The lowdown on breakdown: Open questions in plant proteolysis.

Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and post-harvest regulation), plant responses to environmental signals (endoplasmic-reticulum associated degradation, chloroplast-associated degradation, drought tolerance, the growth-defense tradeoff)), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.

Optimal Transport Distances to Characterize Electronic Excitations.

Understanding the character of electronic excitations is important in computational and reaction mechanistic studies, but their classification from simulations remains an open problem. Distances based on optimal transport have proven very useful in a plethora of classification problems and, therefore, seem a natural tool to try to tackle this challenge. We propose and investigate a new diagnostic Θ based on the Sinkhorn divergence from optimal transport. We evaluate a k-NN classification algorithm on Θ, the popular Λ diagnostic, and their combination, and assess their performance in labeling excitations, finding that (i) the combination only slightly improves the classification, (ii) Rydberg excitations are not separated well in any setting, and (iii) Θ breaks down for charge transfer in small molecules. We then define a length-scale-normalized version of Θ and show that the result correlates closely with Λ for results obtained with Gaussian basis functions. Finally, we discuss the orbital dependence of our approach and explore an orbital-independent version. Using an optimized combination of the optimal transport and overlap diagnostics together with a different metric is in our opinion the most promising for future classification studies.

De novo and inherited monoallelic variants in TUBA4A cause ataxia and spasticity.

Alpha-tubulin 4A encoding gene (TUBA4A) has been associated with familial amyotrophic lateral sclerosis (fALS) and fronto-temporal dementia (FTD), based on identification of likely pathogenic variants in patients from distinct ALS and FTD cohorts. By screening a multicentric French cohort of 448 unrelated probands presenting with cerebellar ataxia, we identified ultra-rare TUBA4A missense variants, all being absent from public databases and predicted pathogenic by multiple in-silico tools. In addition, gene burden analyses in the 100,000 genomes project (100KGP) showed enrichment of TUBA4A rare variants in the inherited ataxia group compared to controls (OR: 57.0847 [10.2- 576.7]; p = 4.02 x10-07). Altogether, we report 12 patients presenting with spasticity and/or cerebellar ataxia and harboring a predicted pathogenic TUBA4A missense mutation, including 5 confirmed de novo cases and a mutation previously reported in a large family presenting with spastic ataxia. Cultured fibroblasts from 3 patients harboring distinct TUBA4A missense showed significant alterations in microtubule organisation and dynamics, providing insight of TUBA4A variants pathogenicity. Our data confirm the identification of a hereditary spastic ataxia disease gene with variable age of onset, expanding the clinical spectrum of TUBA4A associated phenotypes.

Intra-chloroplast proteases: A holistic network view of chloroplast proteolysis.

Different proteases and peptidases are present within chloroplasts and non-photosynthetic plastids to process precursor proteins and to degrade cleaved chloroplast transit peptides and damaged, misfolded, or otherwise unwanted proteins. Collectively, these proteases and peptidases form a proteolysis network, with complementary activities and hierarchies, and build-in redundancies. Furthermore, this network is distributed across the different intra-chloroplast compartments (lumen, thylakoid, stroma, envelope). The challenge is to determine the contributions of each peptidase (system) to this network in chloroplasts and non-photosynthetic plastids. This will require an understanding of substrate recognition mechanisms, degrons, substrate and product size limitations, as well as the capacity and degradation kinetics of each protease. Multiple extra-plastidial degradation pathways complement these intra-chloroplast proteases. This review summarizes our current understanding of these intra-chloroplast proteases in Arabidopsis and crop plants with an emphasis on considerations for building a qualitative and quantitative network view.

Association of CYP2D6 and CYP2C19 metabolizer status with switching and discontinuing antidepressant drugs: an exploratory study.

