Phosphorylation of the alpha-I motif in SYMRK drives root nodule organogenesis.

Symbiosis receptor-like kinase SYMRK is required for root nodule symbiosis between legume plants and nitrogen-fixing bacteria. To understand symbiotic signaling from SYMRK, we determined the crystal structure to 1.95 Å and mapped the phosphorylation sites onto the intracellular domain. We identified four serine residues in a conserved "alpha-I" motif, located on the border between the kinase core domain and the flexible C-terminal tail, that, when phosphorylated, drives organogenesis. Substituting the four serines with alanines abolished symbiotic signaling, while substituting them with phosphorylation-mimicking aspartates induced the formation of spontaneous nodules in the absence of bacteria. These findings show that the signaling pathway controlling root nodule organogenesis is mediated by SYMRK phosphorylation, which may help when engineering this trait into non-legume plants.

Membrane fouling monitoring by 3ω sensing.

Membrane fouling significantly reduces membrane permeability, leading to higher operational expenses. In situ monitoring of membrane fouling can potentially be used to reduce operation cost by optimizing operational parameters and cleaning conditions. In this study, a platinum wire with a diameter of 20 µm was attached to the surface of a ceramic ultrafiltration membrane, and by measuring the voltage across the wire while applying an AC current, the amplitude of the third harmonic wave, the so-called 3ω signal, was obtained. Results showed increasing 3ω signals during formation of fouling layers, which correlates directly to the hydraulic resistance of the formed fouling layer in semi-dead end filtration of polymeric core shell particles and crossflow filtration of diluted milk. This is explained by the insulating effect of the fouling layers which reduces heat convection by crossflow and the different thermal conductivity in the fouling layer compared with the feed. After membrane cleaning, the permeability and the magnitude of the 3ω signal were partly restored, showing that the 3ω method can be used to monitor the effect of cleaning. The frequency of the AC current was varied so it was possible to measure the heat conductivity in the fouling layer (high frequency) and heat convection due to cross-flow (low frequency). This may potentially be used to get information of the type of fouling (heat conductivity) and thickness of the fouling layer (AC frequency where heat conductivity becomes dominating).

A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus.

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro, the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined ß-1,3/ß-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus. The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

Acute exercise of painful muscles does not reduce the hypoalgesic response in young healthy women - a randomized crossover study.

OBJECTIVES: Exercise-induced hypoalgesia (EIH) is characterized by an increase in pain threshold following acute exercise. EIH is reduced in some individuals with chronic musculoskeletal pain, although the mechanisms are unknown. It has been hypothesized that this may relate to whether exercises are performed in painful or non-painful body regions. The primary aim of this randomized experimental crossover study was to investigate whether the presence of pain per se in the exercising muscles reduced the local EIH response. The secondary aim was to investigate if EIH responses were also reduced in non-exercising remote muscles. METHODS: Pain-free women (n=34) participated in three separate sessions. In session 1, the maximal voluntary contraction (MVC) for a single legged isometric knee extension exercise was determined. In sessions 2 and 3, pressure pain thresholds (PPT) were assessed at the thigh and shoulder muscles before and after a 3-min exercise at 30 % of MVC. Exercises were performed with or without thigh muscle pain, which was induced by either a painful injection (hypertonic saline, 5.8 %) or a non-painful injection (isotonic saline, 0.9 %) into the thigh muscle. Muscle pain intensity was assessed with an 11-point numerical rating scale (NRS) at baseline, after injections, during and after exercises. RESULTS: PPTs increased at thigh and shoulder muscles after exercise with painful (14.0-24.9 %) and non-painful (14.3-19.5 %) injections and no significant between-injection EIH differences were observed (p>0.30). Muscle pain intensity was significantly higher following the painful injection compared to the non-painful injection (p<0.001). CONCLUSIONS: Exercising painful muscles did not reduce the local or remote hypoalgesic responses, suggesting that the pain-relieving effects of isometric exercises are not reduced by exercising painful body regions. ETHICAL COMMITTEE NUMBER: S-20210184. TRIAL REGISTRATION NUMBER: NCT05299268.

Structure of the Wnt-Frizzled-LRP6 initiation complex reveals the basis for coreceptor discrimination.

