Fluorescence shadow imaging of Hypsibius exemplaris reveals morphological differences between sucrose- and CaCl2-induced osmobiotes.

Tardigrades are renowned for their ability to survive a wide array of environmental stressors. In particular, tardigrades can curl in on themselves while losing a significant proportion of their internal water content to form a structure referred to as a tun. In surviving varying conditions, tardigrades undergo distinct morphological transformations that could indicate different mechanisms of stress sensing and tolerance specific to the stress condition. Methods to effectively distinguish between morphological transformations, including between tuns induced by different stress conditions, are lacking. Herein, an approach for discriminating between tardigrade morphological states is developed and utilized to compare sucrose- and CaCl2-induced tuns, using the model species Hypsibius exemplaris. A novel approach of shadow imaging with confocal laser scanning microscopy enabled production of three-dimensional renderings of Hys. exemplaris in various physiological states resulting in volume measurements. Combining these measurements with qualitative morphological analysis using scanning electron microscopy revealed that sucrose- and CaCl2-induced tuns have distinct morphologies, including differences in the amount of water expelled during tun formation. Further, varying the concentration of the applied stressor did not affect the amount of water lost, pointing towards water expulsion by Hys. exemplaris being a controlled process that is adapted to the specific stressors.

Chemobiosis reveals tardigrade tun formation is dependent on reversible cysteine oxidation.

Tardigrades, commonly known as 'waterbears', are eight-legged microscopic invertebrates renowned for their ability to withstand extreme stressors, including high osmotic pressure, freezing temperatures, and complete desiccation. Limb retraction and substantial decreases to their internal water stores results in the tun state, greatly increasing their ability to survive. Emergence from the tun state and/or activity regain follows stress removal, where resumption of life cycle occurs as if stasis never occurred. However, the mechanism(s) through which tardigrades initiate tun formation is yet to be uncovered. Herein, we use chemobiosis to demonstrate that tardigrade tun formation is mediated by reactive oxygen species (ROS). We further reveal that tuns are dependent on reversible cysteine oxidation, and that this reversible cysteine oxidation is facilitated by the release of intracellular reactive oxygen species (ROS). We provide the first empirical evidence of chemobiosis and map the initiation and survival of tardigrades via osmobiosis, chemobiosis, and cryobiosis. In vivo electron paramagnetic spectrometry suggests an intracellular release of reactive oxygen species following stress induction; when this release is quenched through the application of exogenous antioxidants, the tardigrades can no longer survive osmotic stress. Together, this work suggests a conserved dependence of reversible cysteine oxidation across distinct tardigrade cryptobioses.

Excess manganese increases photosynthetic activity via enhanced reducing center and antenna plasticity in Chlorella vulgaris.

Photosynthesis relies on many easily oxidizable/reducible transition metals found in the metalloenzymes that make up much of the photosynthetic electron transport chain (ETC). One of these is manganese, an essential cofactor of photosystem II (PSII) and a component of the oxygen-evolving complex, the only biological entity capable of oxidizing water. Additionally, manganese is a cofactor in enzymatic antioxidants, notably the superoxide dismutases-which are localized to the chloroplastic membrane. However, unlike other metals found in the photosynthetic ETC, previous research has shown exposure to excess manganese enhances photosynthetic activity rather than diminishing it. In this study, the impact of PSII heterogeneity on overall performance was investigated using chlorophyll fluorescence, a rapid, non-invasive technique that probed for overall photosynthetic efficiency, reducing site activity, and antenna size and distribution. These measurements unveiled an enhanced plasticity of PSII following excess manganese exposure, in which overall performance and reducing center activity increased while antenna size and proportion of PSIIß centers decreased. This enhanced activity suggests manganese may hold the key to improving photosynthetic efficiency beyond that which is observed in nature.

Quantification of Cannabis in Infused Consumer Products and Their Residues on Skin.

