The neuronal transcription factor MEIS2 is a calpain-2 protease target.

Tight control over transcription factor activity is necessary for a sensible balance between cellular proliferation and differentiation in the embryo and during tissue homeostasis by adult stem cells, but mechanistic details have remained incomplete. The homeodomain transcription factor MEIS2 is an important regulator of neurogenesis in the ventricular-subventricular zone (V-SVZ) adult stem cell niche in mice. We here identify MEIS2 as direct target of the intracellular protease calpain-2 (composed of the catalytic subunit CAPN2 and the regulatory subunit CAPNS1). Phosphorylation at conserved serine and/or threonine residues, or dimerization with PBX1, reduced the sensitivity of MEIS2 towards cleavage by calpain-2. In the adult V-SVZ, calpain-2 activity is high in stem and progenitor cells, but rapidly declines during neuronal differentiation, which is accompanied by increased stability of MEIS2 full-length protein. In accordance with this, blocking calpain-2 activity in stem and progenitor cells, or overexpression of a cleavage-insensitive form of MEIS2, increased the production of neurons, whereas overexpression of a catalytically active CAPN2 reduced it. Collectively, our results support a key role for calpain-2 in controlling the output of adult V-SVZ neural stem and progenitor cells through cleavage of the neuronal fate determinant MEIS2.

IL-3 receptor signalling suppresses chronic intestinal inflammation by controlling mechanobiology and tissue egress of regulatory T cells.

IL-3 has been reported to be involved in various inflammatory disorders, but its role in inflammatory bowel disease (IBD) has not been addressed so far. Here, we determined IL-3 expression in samples from patients with IBD and studied the impact of Il3 or Il3r deficiency on T cell-dependent experimental colitis. We explored the mechanical, cytoskeletal and migratory properties of Il3r -/- and Il3r +/+ T cells using real-time deformability cytometry, atomic force microscopy, scanning electron microscopy, fluorescence recovery after photobleaching and in vitro and in vivo cell trafficking assays. We observed that, in patients with IBD, the levels of IL-3 in the inflamed mucosa were increased. In vivo, experimental chronic colitis on T cell transfer was exacerbated in the absence of Il-3 or Il-3r signalling. This was attributable to Il-3r signalling-induced changes in kinase phosphorylation and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of Tregs from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Tregs and was associated with increased mucosal Treg abundance in patients with IBD. Collectively, our data reveal that IL-3 signaling exerts an important regulatory role at the interface of biophysical and migratory T cell features in intestinal inflammation and suggest that this might be an interesting target for future intervention.

Project DECIDE, part II: decision-making places for people with dementia in Alzheimer's disease: supporting advance decision-making by improving person-environment fit.

BACKGROUND: The UN Convention on the Rights of Persons with Disabilities, and the reformed guardianship law in Germany, require that persons with a disability, including people with dementia in Alzheimer's disease (PwAD), are supported in making self-determined decisions. This support is achieved through communication. While content-related communication is a deficit of PwAD, relational aspects of communication are a resource. Research in supported decision-making (SDM) has investigated the effectiveness of different content-related support strategies for PwAD but has only succeeded in improving understanding, which, although one criterion of capacity to consent, is not sufficient to ensure overall capacity to consent. The aim of the 'spatial intervention study' of the DECIDE project is to examine an innovative resource-oriented SDM approach that focuses on relational aspects. We hypothesise that talking to PwAD in their familiar home setting (as opposed to a clinical setting) will reduce the complexity of the decision-making process and enhance overall capacity to consent. METHODS: People with a suspected or confirmed diagnosis of dementia in Alzheimer's disease will be recruited from two memory clinics (N = 80). We will use a randomised crossover design to investigate the intervention effect of the decision-making place on capacity to consent. Besides reasoning capacity, which is part of overall capacity to consent and will be the primary outcome, various secondary outcomes (e.g., other aspects of capacity to consent, subjective task complexity, decisional conflict) and suspected moderating or mediating variables (e.g., meaning of home, demographic characteristics) will be assessed. DISCUSSION: The results of the study will be used to develop a new SDM strategy that is based on relational resources for PwAD. If a change in location achieves the anticipated improvement in capacity to consent, future research should focus on implementing this SDM strategy in a cost-effective manner in clinical practice. TRIAL REGISTRATION: DRKS00030799 .

Empowering patients with dementia to make legally effective decisions: a randomized controlled trial on enhancing capacity to consent to treatment.