Wnt morphogens are critical for embryonic development and tissue regeneration. Canonical Wnts form ternary receptor complexes composed of tissue-specific Frizzled (Fzd) receptors together with the shared LRP5/6 coreceptors to initiate ß-catenin signaling. The cryo-EM structure of a ternary initiation complex of an affinity-matured XWnt8-Frizzled8-LRP6 complex elucidates the basis of coreceptor discrimination by canonical Wnts by means of their N termini and linker domains that engage the LRP6 E1E2 domain funnels. Chimeric Wnts bearing modular linker "grafts" were able to transfer LRP6 domain specificity between different Wnts and enable non-canonical Wnt5a to signal through the canonical pathway. Synthetic peptides comprising the linker domain serve as Wnt-specific antagonists. The structure of the ternary complex provides a topological blueprint for the orientation and proximity of Frizzled and LRP6 within the Wnt cell surface signalosome.

Synthetic Multivalent Disulfide-Constrained Peptide Agonists Potentiate Wnt1/ß-Catenin Signaling via LRP6 Coreceptor Clustering.

Wnt ligands are critical for tissue homeostasis and form a complex with LRP6 and frizzled coreceptors to initiate Wnt/ß-catenin signaling. Yet, how different Wnts achieve various levels of signaling activation through distinct domains on LRP6 remains elusive. Developing tool ligands that target individual LRP6 domains could help elucidate the mechanism of Wnt signaling regulation and uncover pharmacological approaches for pathway modulation. We employed directed evolution of a disulfide constrained peptide (DCP) to identify molecules that bind to the third ß-propeller domain of LRP6. The DCPs antagonize Wnt3a while sparing Wnt1 signaling. Using PEG linkers with different geometries, we converted the Wnt3a antagonist DCPs to multivalent molecules that potentiated Wnt1 signaling by clustering the LRP6 coreceptor. The mechanism of potentiation is unique as it occurred only in the presence of extracellular secreted Wnt1 ligand. While all DCPs recognized a similar binding interface on LRP6, they displayed different spatial orientations that influenced their cellular activities. Moreover, structural analyses revealed that the DCPs exhibited new folds that were distinct from the parent DCP framework they were evolved from. The multivalent ligand design principles highlighted in this study provide a path for developing peptide agonists that modulate different branches of cellular Wnt signaling.

Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.

Cellular senescence and the senescence-associated secretory phenotype (SASP) are implicated in aging and age-related disease, and SASP-related inflammation is thought to contribute to tissue dysfunction in aging and diseased animals. However, whether and how SASP factors influence the regenerative capacity of tissues remains unclear. Here, using intestinal organoids as a model of tissue regeneration, we show that SASP factors released by senescent fibroblasts deregulate stem cell activity and differentiation and ultimately impair crypt formation. We identify the secreted N-terminal domain of Ptk7 as a key component of the SASP that activates non-canonical Wnt / Ca2+ signaling through FZD7 in intestinal stem cells (ISCs). Changes in cytosolic [Ca2+] elicited by Ptk7 promote nuclear translocation of YAP and induce expression of YAP/TEAD target genes, impairing symmetry breaking and stem cell differentiation. Our study discovers secreted Ptk7 as a factor released by senescent cells and provides insight into the mechanism by which cellular senescence contributes to tissue dysfunction in aging and disease.

Nanobody-driven signaling reveals the core receptor complex in root nodule symbiosis.

Understanding the composition and activation of multicomponent receptor complexes is a challenge in biology. To address this, we developed a synthetic approach based on nanobodies to drive assembly and activation of cell surface receptors and apply the concept by manipulating receptors that govern plant symbiosis with nitrogen-fixing bacteria. We show that the Lotus japonicus Nod factor receptors NFR1 and NFR5 constitute the core receptor complex initiating the cortical root nodule organogenesis program as well as the epidermal program controlling infection. We find that organogenesis signaling is mediated by the intracellular kinase domains whereas infection requires functional ectodomains. Finally, we identify evolutionarily distant barley receptors that activate root nodule organogenesis, which could enable engineering of biological nitrogen-fixation into cereals.

Directed evolution identifies high-affinity cystine-knot peptide agonists and antagonists of Wnt/ß-catenin signaling.