BACKGROUND: Tailoring antidepressant drugs (AD) to patients' genetic drug-metabolism profile is promising. However, literature regarding associations of ADs' treatment effect and/or side effects with drug metabolizing genes CYP2D6 and CYP2C19 has yielded inconsistent results. Therefore, our aim was to longitudinally investigate associations between CYP2D6 (poor, intermediate, and normal) and CYP2C19 (poor, intermediate, normal, and ultrarapid) metabolizer-status, and switching/discontinuing of ADs. Next, we investigated whether the number of perceived side effects differed between metabolizer statuses. METHODS: Data came from the multi-site naturalistic longitudinal cohort Netherlands Study of Depression and Anxiety (NESDA). We selected depression- and/or anxiety patients, who used AD at some point in the course of the 9 years follow-up period (n = 928). Medication use was followed to assess patterns of AD switching/discontinuation over time. CYP2D6 and CYP2C19 alleles were derived using genome-wide data of the NESDA samples and haplotype data from the PharmGKB database. Logistic regression analyses were conducted to investigate the association of metabolizer status with switching/discontinuing ADs. Mann-Whitney U-tests were conducted to compare the number of patient-perceived side effects between metabolizer statuses. RESULTS: No significant associations were observed of CYP metabolizer status with switching/discontinuing ADs, nor with the number of perceived side effects. CONCLUSIONS: We found no evidence for associations between CYP metabolizer statuses and switching/discontinuing AD, nor with side effects of ADs, suggesting that metabolizer status only plays a limited role in switching/discontinuing ADs. Additional studies with larger numbers of PM and UM patients are needed to further determine the potential added value of pharmacogenetics to guide pharmacotherapy.

Exploiting the Hessian for a Better Convergence of the SCF-RDMFT Procedure.

One-body reduced density matrix functional theory provides an alternative to density functional theory, which is able to treat static correlation while keeping a relatively low computation scaling. Its disadvantageous cost comes mainly from a slow convergence of the self-consistent energy optimization. To improve on that problem, we propose in this work the use of the Hessian of the energy, including the coupling term. We show that using the exact Hessian is very effective at reducing the number of iterations. However, since the exact Hessian is too expensive to use in practice, we propose an approximation based on an inexpensive exact part and BFGS updates.

Explanatory and modifying factors of the association between sex and depression onset during adolescence: An exploratory study.

BACKGROUND: The prevalence of Major Depressive Disorder (MDD) is twice as high in women as in men and this difference already emerges during adolescence. Because the mechanisms underlying this sex-difference remain poorly understood, we took a bottom-up approach to identify factors explaining the sex-MDD relationship. METHODS: Data came from the TRacking Adolescents' Individual Lives Survey (TRAILS), a population study investigating youths' development from age 11 into adulthood. We assessed multiple baseline covariates (e.g., demographic, social and psychological) at ages 11-13 years and MDD onset at ages 19 and 25 years. In regression analyses, each covariate's role in the sex-MDD association as an effect modifier or confounder/explanatory variable was investigated. Replicability was evaluated in an independent sample. RESULTS: The analyses identified no effect-modifiers. Baseline internalizing problems, behavioral inhibition, dizziness, comfort in classroom, physical complaints, attention problems, cooperation, self/effortful control, interpersonal life events and computer use partially explained the association between sex and MDD at age 19. The association between sex and MDD at age 25 was explained by largely the same variables, but also by shyness, acne, antisocial behavior, aggression, affection from peers and time spent shopping. The explanatory roles of internalizing problems, behavioral inhibition, negative events involving gossip/rumors and leisure-time spending (computer-use/shopping) were replicated. LIMITATIONS: Potentially important baseline variables were not included or had low response rates. Gender roles or identification were not considered. Baseline MDD was not adjusted for. CONCLUSION: The sex-MDD association is partially explained by sex differences in symptoms and vulnerability factors already present in early adolescence.

High persistence and low treatment rates of metabolic syndrome in patients with mood and anxiety disorders: A naturalistic follow-up study.

