and platelet activation on MF and structured MF. Even though the presented results provide evidence that surface structures on MF appear neither to affect EC viability nor blood coagulation, they shed light on the complexity and challenges when studying biology-material interactions. They thereby contribute to the understanding of EC and blood-material interaction on electrospun membranes.

Cellulose synthase complexes display distinct dynamic behaviors during xylem transdifferentiation.

In plants, plasma membrane-embedded CELLULOSE SYNTHASE (CESA) enzyme complexes deposit cellulose polymers into the developing cell wall. Cellulose synthesis requires two different sets of CESA complexes that are active during cell expansion and secondary cell wall thickening, respectively. Hence, developing xylem cells, which first undergo cell expansion and subsequently deposit thick secondary walls, need to completely reorganize their CESA complexes from primary wall- to secondary wall-specific CESAs. Using live-cell imaging, we analyzed the principles underlying this remodeling. At the onset of secondary wall synthesis, the primary wall CESAs ceased to be delivered to the plasma membrane and were gradually removed from both the plasma membrane and the Golgi. For a brief transition period, both primary wall- and secondary wall-specific CESAs coexisted in banded domains of the plasma membrane where secondary wall synthesis is concentrated. During this transition, primary and secondary wall CESAs displayed discrete dynamic behaviors and sensitivities to the inhibitor isoxaben. As secondary wall-specific CESAs were delivered and inserted into the plasma membrane, the primary wall CESAs became concentrated in prevacuolar compartments and lytic vacuoles. This adjustment in localization between the two CESAs was accompanied by concurrent decreased primary wall CESA and increased secondary wall CESA protein abundance. Our data reveal distinct and dynamic subcellular trafficking patterns that underpin the remodeling of the cellulose biosynthetic machinery, resulting in the removal and degradation of the primary wall CESA complex with concurrent production and recycling of the secondary wall CESAs.

Evolutionary Interplay between Symbiotic Relationships and Patterns of Signal Peptide Gain and Loss.

Can orthologous proteins differ in terms of their ability to be secreted? To answer this question, we investigated the distribution of signal peptides within the orthologous groups of Enterobacterales. Parsimony analysis and sequence comparisons revealed a large number of signal peptide gain and loss events, in which signal peptides emerge or disappear in the course of evolution. Signal peptide losses prevail over gains, an effect which is especially pronounced in the transition from the free-living or commensal to the endosymbiotic lifestyle. The disproportionate decline in the number of signal peptide-containing proteins in endosymbionts cannot be explained by the overall reduction of their genomes. Signal peptides can be gained and lost either by acquisition/elimination of the corresponding N-terminal regions or by gradual accumulation of mutations. The evolutionary dynamics of signal peptides in bacterial proteins represents a powerful mechanism of functional diversification.