Objectives: As our society ages, the incidence of age-related diseases increases and with it the number of medical treatments that require informed consent. Capacity to consent is often categorically questioned in persons with dementia (PwD) without appropriate assessment, depriving them of their right to autonomous decision-making. Supportive structures for PwD that comply with legal requirements are lacking. The EmMa project tried to overcome this shortcoming by developing and testing possible supportive measures to enhance the informed consent process for PwD.Method: These enhanced consent procedures (ECPs) were tested in a randomized controlled trial with 40 PwD. It was hypothesized that strengths-based ECPs could improve capacity to consent to a drug treatment in PwD as measured with a semi-structured interview.Results: Against the expectations, no effect of the ECPs on capacity to consent could be found, but the ECPs improved understanding of information in PwD.Conclusion: To empower PwD in clinical settings, however, all aspects of capacity to consent should be targeted with specific aids that are implemented carefully and selectively. More research on possible aids for ECPs is urgently needed in order to enable ethically and legally robust informed consent. In particular, effective ways to improve both reasoning and appreciation are yet to be found.

Corporate sector engagement in contemporary 'crises': the case of refugee integration in Germany.

Refugee integration is one of the main global challenges of the present, at a time when the corporate sector is regarded as a key actor in multi-stakeholder partnerships through the 2030 Agenda for Sustainable Development. This paper examines its role as a partner of the state in addressing the movement of refugees into Germany from 2015 onwards. Based on interview data and informal conversations with members of Wir Zusammen, an integration initiative, and supplemented by a review of business reports and media documentation, it discusses the multifaceted engagements by parts of the corporate sector in Germany with refugee integration. These are analysed as 'thin' and 'thick', and as following different institutional logics. The paper adds to understanding of the political dimensions of corporate responses, their potential to challenge the status quo, and their pitfalls. Ultimately, it argues that corporate involvement with humanitarian and development challenges works best when embedded locally and is context specific.

Residual homing of α4ß7-expressing ß1+PI16+ regulatory T cells with potent suppressive activity correlates with exposure-efficacy of vedolizumab.

OBJECTIVE: The anti-α4ß7 integrin antibody vedolizumab is administered at a fixed dose for the treatment of IBDs. This leads to a wide range of serum concentrations in patients and previous studies had suggested that highest exposure levels are associated with suboptimal clinical response. We aimed to determine the mechanisms underlying these non-linear exposure-efficacy characteristics of vedolizumab. DESIGN: We characterised over 500 samples from more than 300 subjects. We studied the binding of vedolizumab to T cells and investigated the functional consequences for dynamic adhesion, transmigration, gut homing and free binding sites in vivo. Employing single-cell RNA sequencing, we characterised α4ß7 integrin-expressing T cell populations 'resistant' to vedolizumab and validated our findings in vitro and in samples from vedolizumab-treated patients with IBD. We also correlated our findings with a post-hoc analysis of the Gemini II and III studies. RESULTS: Regulatory T (TReg) cells exhibited a right-shifted vedolizumab binding profile compared with effector T (TEff) cells. Consistently, in a certain concentration range, the residual adhesion, transmigration, homing of and availability of functional α4ß7 on TReg cells in vivo was higher than that of/on TEff cells. We identified a vedolizumab-'resistant' α4ß7-expressing ß1+PI16+ TReg cell subset with pronounced regulatory properties as the substrate for this effect. Our observations correlated with exposure-efficacy data from Gemini II and III trials. CONCLUSION: Completely blocking TEff cell trafficking with vedolizumab, while simultaneously permitting residual homing of powerful TReg cells in an optimal 'therapeutic window' based on target exposure levels might be a strategy to optimise treatment outcomes in patients with IBD.

Project DECIDE, part 1: increasing the amount of valid advance directives in people with Alzheimer's disease by offering advance care planning-a prospective double-arm intervention study.