BACKGROUND: Patients with affective and anxiety disorders are at risk of metabolic syndrome (MetS) and, consequently, cardiovascular disease and premature death. In this study, the course and treatment of MetS was investigated using longitudinal data from a naturalistic sample of affective- and anxiety-disordered outpatients (Monitoring Outcome of psychiatric PHARmacotherapy [MOPHAR]). METHODS: Demographics, clinical characteristics, medication use, and MetS components were obtained for n = 2098 patients at baseline and, in a FU-subsample of n = 507 patients, after a median follow-up (FU) of 11 months. Furthermore, pharmacological treatment rates of MetS were investigated at baseline and FU. Finally, demographic and clinical determinants of change in MetS (component) scores were investigated. RESULTS: At baseline, 34.6 % of n = 2098 patients had MetS, 41.4 % of whom received treatment. Of patients with persisting MetS, 46.1 % received treatment for one (or more) MetS component(s) at baseline, and 56.6 % received treatment at FU. Treatment rates of solely elevated blood pressure and reduced HDL-cholesterol did significantly, but modestly, improve. Higher age, male sex, smoking behavior, low education, diabetes, and depressive versus anxiety disorder were predictors of worse outcome at FU on at least one MetS component. LIMITATIONS: We did not have data on lifestyle interventions as a form of treatment, which might partly have explained the observed low pharmacotherapeutic treatment rates. CONCLUSION: MetS (components) show high persistence rates in affective- and anxiety-disordered patients, and are, despite adequate monitoring, undertreated over time. This indicates that adherence and implementation of monitoring protocols should be crucially improved in psychiatric outpatients in secondary care.

Effects of right prefrontal theta-burst transcranial magnetic stimulation or transcranial direct current stimulation on apathy in patients with schizophrenia: A multicenter RCT.

Apathy is a core negative symptom associated with an unfavorable functional outcome. Noninvasive brain stimulation has shown promise in the treatment of schizophrenia but has not been tested specifically for apathy. We conducted a randomized controlled trial of intermittent theta-burst (iTBS) transcranial magnetic stimulation and transcranial direct current stimulation (tDCS) targeted at the right dorsolateral prefrontal cortex (DLPFC) in patients diagnosed with a psychotic disorder suffering from apathy. The study was a multicenter, randomized, placebo-controlled, and rater-blinded trial. Patients (N = 88) were randomized into active iTBS, active tDCS, sham iTBS or sham tDCS treatment, daily for two weeks (excluding weekends). Effects were measured post-treatment and at four week and ten week follow-up. Primary outcome was apathy severity (Apathy Evaluation Scale, clinician-rated). Additional measures included assessment of negative symptoms, depression, anhedonia and quality of life. No significant difference in improvement of apathy or negative symptoms was observed for real versus sham treatment with either iTBS or tDCS, though all groups improved to a small extent. We conclude that two weeks of brain stimulation over the right DLPFC with either iTBS or tDCS is not effective for improving apathy or negative symptoms. Longer and more intensive protocols may yield different results.

Detection and editing of the updated Arabidopsis plastid- and mitochondrial-encoded proteomes through PeptideAtlas.

Arabidopsis (Arabidopsis thaliana) ecotype Col-0 has plastid and mitochondrial genomes encoding over 100 proteins. Public databases (e.g. Araport11) have redundancy and discrepancies in gene identifiers for these organelle-encoded proteins. RNA editing results in changes to specific amino acid residues or creation of start and stop codons for many of these proteins, but the impact of RNA editing at the protein level is largely unexplored due to the complexities of detection. Here, we assembled the nonredundant set of identifiers, their correct protein sequences, and 452 predicted nonsynonymous editing sites of which 56 are edited at lower frequency. We then determined accumulation of edited and/or unedited proteoforms by searching ∼259 million raw tandem MS spectra from ProteomeXchange, which is part of PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/). We identified all mitochondrial proteins and all except 3 plastid-encoded proteins (NdhG/Ndh6, PsbM, and Rps16), but no proteins predicted from the 4 ORFs were identified. We suggest that Rps16 and 3 of the ORFs are pseudogenes. Detection frequencies for each edit site and type of edit (e.g. S to L/F) were determined at the protein level, cross-referenced against the metadata (e.g. tissue), and evaluated for technical detection challenges. We detected 167 predicted edit sites at the proteome level. Minor frequency sites were edited at low frequency at the protein level except for cytochrome C biogenesis 382 at residue 124 (Ccb382-124). Major frequency sites (>50% editing of RNA) only accumulated in edited form (>98% to 100% edited) at the protein level, with the exception of Rpl5-22. We conclude that RNA editing for major editing sites is required for stable protein accumulation.

