Integrating Phenotypic and Chemoproteomic Approaches to Identify Covalent Targets of Dietary Electrophiles in Platelets.

A large variety of dietary phytochemicals has been shown to improve thrombosis and stroke outcomes in preclinical studies. Many of these compounds feature electrophilic functionalities that potentially undergo covalent addition to the sulfhydryl side chain of cysteine residues within proteins. However, the impact of such covalent modifications on the platelet activity and function remains unclear. This study explores the irreversible engagement of 23 electrophilic phytochemicals with platelets, unveiling the unique antiplatelet selectivity of sulforaphane (SFN). SFN impairs platelet responses to adenosine diphosphate (ADP) and a thromboxane A2 receptor agonist while not affecting thrombin and collagen-related peptide activation. It also substantially reduces platelet thrombus formation under arterial flow conditions. Using an alkyne-integrated probe, protein disulfide isomerase A6 (PDIA6) was identified as a rapid kinetic responder to SFN. Mechanistic profiling studies revealed SFN's nuanced modulation of PDIA6 activity and substrate specificity. In an electrolytic injury model of thrombosis, SFN enhanced the thrombolytic activity of recombinant tissue plasminogen activator (rtPA) without increasing blood loss. Our results serve as a catalyst for further investigations into the preventive and therapeutic mechanisms of dietary antiplatelets, aiming to enhance the clot-busting power of rtPA, currently the only approved therapeutic for stroke recanalization that has significant limitations.

Intermittent hypoxia enhances the expression of hypoxia inducible factor HIF1A through histone demethylation.

The cellular response to hypoxia is regulated through enzymatic oxygen sensors, including the prolyl hydroxylases, which control degradation of the well-known hypoxia inducible factors (HIFs). Other enzymatic oxygen sensors have been recently identified, including members of the KDM histone demethylase family. Little is known about how different oxygen-sensing pathways interact and if this varies depending on the form of hypoxia, such as chronic or intermittent. In this study, we investigated how two proposed cellular oxygen-sensing systems, HIF-1 and KDM4A, KDM4B, and KDM4C, respond in cells exposed to rapid forms of intermittent hypoxia (minutes) and compared to chronic hypoxia (hours). We found that intermittent hypoxia increases HIF-1α protein through a pathway distinct from chronic hypoxia, involving the KDM4A, KDM4B, and KDM4C histone lysine demethylases. Intermittent hypoxia increases the quantity and activity of KDM4A, KDM4B, and KDM4C, resulting in a decrease in histone 3 lysine 9 (H3K9) trimethylation near the HIF1A locus. We demonstrate that this contrasts with chronic hypoxia, which decreases KDM4A, KDM4B, and KDM4C activity, leading to hypertrimethylation of H3K9 globally and at the HIF1A locus. Altogether, we found that demethylation of histones bound to the HIF1A gene in intermittent hypoxia increases HIF1A mRNA expression, which has the downstream effect of increasing overall HIF-1 activity and expression of HIF target genes. This study highlights how multiple oxygen-sensing pathways can interact to regulate and fine tune the cellular hypoxic response depending on the period and length of hypoxia.

An integrated platform approach enables discovery of potent, selective and ligand-competitive cyclic peptides targeting the GIP receptor.

In any drug discovery effort, the identification of hits for further optimisation is of crucial importance. For peptide therapeutics, display technologies such as mRNA display have emerged as powerful methodologies to identify these desired de novo hit ligands against targets of interest. The diverse peptide libraries are genetically encoded in these technologies, allowing for next-generation sequencing to be used to efficiently identify the binding ligands. Despite the vast datasets that can be generated, current downstream methodologies, however, are limited by low throughput validation processes, including hit prioritisation, peptide synthesis, biochemical and biophysical assays. In this work we report a highly efficient strategy that combines bioinformatic analysis with state-of-the-art high throughput peptide synthesis to identify nanomolar cyclic peptide (CP) ligands of the human glucose-dependent insulinotropic peptide receptor (hGIP-R). Furthermore, our workflow is able to discriminate between functional and remote binding non-functional ligands. Efficient structure-activity relationship analysis (SAR) combined with advanced in silico structural studies allow deduction of a thorough and holistic binding model which informs further chemical optimisation, including efficient half-life extension. We report the identification and design of the first de novo, GIP-competitive, incretin receptor family-selective CPs, which exhibit an in vivo half-life up to 10.7 h in rats. The workflow should be generally applicable to any selection target, improving and accelerating hit identification, validation, characterisation, and prioritisation for therapeutic development.