Cannabis consumer products are a $4.6 billion industry in the U.S. that is projected to exceed $14 billion by 2025. Despite an absence of U.S. Food and Drug Administration (FDA) regulation or clinical data, thousands of nutraceuticals, topical consumer products, and beauty products claim benefits of hemp or cannabidiol. However, a lack of required quality control measures prevents consumers from knowing the true concentration or purities of cannabis-labeled products. Thirteen over-the-counter consumer products were examined for the presence of cannabidiol (CBD), cannabinol (CBN), Δ9-tetrahydrocannabinol (THC), cannabidiolic acid (CBDA), and Δ9-tetrahydrocannabinolic acid A (THCA). Additionally, the efficacy of topical applications was investigated using a porcine skin model, in which particle size and zeta potential relate to skin permeability. Skin permeation was correlated to particle size and relative stability in skin-like conditions but not directly related to the CBD content, suggesting that topical products can be designed to enhance overall skin permeation. Of the products analyzed, all products have some traceable amount of cannabinoids, while seven products had multiple cannabinoids with quantifiable amounts. Overall, the need for further regulation is clear, as most products have apparent distinctions between their true and labeled contents.

Abscisic acid-controlled redox proteome of Arabidopsis and its regulation by heterotrimeric Gß protein.

The plant hormone abscisic acid (ABA) plays crucial roles in regulation of stress responses and growth modulation. Heterotrimeric G-proteins are key mediators of ABA responses. Both ABA and G-proteins have also been implicated in intracellular redox regulation; however, the extent to which reversible protein oxidation manipulates ABA and/or G-protein signaling remains uncharacterized. To probe the role of reversible protein oxidation in plant stress response and its dependence on G-proteins, we determined the ABA-dependent reversible redoxome of wild-type and Gß-protein null mutant agb1 of Arabidopsis. We quantified 6891 uniquely oxidized cysteine-containing peptides, 923 of which show significant changes in oxidation following ABA treatment. The majority of these changes required the presence of G-proteins. Divergent pathways including primary metabolism, reactive oxygen species response, translation and photosynthesis exhibited both ABA- and G-protein-dependent redox changes, many of which occurred on proteins not previously linked to them. We report the most comprehensive ABA-dependent plant redoxome and uncover a complex network of reversible oxidations that allow ABA and G-proteins to rapidly adjust cellular signaling to adapt to changing environments. Physiological validation of a subset of these observations suggests that functional G-proteins are required to maintain intracellular redox homeostasis and fully execute plant stress responses.

Crosslinking mass spectrometry unveils novel interactions and structural distinctions in the model green alga Chlamydomonas reinhardtii.

Interactomics is an emerging field that seeks to identify both transient and complex-bound protein interactions that are essential for metabolic functions. Crosslinking mass spectrometry (XL-MS) has enabled untargeted global analysis of these protein networks, permitting largescale simultaneous analysis of protein structure and interactions. Increased commercial availability of highly specific, cell permeable crosslinkers has propelled the study of these critical interactions forward, with the development of MS-cleavable crosslinkers further increasing confidence in identifications. Herein, the global interactome of the unicellular alga Chlamydomonas reinhardtii was analyzed via XL-MS by implementing the MS-cleavable disuccinimidyl sulfoxide (DSSO) crosslinker and enriching for crosslinks using strong cation exchange chromatography. Gentle lysis via repeated freeze-thaw cycles facilitated in vitro analysis of 157 protein-protein crosslinks (interlinks) and 612 peptides linked to peptides of the same protein (intralinks) at 1% FDR throughout the C. reinhardtii proteome. The interlinks confirmed known protein relationships across the cytosol and chloroplast, including coverage on 42% and 38% of the small and large ribosomal subunits, respectively. Of the 157 identified interlinks, 92% represent the first empirical evidence of interaction observed in C. reinhardtii. Several of these crosslinks point to novel associations between proteins, including the identification of a previously uncharacterized Mg-chelatase associated protein (Cre11.g477733.t1.2) bound to seven distinct lysines on Mg-chelatase (Cre06.g306300.t1.2). Additionally, the observed intralinks facilitated characterization of novel protein structures across the C. reinhardtii proteome. Together, these data establish a framework of protein-protein interactions that can be further explored to facilitate understanding of the dynamic protein landscape in C. reinhardtii.

Inositol polyphosphates and target of rapamycin kinase signalling govern photosystem II protein phosphorylation and photosynthetic function under light stress in Chlamydomonas.