Developing peptide-based tools to fine-tune growth signaling pathways, in particular molecules with exquisite selectivity and high affinities, opens up opportunities for cellular reprogramming in tissue regeneration. Here, we present a library based on cystine-knot peptides (CKPs) that incorporate multiple loops for randomization and selection via directed evolution. Resulting binders could be assembled into multimeric structures to fine-tune cellular signaling. An example is presented for the Wnt pathway, which plays a key role in the homeostasis and regeneration of tissues such as lung, skin, and intestine. We discovered picomolar affinity CKP agonists of the human LPR6 receptor by exploring the limits of the topological manipulation of LRP6 dimerization. Structural analyses revealed that the agonists bind at the first ß-propeller domain of LRP6, mimicking the natural Wnt inhibitors DKK1 and SOST. However, the CKP agonists exhibit a different mode of action as they amplify the signaling of natural Wnt ligands but do not activate the pathway by themselves. In an alveolosphere organoid model, the CKP agonists induced alveolar stem cell activity. They also stimulated growth in primary human intestinal organoids. The approach described here advances the important frontier of next-generation agonist design and could be applied to other signaling pathways to discover tunable agonist ligands.

Introducing Cysteines into Nanobodies for Site-Specific Labeling.

We have developed a generally applicable methodology for cysteine mutagenesis of nanobody (Nb) framework region serine residues. This strategy allows for subsequent labeling with thiol-reactive compounds without disrupting Nb antigen binding. We provide a protocol for production, labeling, and affinity determination of cysteine-engineered Nbs (cys-Nbs) with Alexa Fluor 488-maleimide and the mercury compound para-chloromercuribenzoic acid (PCMB). Alexa Fluor 488- and PCMB-labeled cys-Nbs can be used for immunofluorescence microscopy and experimental phasing in crystallography, respectively.

Hypoalgesia after exercises with painful vs. non-painful muscles in healthy subjects - a randomized cross-over study.

OBJECTIVES: Exercise-induced hypoalgesia (EIH) is a decrease in the pain sensitivity after exercise. Individuals with chronic pain show less EIH after one exercise session compared with pain-free individuals possibly due to pain in exercising muscles. The primary aim of this randomized controlled cross-over study was to compare the EIH response at the exercising thigh muscle following exercises performed with painful vs. non-painful muscles. Secondary aims were to explore if a reduced EIH response was confined to the painful muscle, and whether the muscle pain intensity and the EIH responses were negatively associated. METHODS: In two sessions, 34 pain-free participants received a painful (hypertonic saline, 5.8%) injection and a control (isotonic saline, 0.9%) injection in the right thigh muscle before performing a 3 min isometric wall squat exercise. Pressure pain thresholds (PPTs) were assessed at both thighs and the left neck/shoulder at baseline, after injections and after exercise. Pain intensities in the thighs were rated on numerical rating scales (NRS: 0-10). RESULTS: Hypertonic saline induced moderate thigh pain at rest (NRS: 4.6 ± 2.1) compared to the control injection (NRS: 0.3 ± 0.4; p<0.001). EIH at the thighs and neck/shoulder were not different between sessions (Injected thigh: 0 kPa; 95% CI: -51 to 52; Contralateral thigh: -6 kPa; 95% CI: -42 to 30; neck/shoulder: 19 kPa; 95% CI: -6 to 44). No significant associations between pain intensity ratings immediately after the Painful injection and EIH responses at any assessment sites were found (right thigh: ß=0.08, 95% CI: -12.95 to 20.64, p=0.64, left thigh: ß=-0.33, 95% CI: -27.86 to 0.44, p=0.06; neck/shoulder: ß=-0.18, 95% CI: -15.11 to 4.96, p=0.31). CONCLUSIONS: Pain in the area of an exercising muscle did not reduce local or systemic EIH responses. TRIAL REGISTRATION NUMBER: NCT04354948.

Distinct conformations of the HIV-1 V3 loop crown are targetable for broad neutralization.

The V3 loop of the HIV-1 envelope (Env) protein elicits a vigorous, but largely non-neutralizing antibody response directed to the V3-crown, whereas rare broadly neutralizing antibodies (bnAbs) target the V3-base. Challenging this view, we present V3-crown directed broadly neutralizing Designed Ankyrin Repeat Proteins (bnDs) matching the breadth of V3-base bnAbs. While most bnAbs target prefusion Env, V3-crown bnDs bind open Env conformations triggered by CD4 engagement. BnDs achieve breadth by focusing on highly conserved residues that are accessible in two distinct V3 conformations, one of which resembles CCR5-bound V3. We further show that these V3-crown conformations can, in principle, be attacked by antibodies. Supporting this conclusion, analysis of antibody binding activity in the Swiss 4.5 K HIV-1 cohort (n = 4,281) revealed a co-evolution of V3-crown reactivities and neutralization breadth. Our results indicate a role of V3-crown responses and its conformational preferences in bnAb development to be considered in preventive and therapeutic approaches.