BACKGROUND: Everybody has the right to decide whether to receive specific medical treatment or not and to provide their free, prior and informed consent to do so. As dementia progresses, people with Alzheimer's dementia (PwAD) can lose their capacity to provide informed consent to complex medical treatment. When the capacity to consent is lost, the autonomy of the affected person can only be guaranteed when an interpretable and valid advance directive exists. Advance directives are not yet common in Germany, and their validity is often questionable. Once the dementia diagnosis has been made, it is assumed to be too late to write an advance directive. One approach used to support the completion of advance directives is 'Respecting Choices'®-an internationally recognised, evidence-based model of Advance Care Planning (ACP), which, until now, has not been evaluated for the target group of PwAD. This study's aims include (a) to investigate the proportion of valid advance directives in a memory clinic population of persons with suspected AD, (b) to determine the predictors of valid advance directives, and (c) to examine whether the offer of ACP can increase the proportion of valid advance directives in PwAD. METHOD: We intend to recruit at least N = 250 participants from two memory clinics in 50 consecutive weeks. Of these, the first 25 weeks constitute the baseline phase (no offer of ACP), the following 25 weeks constitute the intervention phase (offer of ACP). The existence and validity of an advance directive will be assessed twice (before and after the memory clinic appointment). Moreover, potential predictors of valid advance directives are assessed. DISCUSSION: The results of this study will enhance the development of consent procedures for advance directives of PwAD based on the ACP/Respecting Choices (R) approach. Therefore, this project contributes towards increasing the autonomy and inclusion of PwAD and the widespread acceptance of valid advance directives in PwAD. Trial Registration DRKS, DRKS00026691, registered 15th of October 2021, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00026691.

Clinical experiences and predictors of success of treatment with vedolizumab in IBD patients: a cohort study.

BACKGROUND: Vedolizumab has become a standard treatment for the inflammatory bowel diseases ulcerative colitis (UC) and Crohn's disease (CD). However, there is an ongoing debate on the ideal individual treatment algorithms and means to predict treatment response are not routinely established. AIMS: We aimed to describe our experiences with vedolizumab at a large German tertiary referral center and to identify clinical predictors of success of vedolizumab treatment. METHODS: We performed a retrospective single-center cohort study employing univariable and multivariable analyses as well as Kaplan-Meier analyses of persistence on treatment. RESULTS: 36% and 35% of the patients with UC and CD, respectively, reached clinical remission after 17 weeks. Patients with lower clinical disease activity were more likely to achieve remission. The median persistence on treatment was 33 months for UC and 29 months for CD. CONCLUSION: Our study confirms that vedolizumab is an efficient option for the treatment of UC and CD. Clinical parameters of disease activity may help to predict the success of treatment.

A Multiple Piccolino-RIBEYE Interaction Supports Plate-Shaped Synaptic Ribbons in Retinal Neurons.

Active zones at chemical synapses are highly specialized sites for the regulated release of neurotransmitters. Despite a high degree of active zone protein conservation in vertebrates, every type of chemical synapse expresses a given set of protein isoforms and splice variants adapted to the demands on neurotransmitter release. So far, we know little about how specific active zone proteins contribute to the structural and functional diversity of active zones. In this study, we explored the nanodomain organization of ribbon-type active zones by addressing the significance of Piccolino, the ribbon synapse-specific splice variant of Piccolo, for shaping the ribbon structure. We followed up on previous results, which indicated that rod photoreceptor synaptic ribbons lose their structural integrity in a knockdown of Piccolino. Here, we demonstrate an interaction between Piccolino and the major ribbon component RIBEYE that supports plate-shaped synaptic ribbons in retinal neurons. In a detailed ultrastructural analysis of three different types of retinal ribbon synapses in Piccolo/Piccolino-deficient male and female rats, we show that the absence of Piccolino destabilizes the superstructure of plate-shaped synaptic ribbons, although with variable manifestation in the cell types examined. Our analysis illustrates how the expression of a specific active zone protein splice variant (e.g., Piccolino) contributes to structural diversity of vertebrate active zones.SIGNIFICANCE STATEMENT Retinal ribbon synapses are a specialized type of chemical synapse adapted for the regulated fast and tonic release of neurotransmitter. The hallmark of retinal ribbon synapses is the plate-shaped synaptic ribbon, which extends from the release site into the terminals' cytoplasm and tethers hundreds of synaptic vesicles. Here, we show that Piccolino, the synaptic ribbon specific splice variant of Piccolo, interacts with RIBEYE, the main component of synaptic ribbons. This interaction occurs via several PxDLS-like motifs located at the C terminus of Piccolino, which can connect multiple RIBEYE molecules. Loss of Piccolino disrupts the characteristic plate-shaped structure of synaptic ribbons, indicating a role of Piccolino in synaptic ribbon assembly.

COVID-19 and the case for global development.