Stress and nutrient availability influence cell proliferation through complex intracellular signalling networks. In a previous study it was found that pyro-inositol polyphosphates (InsP7 and InsP8 ) produced by VIP1 kinase, and target of rapamycin (TOR) kinase signalling interacted synergistically to control cell growth and lipid metabolism in the green alga Chlamydomonas reinhardtii. However, the relationship between InsPs and TOR was not completely elucidated. We used an in vivo assay for TOR activity together with global proteomic and phosphoproteomic analyses to assess differences between wild-type and vip1-1 in the presence and absence of rapamycin. We found that TOR signalling is more severely affected by the inhibitor rapamycin in a vip1-1 mutant compared with wild-type, indicating that InsP7 and InsP8 produced by VIP1 act independently but also coordinately with TOR. Additionally, among hundreds of differentially phosphorylated peptides detected, an enrichment for photosynthesis-related proteins was observed, particularly photosystem II proteins. The significance of these results was underscored by the finding that vip1-1 strains show multiple defects in photosynthetic physiology that were exacerbated under high light conditions. These results suggest a novel role for inositol pyrophosphates and TOR signalling in coordinating photosystem phosphorylation patterns in Chlamydomonas cells in response to light stress and possibly other stresses.

Mapping the plant proteome: tools for surveying coordinating pathways.

Plants rapidly respond to environmental fluctuations through coordinated, multi-scalar regulation, enabling complex reactions despite their inherently sessile nature. In particular, protein post-translational signaling and protein-protein interactions combine to manipulate cellular responses and regulate plant homeostasis with precise temporal and spatial control. Understanding these proteomic networks are essential to addressing ongoing global crises, including those of food security, rising global temperatures, and the need for renewable materials and fuels. Technological advances in mass spectrometry-based proteomics are enabling investigations of unprecedented depth, and are increasingly being optimized for and applied to plant systems. This review highlights recent advances in plant proteomics, with an emphasis on spatially and temporally resolved analysis of post-translational modifications and protein interactions. It also details the necessity for generation of a comprehensive plant cell atlas while highlighting recent accomplishments within the field.

Implementation of Microfluidics for Antimicrobial Susceptibility Assays: Issues and Optimization Requirements.

Despite the continuous emergence of multi-drug resistant pathogens, the number of new antimicrobials reaching the market is critically low. Natural product peptides are a rich source of bioactive compounds, and advances in mass spectrometry have achieved unprecedented capabilities for the discovery and characterization of novel molecular species. However, traditional bioactivity assay formats hinder the discovery and biochemical characterization of natural product antimicrobial peptides (AMPs), necessitating large sample quantities and significant optimization of experimental parameters to achieve accurate/consistent activity measurements. Microfluidic devices offer a promising alternative to bulk assay systems. Herein, a microfluidics-based bioassay was compared to the traditional 96-well plate format in respective commercially-available hardware. Bioactivity in each assay type was compared using a Viola inconspicua peptide library screened against E. coli ATCC 25922. Brightfield microcopy was used to determine bioactivity in microfluidic channels while both common optical and fluorescence-based measurements of cell viability were critically assessed in plate-based assays. Exhibiting some variation in optical density and fluorescence-based measurements, all plate-based assays conferred bioactivity in late eluting V. inconspicua library fractions. However, significant differences in the bioactivity profiles of plate-based and microfluidic assays were found, and may be derived from the materials comprising each assay device or the growth/assay conditions utilized in each format. While new technologies are necessary to overcome the limitations of traditional bioactivity assays, we demonstrate that off-the-shelf implementation of microfluidic devices is non-trivial and significant method development/optimization is required before conventional use can be realized for sensitive and rapid detection of AMPs in natural product matrices.

Maleimide-Based Chemical Proteomics for Quantitative Analysis of Cysteine Reactivity.

Cysteine is the most intrinsically nucleophilic residue in proteins and serves as a mediator against increasing reactive oxygen species (ROS) via reversible thiol oxidation. Despite the importance of cysteine oxidation in understanding biological stress response, cysteine sites most reactive toward ROS remain largely unknown and are a major analytical challenge. Herein, a chemical proteomic method to quantify site-specific cysteine reactivity using a maleimide-activated, thiol-reactive probe (N-propargylmaleimide, NPM) is described. Implementation of a gel-based approach via conjugation of rhodamine-azide to NPM-labeled cysteine residues by copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry allowed simple and highly sensitive fluorescence profiling. Relative quantification of >1500 unique cysteine sites from greater than 800 proteins was achieved by conjugating dialkoxydiphenylsilane (DADPS) biotin-azide by the CuAAC reaction and subsequently performing biotin-streptavidin affinity purification and mass-spectrometry-based proteomics. Taken together, this work defines a novel role for the NPM probe in chemical proteomics and presents a robust method for determination of cysteine reactivity during oxidative stress response.