Transfer of training-Virtual reality training with augmented multisensory cues improves user experience during training and task performance in the real world.

Virtual reality (VR) can create safe, cost-effective, and engaging learning environments. It is commonly assumed that improvements in simulation fidelity lead to better learning outcomes. Some aspects of real environments, for example vestibular or haptic cues, are difficult to recreate in VR, but VR offers a wealth of opportunities to provide additional sensory cues in arbitrary modalities that provide task relevant information. The aim of this study was to investigate whether these cues improve user experience and learning outcomes, and, specifically, whether learning using augmented sensory cues translates into performance improvements in real environments. Participants were randomly allocated into three matched groups: Group 1 (control) was asked to perform a real tyre change only. The remaining two groups were trained in VR before performance was evaluated on the same, real tyre change task. Group 2 was trained using a conventional VR system, while Group 3 was trained in VR with augmented, task relevant, multisensory cues. Objective performance, time to completion and error number, subjective ratings of presence, perceived workload, and discomfort were recorded. The results show that both VR training paradigms improved performance for the real task. Providing additional, task-relevant cues during VR training resulted in higher objective performance during the real task. We propose a novel method to quantify the relative performance gains between training paradigms that estimates the relative gain in terms of training time. Systematic differences in subjective ratings that show comparable workload ratings, higher presence ratings and lower discomfort ratings, mirroring objective performance measures, were also observed. These findings further support the use of augmented multisensory cues in VR environments as an efficient method to enhance performance, user experience and, critically, the transfer of training from virtual to real environment scenarios.

Variation in Urinary Stone Parameters Throughout the Day and the Effect of Increased Fluid and Citrate Supplementation.

Background: Because 24-hour urine collections are cumbersome, many studies have evaluated the use of spot urine samples as a substitute, mostly finding poor concordance between the two. Daily variation in stone parameters probably contributes to the lack of concordance, but specific variation in various stone parameters is not well delineated. The variations likely lead to peaks and troughs, which can increase the risk of stone formation. Methods: We prospectively recruited 20 nonstone-forming patients, recording their total fluid intake over 24 hours and collecting voids at first morning, 9 to 10 A.M., 1 to 2 P.M., and 4 to 5 P.M. for evaluation of pH, specific gravity, calcium, citrate, and creatinine. Participants were then asked to double their fluid intake and take a daily True Lemon supplement over the course of the next 3 days. Urine was recollected postintervention. Results: Baseline [citrate]/[creatinine] increased throughout the day such that the 5 P.M. level was significantly higher compared with first void (0.58 vs 0.42, p = 0.027); [calcium]/[creatinine] daily variation was not statistically significant, but showed a distinct pattern that was present in both sets of collections. Daily [calcium]/[citrate] variation was significantly (p = 0.004) and consistently highest in the early morning on both day 1 (0.43) and day 4 (0.45). There was no significant variation in specific gravity and pH. Increasing fluid intake and citrate supplementation increase the daily variation in pH and [citrate]/[creatinine], but did not increase the values compared with their respective preintervention void times. There was also no detectable postintervention effect on [Ca]/[creatinine] or specific gravity. Conclusions: Urinary citrate concentration follows a circadian pattern, while urinary calcium has a diurnal excretion pattern. [Calcium]:[citrate] is highest in the early morning, indicating a high-risk time of day for stone formation. Spot urine samples identify a key time of day, which 24-hour urine collections may miss, for clinical monitoring.

Structures of Arabidopsis thaliana oxygen-sensing plant cysteine oxidases 4 and 5 enable targeted manipulation of their activity.