COVID-19 accentuates the case for a global, rather than an international, development paradigm. The novel disease is a prime example of a development challenge for all countries, through the failure of public health as a global public good. The COVID-19 pandemic has highlighted the falsity of any assumption that the global North has all the expertise and solutions to tackle global challenges, and has further highlighted the need for multi-directional learning and transformation in all countries towards a more sustainable and equitable world. We illustrate our argument for a global development paradigm by examining the implications of the COVID-19 pandemic across four themes or 'vignettes': global value chains, digitalisation, debt, and climate change. We conclude that development studies must adapt to a very different context from when the field emerged in the mid-20th century.

Analysis of RIM Expression and Function at Mouse Photoreceptor Ribbon Synapses.

RAB3A-interacting molecule (RIM) proteins are important regulators of transmitter release from active zones. At conventional chemical synapses, RIMs contribute substantially to vesicle priming and docking and their loss reduces the readily releasable pool of synaptic vesicles by up to 75%. The priming function of RIMs is mediated via the formation of a tripartite complex with Munc13 and RAB3A, which brings synaptic vesicles in close proximity to Ca2+ channels and the fusion site and activates Munc13. We reported previously that, at mouse photoreceptor ribbon synapses, vesicle priming is Munc13 independent. In this study, we examined RIM expression, distribution, and function at male and female mouse photoreceptor ribbon synapses. We provide evidence that RIM1α and RIM1ß are highly likely absent from mouse photoreceptors and that RIM2α is the major large RIM isoform present at photoreceptor ribbon synapses. We show that mouse photoreceptors predominantly express RIM2 variants that lack the interaction domain for Munc13. Loss of full-length RIM2α in a RIM2α mutant mouse only marginally perturbs photoreceptor synaptic transmission. Our findings therefore strongly argue for a priming mechanism at the photoreceptor ribbon synapse that is independent of the formation of a RIM-Munc13-RAB3A complex and thus provide further evidence for a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses in synaptic vesicle exocytosis.SIGNIFICANCE STATEMENT RAB3A-interacting molecules 1 and 2 (RIM1/2) are essential regulators of exocytosis. At conventional chemical synapses, their function involves Ca2+ channel clustering and synaptic vesicle priming and docking through interactions with Munc13 and RAB3A, respectively. Examining wild-type and RIM2 mutant mice, we show here that the sensory photoreceptor ribbon synapses most likely lack RIM1 and predominantly express RIM2 variants that lack the interaction domain for Munc13. Our findings demonstrate that the photoreceptor-specific RIM variants are not essential for synaptic vesicle priming at photoreceptor ribbon synapses, which represents a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses with respect to synaptic vesicle priming mechanisms.

[Capacity to consent to treatment in dementia patients : Interdisciplinary perspectives]. / Einwilligungsfähigkeit bei Demenz : Interdisziplinäre Perspektiven.

BACKGROUND: Currently, there are no concrete, evidence-based default procedures to adequately assess the capacity to consent to medical treatment. This explains why different raters use individual heuristics, differing both within and between disciplines. The lack of a procedure that is standardized, validated, and equally accepted across disciplines to assess the capacity to consent to treatment has led to an uncertainty in legal and medical practice regarding appropriate action and decision making. Due to the dramatic increase in the number of patients with dementia and multimorbidity, who have to regularly consent to various medical treatments but whose capacity to consent is hindered due to cognitive impairments, this topic is particularly timely. OBJECTIVE: The present study aims to investigate how experts of different disciplines (law, medicine, ethics) assess the capacity to consent on the basis of a case study involving a patient with dementia. MATERIALS AND METHODS: The judgements and judgement criteria of 41 experts from various disciplines were surveyed via a self-administered questionnaire that was distributed via postal mail. RESULTS: The results show that experts from various disciplines come to different conclusions regarding the capacity of the patient with dementia to consent. CONCLUSION: These observations lead to the conclusion that there is an urgent need for an interdisciplinary guideline for the assessment of the capacity to consent to treatment, for interdisciplinary training, and for more interdisciplinary exchange in the assessment process.

Compliance with CBT referral in nursing home residents diagnosed with depression: Results from a feasibility study.