Photosynthetic Metabolism and Nitrogen Reshuffling Are Regulated by Reversible Cysteine Thiol Oxidation Following Nitrogen Deprivation in Chlamydomonas.

As global temperatures climb to historic highs, the far-reaching effects of climate change have impacted agricultural nutrient availability. This has extended to low latitude oceans, where a deficit in both nitrogen and phosphorus stores has led to dramatic decreases in carbon sequestration in oceanic phytoplankton. Although Chlamydomonas reinhardtii, a freshwater model green alga, has shown drastic systems-level alterations following nitrogen deprivation, the mechanisms through which these alterations are triggered and regulated are not fully understood. This study examined the role of reversible oxidative signaling in the nitrogen stress response of C. reinhardtii. Using oxidized cysteine resin-assisted capture enrichment coupled with label-free quantitative proteomics, 7889 unique oxidized cysteine thiol identifiers were quantified, with 231 significantly changing peptides from 184 proteins following 2 h of nitrogen deprivation. These results demonstrate that the cellular response to nitrogen assimilation, photosynthesis, pigment biosynthesis, and lipid metabolism are regulated by reversible oxidation. An enhanced role of non-damaging oxidative pathways is observed throughout the photosynthetic apparatus that provides a framework for further analysis in phototrophs.

Inhibition of TOR in Chlamydomonas reinhardtii Leads to Rapid Cysteine Oxidation Reflecting Sustained Physiological Changes.

The target of rapamycin (TOR) kinase is a master metabolic regulator with roles in nutritional sensing, protein translation, and autophagy. In Chlamydomonas reinhardtii, a unicellular green alga, TOR has been linked to the regulation of increased triacylglycerol (TAG) accumulation, suggesting that TOR or a downstream target(s) is responsible for the elusive "lipid switch" in control of increasing TAG accumulation under nutrient limitation. However, while TOR has been well characterized in mammalian systems, it is still poorly understood in photosynthetic systems, and little work has been done to show the role of oxidative signaling in TOR regulation. In this study, the TOR inhibitor AZD8055 was used to relate reversible thiol oxidation to the physiological changes seen under TOR inhibition, including increased TAG content. Using oxidized cysteine resin-assisted capture enrichment coupled with label-free quantitative proteomics, 401 proteins were determined to have significant changes in oxidation following TOR inhibition. These oxidative changes mirrored characterized physiological modifications, supporting the role of reversible thiol oxidation in TOR regulation of TAG production, protein translation, carbohydrate catabolism, and photosynthesis through the use of reversible thiol oxidation. The delineation of redox-controlled proteins under TOR inhibition provides a framework for further characterization of the TOR pathway in photosynthetic eukaryotes.

Characterizing the effect of Poast on Chlorella vulgaris, a non-target organism.

Herbicides may cause unexpected damage to non-target organisms as it is challenging to predict undesirable biotic interactions. Poast is a widely used herbicide formulation that contains sethoxydim and targets the acetyl-CoA carboxylase of perennial grasses. In this study, Chlorella vulgaris, a unicellular green microalga, was exposed to a 0.08% working concentration of Poast and the physiological and biochemical changes that took place were monitored using biochemical assays, fluorometry, oximetry, and immunoblotting. Within 15 min, severe photosynthetic damage was observed through a reduction in oxygen production and a reduced rate of electron transfer beyond photosystem II. In addition to direct damage to the photosynthetic machinery, it was shown that cells experienced membrane fragmentation. Within 30 min, over 90% of the exposed cells were nonviable. However, sethoxydim, the active ingredient, did not cause detrimental effects when applied along with mineral spirits, the primary solvent of the formulation. A synergistic or additive effect between sethoxydim and the formulation components cannot be ruled out. This data suggests that Poast has the potential to cause severe harm to unicellular phototrophs in the case of herbicide over application or runoff.