[Management of antipsychotic-induced metabolic side effects].

Antipsychotics are associated with significant weight gain and other metabolic side effects. There are, however, substantial differences in their propensity for causing metabolic side effects as summarised in this review. These differences are important to consider when deciding which antipsychotic to use. Given the risk of metabolic side effects, patients should be closely monitored regarding anthropometric measures and metabolic parameters. Moreover, both non-pharmacological and pharmacological interventions should be considered for treatment of antipsychotic-induced weight gain.

MANAGEMENT OF ENDOCRINE DISEASE: Glucocorticoid-induced adrenal insufficiency: replace while we wait for evidence?

Glucocorticoids are, besides non-steroidal anti-inflammatory drugs, the most widely used anti-inflammatory medications. Prevalence studies indicate substantial use of both systemic and locally acting agents. A recognised adverse effect of glucocorticoid treatment is adrenal insufficiency, which is highly prevalent based on biochemical testing, but its clinical implications are poorly understood. Current evidence, including randomised trials and observational studies, indicates substantial variation among patients in both risk and course of glucocorticoid-induced adrenal insufficiency, but both are currently unpredictable. Oral and intra-articular formulations, as well as long-term and high-dose treatments, carry the highest risk of glucocorticoid-induced adrenal insufficiency defined by biochemical tests. However, no route of administration, treatment duration, or dose can be considered without risk. More research is needed to estimate the risk and temporal pattern of glucocorticoid-induced adrenal insufficiency, to investigate its clinical implications, and to identify predictors of risk and prognosis. Randomized trials are required to evaluate whether hydrocortisone replacement therapy mitigates risk and symptoms of glucocorticoid-induced adrenal insufficiency in patients discontinuing glucocorticoid treatment. This review aims to provide an overview of the available evidence, pointing to knowledge gaps and unmet needs.

Modulation of Exercise-Induced Hypoalgesia Following an Exercise Intervention in Healthy Subjects.

BACKGROUND: Exercise is recommended to promote and maintain health and as treatment for more than 25 diseases and pain conditions. Exercise-induced hypoalgesia (EIH), a measure of descending pain inhibitory control, has been found to be impaired in some chronic pain conditions, but it is currently unclear if EIH is modifiable. This study investigated whether a long-term exercise intervention could modulate EIH in healthy subjects. METHODS: In 38 healthy subjects, EIH was assessed as change in pressure pain threshold (PPT) after a three-minute isometric wall squat within the first week and after approximately seven weeks of military training (MT). Further, temporal summation of pain (TSP) and Knee injury and Osteoarthritis Outcome Score (KOOS) were assessed. Physical performance capacity was assessed using the Endurance 20-m shuttle run fitness test (20MSR). Hypoalgesic (EIH > 0.0 kPa) and hyperalgesic (EIH ≤ 0.0 kPa) subgroups were defined based on baseline EIH. Change in EIH following MT was used as the primary outcome. RESULTS: Increased EIH (P = 0.008), PPT (P < 0.003), and 20MSR (P < 0.001) were found following MT, with no changes in TSP and KOOS (P > 0.05). Subjects with a hyperalgesic EIH response at baseline (26% of the participants) presented significantly improved EIH following MT (P = 0.010). Finally, an association between 20MRS change and EIH change was found (r = 0.369, P = 0.023). CONCLUSIONS: MT increased EIH, especially in subjects who demonstrated a hyperalgesic response at baseline. Improvement in physical performance capacity was associated with an improvement in EIH, indicating that improvement in physical performance capacity may improve central pain mechanisms.

Ligand-recognizing motifs in plant LysM receptors are major determinants of specificity.

Plants evolved lysine motif (LysM) receptors to recognize and parse microbial elicitors and drive intracellular signaling to limit or facilitate microbial colonization. We investigated how chitin and nodulation (Nod) factor receptors of Lotus japonicus initiate differential signaling of immunity or root nodule symbiosis. Two motifs in the LysM1 domains of these receptors determine specific recognition of ligands and discriminate between their in planta functions. These motifs define the ligand-binding site and make up the most structurally divergent regions in cognate Nod factor receptors. An adjacent motif modulates the specificity for Nod factor recognition and determines the selection of compatible rhizobial symbionts in legumes. We also identified how binding specificities in LysM receptors can be altered to facilitate Nod factor recognition and signaling from a chitin receptor, advancing the prospects of engineering rhizobial symbiosis into nonlegumes.