Objectives: Patient-level factors that influence compliance with a recommendation for CBT in nursing home residents diagnosed with depression were identified. Methods: Within a cluster-randomized trial on stepped care for depression in nursing homes (DAVOS-study, Trial registration: DRKS00015686), participants received an intake interview administered by a licensed psychotherapist. If psychotherapy was required, patients were offered a referral for CBT. Sociodemographic characteristics, severity of depression, loneliness, physical health, antidepressant medication, prior experience with psychotherapy, and attitudes towards own aging were assessed. A binary regression determined predictors of compliance with referral. Results: Of 123 residents receiving an intake interview, 80 were recommended a CBT. Forty-seven patients (58.8 %) followed the recommendation. The binary logistic regression model on compliance with recommended CBT was significant, χ2(9) = 21.64, p = .010. Significant predictors were age (Odds Ratio (OR) = 0.9; 95 % Confidence Interval (CI) = 0.82, 0.99; p = .024) and depression (OR = 1.33; 95 % CI = 1.08, 1.65; p = .008). Conclusion: Within the implemented setting compliance rate was comparable to other age groups. Future interventions should include detailed psychoeducation on the benefits of psychotherapy on mild depressive symptoms in older age and evidence-based interventions to address the stigma of depression. Interventions such as reminiscence-based methods or problem-solving could be useful to increase compliance with referral, especially in very old patients (80+). Language barriers and a culturally sensitive approach should be considered when screening residents.

Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies.

Monocytes, as well as downstream macrophages and dendritic cells, are essential players in the immune system, fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies on reporter models, mouse monocytes are categorized into a classical and a non-classical subset based on their inversely correlated surface expression of Ly6C/CCR2 and CX3CR1. Here, we aimed to challenge this concept by antibody staining and reporter mouse models. Therefore, we took advantage of Cx3cr1GFP and Ccr2RFP reporter mice, in which the respective gene was replaced by a fluorescent reporter protein gene. We analyzed the expression of CX3CR1 and CCR2 by flow cytometry using several validated fluorochrome-coupled antibodies and compared them with the reporter gene signal in these reporter mouse strains. Although we were able to validate the specificity of the fluorochrome-coupled flow cytometry antibodies, mouse Ly6Chigh classical and Ly6Clow non-classical monocytes showed no differences in CX3CR1 expression levels in the peripheral blood and spleen when stained with these antibodies. On the contrary, in Cx3cr1GFP reporter mice, we were able to reproduce the inverse correlation of the CX3CR1 reporter gene signal and Ly6C surface expression. Furthermore, differential CCR2 surface expression correlating with the expression of Ly6C was observed by antibody staining, but not in Ccr2RFP reporter mice. In conclusion, our data suggest that phenotyping strategies for mouse monocyte subsets should be carefully selected. In accordance with the literature, the suitability of CX3CR1 antibody staining is limited, whereas for CCR2, caution should be applied when using reporter mice.

Etrolizumab-s fails to control E-Cadherin-dependent co-stimulation of highly activated cytotoxic T cells.

Despite promising preclinical and earlier clinical data, a recent phase III trial on the anti-ß7 integrin antibody etrolizumab in Crohn's disease (CD) did not reach its primary endpoint. The mechanisms leading to this outcome are not well understood. Here we characterize the ß7+ T cell compartment from patients with CD in comparison to cells from individuals without inflammatory bowel disease. By flow cytometric, transcriptomic and functional profiling of circulating T cells, we find that triple-integrin-expressing (α4+ß7+ß1hi) T cells have the potential to home to the gut despite α4ß7 blockade and have a specific cytotoxic signature. A subset of triple-integrin-expressing cells readily acquires αE expression and could be co-stimulated via E-Cadherin-αEß7 interactions in vitro. Etrolizumab-s fails to block such αEß7 signalling at high levels of T cell stimulation. Consistently, in CD patients treated with etrolizumab, T cell activation correlates with cytotoxic signatures. Collectively, our findings might add one important piece to the puzzle to explain phase III trial results with etrolizumab, while they also highlight that αEß7 remains an interesting target for future therapeutic approaches in inflammatory bowel disease.

Blocking GPR15 counteracts integrin-dependent T cell gut homing in vivo.