In higher plants, molecular responses to exogenous hypoxia are driven by group VII ethylene response factors (ERF-VIIs). These transcriptional regulators accumulate in the nucleus under hypoxia to activate anaerobic genes but are destabilized in normoxic conditions through the action of oxygen-sensing plant cysteine oxidases (PCOs). The PCOs catalyze the reaction of oxygen with the conserved N-terminal cysteine of ERF-VIIs to form cysteine sulfinic acid, triggering degradation via the Cys/Arg branch of the N-degron pathway. The PCOs are therefore a vital component of the plant oxygen signaling system, connecting environmental stimulus with cellular and physiological response. Rational manipulation of PCO activity could regulate ERF-VII levels and improve flood tolerance, but requires detailed structural information. We report crystal structures of the constitutively expressed PCO4 and PCO5 from Arabidopsis thaliana to 1.24 and 1.91 Å resolution, respectively. The structures reveal that the PCOs comprise a cupin-like scaffold, which supports a central metal cofactor coordinated by three histidines. While this overall structure is consistent with other thiol dioxygenases, closer inspection of the active site indicates that other catalytic features are not conserved, suggesting that the PCOs may use divergent mechanisms to oxidize their substrates. Conservative substitution of two active site residues had dramatic effects on PCO4 function both in vitro and in vivo, through yeast and plant complementation assays. Collectively, our data identify key structural elements that are required for PCO activity and provide a platform for engineering crops with improved hypoxia tolerance.

Readability Metrics of Provider Postoperative Handouts in Urology.

OBJECTIVE: To examine the readability of postoperative urology handouts and assess for areas of improvement. We hypothesize that the majority of provider handouts exceed the National Institutes of Health recommendation of writing at a sixth-grade reading level. METHODS: We reviewed 238 postoperative patient handouts in the public domain representing United States academic and private practices. All handouts were categorized and re-formatted into text-only using Microsoft Word. A median reading grade was calculated using the Readability.io web application using Flesch-Kincaid Grade Level, Gunning Fog index, Coleman-Liau index, Simple Measure of Gobbledygook, and Automated-Reading Index. Word count was also assessed. RESULTS: Provider handouts were written at a median 9.3 grade reading level (range 5.8-14, IQR 8.45-10). A total of 15 (6.8%) handouts were written at a sixth-grade reading level, with only 1 (0.4%) handout written below the target. Six (2.7%) handouts were written at college-level. There were no significant differences between different subspecialties. Median word count was 509 (range 90-3796, IQR 361-738). Although a high word count may make it more difficult for patients to follow suggestions, the readability of each handout did not correlate with word count. CONCLUSIONS: Our data show that over 93% of analyzed handouts failed to meet National Institutes of Health recommendations for grade level. Longer word counts did not correlate with higher reading levels. It will be important to assess patient satisfaction with handouts and to correlate the complexity of postoperative handouts with outcome, such as unplanned phone calls and unscheduled visits.

Quantification of CO2 Mineralization at the Wallula Basalt Pilot Project.

In 2013, the Pacific Northwest National Laboratory led a geologic carbon sequestration field demonstration where ∼1000 tonnes of CO2 was injected into several deep Columbia River Basalt zones near Wallula, Washington. Rock core samples extracted from the injection zone two years after CO2 injection revealed nascent carbonate mineralization that was qualitatively consistent with expectations from laboratory experiments and reactive transport modeling. Here, we report on a new detailed analysis of the 2012 pre-injection and 2015 post-injection hydrologic tests that capitalizes on the difference in fluid properties between scCO2 and water to assess changes in near-field, wellbore, and reservoir conditions that are apparent approximately two years following the end of injection. This comparative hydrologic test analysis method provides a new way to quantify the amount of injected CO2 that was mineralized in the field test. Modeling results indicate that approximately 60% of the injected CO2 was sequestered via mineralization within two years, with the resulting carbonates occupying ∼4% of the available reservoir pore space. The method presented here provides a new monitoring tool to assess the fate of CO2 injected into chemically reactive basalt formations but could also be adapted for long-term monitoring and verification within more traditional subsurface carbon storage reservoirs.

Conserved N-terminal cysteine dioxygenases transduce responses to hypoxia in animals and plants.