Individual differences in learning during decision-making may predict specific harms associated with gambling.

Problem gambling has been linked to impairments in decision-making under uncertainty. Problem gamblers are more likely to favor high-risk, high-reward, and short-term gains over more advantageous choice alternatives, and this preference has been linked to impaired learning about decision outcomes. In this paper we link specific learning processes in decision-making to specific harms related to problem gambling. We asked a group of 140 casual gamblers to 1) perform a canonical decision-making task (the Iowa Gambling Task) online, and 2) to complete a self-report survey (the GamTest) designed to measure self-perceived harm caused by their gambling. We used a reinforcement learning model to explain individual differences in the decision task, and related individuals' model parameters to the specific problem areas reported using the questionnaire. We found that people who learned more from gains than from losses on the task were more likely to report overall gambling problems, and problems specifically related to money. We also found that people whose learning was more driven by the frequency of rewards were more likely to report problems related to the amount of time spent gambling, as well as social problems. We discuss possible psychological and neural processes mediating learning and gambling related harms, and we discuss the relevance of our approach to the diagnosis of problem gambling and its consequences.

It Takes Two to Regenerate: Optimizing Custom Wnt Surrogates.

In this issue of Cell Chemical Biology, Chen et al. (2020) present an antibody-based platform to generate Wnt agonists, offering multiple design principles for systematic investigation of Wnt activation. This study lays the groundwork to develop potent Wnt agonists for applications in regenerative medicine.

Pretreatment Exercise-induced Hypoalgesia is Associated With Change in Pain and Function After Standardized Exercise Therapy in Painful Knee Osteoarthritis.

OBJECTIVES: Exercise-induced hypoalgesia (EIH), a measure of descending pain inhibitory control, has been found hyperalgesic in subgroups of painful knee osteoarthritis (KOA) patients. The effect of standardized exercise therapy (ET) on clinical pain intensity in KOA has been demonstrated. However, the prognostic value of EIH in KOA patients completing an ET program has not been investigated. This study investigated the prognostic value of EIH on pain relief following ET in KOA patients. MATERIALS AND METHODS: In 24 painful KOA patients (Numerical Rating Scale, 0 to 10 ≥3), EIH was assessed as change in pressure pain threshold after 2-minute "lateral raises" before and after ET in this observational study. In addition, temporal summation of pain, clinical pain scores (Numerical Rating Scale, Knee injury and Osteoarthritis Outcome Score [KOOS], and PainDETECT Questionnaire) were assessed before and after ET. The KOOS-4 is defined by the KOOS subscale scores for Pain, Symptoms, Activities of Daily Living, and Quality of Life and was used as the primary outcome. RESULTS: Following ET, all clinical pain scores improved (P<0.01) but no changes in pressure pain threshold, temporal summation of pain, or EIH were found (P>0.05). Linear regression models identified pretreatment EIH (ß=0.59, P<0.005) and PainDETECT Questionnaire (ß=0.57, P<0.005) as independent factors for relative change in KOOS-4 after ET (adjusted R=46.8%). DISCUSSION: These preliminary and exploratory results suggest that patients with a high EIH response before a standardized ET program may be associated with a large improvement in pain after treatment. This measure may potentially help clinicians as a prognostic tool for outcome prediction following ET in KOA patients.

Lead Optimization Yields High Affinity Frizzled 7-Targeting Peptides That Modulate Clostridium difficile Toxin B Pathogenicity in Epithelial Cells.

Frizzled 7 (FZD7) receptors have been shown to play a central role in intestinal stem cell regeneration and, more recently, in Clostridium difficile pathogenesis. Yet, targeting FZD7 receptors with small ligands has not been explored as an approach to block C. difficile pathogenesis. Here, we report the discovery of high affinity peptides that selectively bind to FZD7 receptors. We describe an integrated approach for lead optimization, utilizing structure-based rational design and directed evolution, to enhance the peptide binding affinity while still maintaining FZD7 receptor selectivity. This work yielded new peptide leads with picomolar binding constants to FZD7 as measured by biophysical methods. The new peptides block the interaction between C. difficile toxin B (TcdB) and FZD receptors and perturb C. difficile pathogenesis in epithelial cells. As such, our findings provide a proof of concept that targeting FZD receptors could be a viable pharmacological approach to protect epithelial cells from TcdB pathogenicity.