BACKGROUND AND AIMS: The G protein coupled receptor GPR15 is expressed on and functionally important for T cells homing to the large intestine. However, the precise mechanisms by which GPR15 controls gut homing have been unclear. Thus, we aimed to elucidate these mechanisms as well as to explore the potential of targeting GPR15 for interfering with T cell recruitment to the colon in IBD. METHODS: We used dynamic adhesion and transmigration assays as well as a humanized in vivo model of intestinal cell trafficking to study GPR15-dependent effects on gut homing. Moreover, we analysed GPR15 and integrin expression in patients with and without IBD cross-sectionally and longitudinally. RESULTS: GPR15 controlled T cell adhesion to MAdCAM-1 and VCAM-1 upstream of α4ß7 and α4ß1 integrin, respectively. Consistently, high co-expression of these integrins with GPR15 was found on T cells from patients with IBD and GPR15 also promoted T cell recruitment to the colon in humanized mice. Anti-GPR15 antibodies effectively blocked T cell gut homing in vitro and in vivo. In vitro data as well as observations in a cohort of patients treated with vedolizumab suggest that this might be more effective than inhibiting α4ß7. CONCLUSIONS: GPR15 seems to have a broad, but organ-selective impact on T cell trafficking and is therefore a promising target for future therapy of IBD. Further studies are needed.

'The Ethiopian famine' revisited: band aid and the antipolitics of celebrity humanitarian action.

In many ways the Ethiopian famine of 1983-85 has served as a watershed with respect to humanitarian action. One of its lasting legacies has been the emergence of Band Aid and the subsequent increase in celebrity humanitarianism. A revisiting of the events of 1983-85 occurred in 2010 during a dispute in which it was alleged that a portion of the donations of Band Aid were spent on arms purchases. This paper takes this controversy as its starting point. It goes on to use the theoretical reflections of Giorgio Agamben to consider the dynamics that unfolded during the Ethiopian famine of 1983-85 and to analyse the underlying conceptualisation behind the emergence of Band Aid-type celebrity humanitarianism. The paper concludes with some wider thoughts on how the in essence antipolitical agenda of celebrity humanitarian action is transported into the everyday understanding of 'African disaster', resulting ultimately in the perpetuation of hegemonic control by the global North.

Manual cell selection in single cell transcriptomics using scSELpy supports the analysis of immune cell subsets.

Introduction: Single cell RNA sequencing plays an increasing and indispensable role in immunological research such as in the field of inflammatory bowel diseases (IBD). Professional pipelines are complex, but tools for the manual selection and further downstream analysis of single cell populations are missing so far. Methods: We developed a tool called scSELpy, which can easily be integrated into Scanpy-based pipelines, allowing the manual selection of cells on single cell transcriptomic datasets by drawing polygons on various data representations. The tool further supports the downstream analysis of the selected cells and the plotting of results. Results: Taking advantage of two previously published single cell RNA sequencing datasets we show that this tool is useful for the positive and negative selection of T cell subsets implicated in IBD beyond standard clustering. We further demonstrate the feasibility for subphenotyping T cell subsets and use scSELpy to corroborate earlier conclusions drawn from the dataset. Moreover, we also show its usefulness in the context of T cell receptor sequencing. Discussion: Collectively, scSELpy is a promising additive tool fulfilling a so far unmet need in the field of single cell transcriptomic analysis that might support future immunological research.

Prediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering.

A significant number of proteins are annotated as functionally uncharacterized proteins. Within this protocol, we describe how to use protein family multiple sequence alignments and structural bioinformatics resources to design loss-of-function mutations of previously uncharacterized proteins within the glycosyltransferase family. We detail approaches to determine target protein active sites using three-dimensional modeling. We generate active site mutants and quantify any changes in enzymatic function by a glycosyltransferase assay. With modifications, this protocol could be applied to other metal-dependent enzymes. For complete details on the use and execution of this protocol, please refer to Ilina et al. (2022).1.

Computational mechanisms underlying the dynamics of physical and cognitive fatigue.

The willingness to exert effort for reward is essential but comes at the cost of fatigue. Theories suggest fatigue increases after both physical and cognitive exertion, subsequently reducing the motivation to exert effort. Yet a mechanistic understanding of how this happens on a moment-to-moment basis, and whether mechanisms are common to both mental and physical effort, is lacking. In two studies, participants reported momentary (trial-by-trial) ratings of fatigue during an effort-based decision-making task requiring either physical (grip-force) or cognitive (mental arithmetic) effort. Using a novel computational model, we show that fatigue fluctuates from trial-to-trial as a function of exerted effort and predicts subsequent choices. This mechanism was shared across the domains. Selective to the cognitive domain, committing errors also induced momentary increases in feelings of fatigue. These findings provide insight into the computations underlying the influence of effortful exertion on fatigue and motivation, in both physical and cognitive domains.