Organisms must respond to hypoxia to preserve oxygen homeostasis. We identify a thiol oxidase, previously assigned as cysteamine (2-aminoethanethiol) dioxygenase (ADO), as a low oxygen affinity (high-K mO2) amino-terminal cysteine dioxygenase that transduces the oxygen-regulated stability of proteins by the N-degron pathway in human cells. ADO catalyzes the conversion of amino-terminal cysteine to cysteine sulfinic acid and is related to the plant cysteine oxidases that mediate responses to hypoxia by an identical posttranslational modification. We show in human cells that ADO regulates RGS4/5 (regulator of G protein signaling) N-degron substrates, modulates G protein-coupled calcium ion signals and mitogen-activated protein kinase activity, and that its activity extends to other N-cysteine proteins including the angiogenic cytokine interleukin-32. Identification of a conserved enzymatic oxygen sensor in multicellular eukaryotes opens routes to better understanding and therapeutic targeting of adaptive responses to hypoxia.

The UbiX flavin prenyltransferase reaction mechanism resembles class I terpene cyclase chemistry.

The UbiX-UbiD enzymes are widespread in microbes, acting in concert to decarboxylate alpha-beta unsaturated carboxylic acids using a highly modified flavin cofactor, prenylated FMN (prFMN). UbiX serves as the flavin prenyltransferase, extending the isoalloxazine ring system with a fourth non-aromatic ring, derived from sequential linkage between a dimethylallyl moiety and the FMN N5 and C6. Using structure determination and solution studies of both dimethylallyl monophosphate (DMAP) and dimethyallyl pyrophosphate (DMAPP) dependent UbiX enzymes, we reveal the first step, N5-C1' bond formation, is contingent on the presence of a dimethylallyl substrate moiety. Hence, an SN1 mechanism similar to other prenyltransferases is proposed. Selected variants of the (pyro)phosphate binding site are unable to catalyse subsequent Friedel-Crafts alkylation of the flavin C6, but can be rescued by addition of (pyro)phosphate. Thus, retention of the (pyro)phosphate leaving group is required for C6-C3' bond formation, resembling pyrophosphate initiated class I terpene cyclase reaction chemistry.

Zinc Excess Induces a Hypoxia-Like Response by Inhibiting Cysteine Oxidases in Poplar Roots.

Poplar (Populus spp.) is a tree species considered for the remediation of soil contaminated by metals, including zinc (Zn). To improve poplar's capacity for Zn assimilation and compartmentalization, it is necessary to understand the physiological and biochemical mechanisms that enable these features as well as their regulation at the molecular level. We observed that the molecular response of poplar roots to Zn excess overlapped with that activated by hypoxia. Therefore, we tested the effect of Zn excess on hypoxia-sensing components and investigated the consequence of root hypoxia on poplar fitness and Zn accumulation capacity. Our results suggest that high intracellular Zn concentrations mimic iron deficiency and inhibit the activity of the oxygen sensors Plant Cysteine Oxidases, leading to the stabilization and activation of ERF-VII transcription factors, which are key regulators of the molecular response to hypoxia. Remarkably, excess Zn and waterlogging similarly decreased poplar growth and development. Simultaneous excess Zn and waterlogging did not exacerbate these parameters, although Zn uptake was limited. This study unveils the contribution of the oxygen-sensing machinery to the Zn excess response in poplar, which may be exploited to improve Zn tolerance and increase Zn accumulation capacity in plants.

Oxygen-dependent proteolysis regulates the stability of angiosperm polycomb repressive complex 2 subunit VERNALIZATION 2.

The polycomb repressive complex 2 (PRC2) regulates epigenetic gene repression in eukaryotes. Mechanisms controlling its developmental specificity and signal-responsiveness are poorly understood. Here, we identify an oxygen-sensitive N-terminal (N-) degron in the plant PRC2 subunit VERNALIZATION(VRN) 2, a homolog of animal Su(z)12, that promotes its degradation via the N-end rule pathway. We provide evidence that this N-degron arose early during angiosperm evolution via gene duplication and N-terminal truncation, facilitating expansion of PRC2 function in flowering plants. We show that proteolysis via the N-end rule pathway prevents ectopic VRN2 accumulation, and that hypoxia and long-term cold exposure lead to increased VRN2 abundance, which we propose may be due to inhibition of VRN2 turnover via its N-degron. Furthermore, we identify an overlap in the transcriptional responses to hypoxia and prolonged cold, and show that VRN2 promotes tolerance to hypoxia. Our work reveals a mechanism for post-translational regulation of VRN2 stability that could potentially link environmental inputs to the epigenetic control of plant development.

Perceptual assessment of environmental stability modulates postural sway.

We actively maintain postural equilibrium in everyday life, and, although we are unaware of the underlying processing, there is increasing evidence for cortical involvement in this postural control. Converging evidence shows that we make appropriate use of 'postural anchors', for example static objects in the environment, to stabilise our posture. Visually evoked postural responses (VEPR) that are caused when we counteract the illusory perception of self-motion in space (vection) are modulated in the presence of postural anchors and therefore provide a convenient behavioural measure. The aim of this study is to evaluate the factors influencing visual appraisal of the suitability of postural anchors. We are specifically interested in the effect of perceived 'reality' in VR the expected 'stability' of visual anchors. To explore the effect of 'reality' we introduced an accommodation-vergence conflict. We show that VEPR are appropriately modulated only when virtual visual 'anchors' are rendered such that vergence and accommodation cues are consistent. In a second experiment we directly test whether cognitive assessment of the likely stability of real perceptual anchors (we contrast a 'teapot on a stand' and a 'helium balloon') affects VEPR. We show that the perceived positional stability of environmental anchors modulate postural responses. Our results confirm previous findings showing that postural sway is modulated by the configuration of the environment and further show that an assessment of the stability and reality of the environment plays an important role in this process. On this basis we propose design guidelines for VR systems, in particular we argue that accommodation-vergence conflicts should be minimised and that high quality motion tracking and rendering are essential for high fidelity VR.

The plant cysteine oxidases from Arabidopsis thaliana are kinetically tailored to act as oxygen sensors.

Group VII ethylene response factors (ERF-VIIs) regulate transcriptional adaptation to flooding-induced hypoxia in plants. ERF-VII stability is controlled in an O2-dependent manner by the Cys/Arg branch of the N-end rule pathway whereby oxidation of a conserved N-terminal cysteine residue initiates target degradation. This oxidation is catalyzed by plant cysteine oxidases (PCOs), which use O2 as cosubstrate to generate Cys-sulfinic acid. The PCOs directly link O2 availability to ERF-VII stability and anaerobic adaptation, leading to the suggestion that they act as plant O2 sensors. However, their ability to respond to fluctuations in O2 concentration has not been established. Here, we investigated the steady-state kinetics of Arabidopsis thaliana PCOs 1-5 to ascertain whether their activities are sensitive to O2 levels. We found that the most catalytically competent isoform is AtPCO4, both in terms of responding to O2 and oxidizing AtRAP2.2/2,12 (two of the most prominent ERF-VIIs responsible for promoting the hypoxic response), which suggests that AtPCO4 plays a central role in ERF-VII regulation. Furthermore, we found that AtPCO activity is susceptible to decreases in pH and that the hypoxia-inducible AtPCOs 1/2 and the noninducible AtPCOs 4/5 have discrete AtERF-VII substrate preferences. Pertinently, the AtPCOs had Km(O2)app values in a physiologically relevant range, which should enable them to sensitively react to changes in O2 availability. This work validates an O2-sensing role for the PCOs and suggests that differences in expression pattern, ERF-VII selectivity, and catalytic capability may enable the different isoforms to have distinct biological functions. Individual PCOs could therefore be targeted to manipulate ERF-VII levels and improve stress tolerance in plants.

Plant cysteine oxidases are dioxygenases that directly enable arginyl transferase-catalysed arginylation of N-end rule targets.

Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilization in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This provides molecular evidence of N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and a substrate of ATE1 in plants. The PCOs and ATE1 may be viable intervention targets to stabilize N-end rule substrates, including ERF-VIIs, to enhance submergence tolerance in agriculture.