Detection of the Arabidopsis Proteome and Its Post-translational Modifications and the Nature of the Unobserved (Dark) Proteome in PeptideAtlas.

This study describes a new release of the Arabidopsis thaliana PeptideAtlas proteomics resource (build 2023-10) providing protein sequence coverage, matched mass spectrometry (MS) spectra, selected post-translational modifications (PTMs), and metadata. 70 million MS/MS spectra were matched to the Araport11 annotation, identifying ∼0.6 million unique peptides and 18,267 proteins at the highest confidence level and 3396 lower confidence proteins, together representing 78.6% of the predicted proteome. Additional identified proteins not predicted in Araport11 should be considered for the next Arabidopsis genome annotation. This release identified 5198 phosphorylated proteins, 668 ubiquitinated proteins, 3050 N-terminally acetylated proteins, and 864 lysine-acetylated proteins and mapped their PTM sites. MS support was lacking for 21.4% (5896 proteins) of the predicted Araport11 proteome: the "dark" proteome. This dark proteome is highly enriched for E3 ligases, transcription factors, and for certain (e.g., CLE, IDA, PSY) but not other (e.g., THIONIN, CAP) signaling peptides families. A machine learning model trained on RNA expression data and protein properties predicts the probability that proteins will be detected. The model aids in discovery of proteins with short half-life (e.g., SIG1,3 and ERF-VII TFs) and for developing strategies to identify the missing proteins. PeptideAtlas is linked to TAIR, tracks in JBrowse, and several other community proteomics resources.

Relating stability of individual dynamical networks to change in psychopathology.

One hypothesis flowing from the network theory of psychopathology is that symptom network structure is associated with psychopathology severity and in turn, one may expect that individual network structure changes with the level of psychopathology severity. However, this expectation has rarely been addressed directly. This study aims to examine (1) the stability of individual contemporaneous symptom networks over a one-year period and (2) whether network stability is associated with a change in psychopathology. We used daily diary data of n = 66 individuals, located along the psychosis severity continuum, from two separate 90-day periods, one year apart (t = 180). Based on the newly developed Individual Network Invariance Test (INIT) to assess symptom-network stability, participants were divided into two groups with stable and unstable networks and we tested whether these groups differed in their absolute change in psychopathology severity. The majority of the sample (n = 51, 77.3%) showed a stable network over time while most individuals showed a decrease in psychopathological severity. We found no significant association between a change in psychopathology severity and individual network stability. Our results call for further critical evaluation of the association between networks and psychopathology to optimize the implementation of clinical applications based on current methods.

A blended module (STAIRS) to promote functional and personal recovery in patients with a major depressive disorder in remission: study protocol of a concurrent mixed methods randomized controlled trial.

BACKGROUND: Despite the availability of a wide variety of evidence-based treatments for major depressive disorder (MDD), many patients still experience impairments in their lives after remission. Programs are needed that effectively support patients in coping with these impairments. The program Storytelling and Training to Advance Individual Recovery Skills (STAIRS) was developed to address this need and combines the use of peer contact, expert-by-experience guidance, family support and professional blended care. The aim of the planned study is (1) to assess the efficacy of the STAIRS program in patients with remitted MDD, (2) to investigate patients' subjective experiences with STAIRS, and (3) to evaluate the program's cost-effectiveness. METHODS: A concurrent mixed-methods randomized controlled trial design will be used. Patients aged between 18 and 65 years with remitted MDD (N = 140) will be randomized to either a group receiving care as usual (CAU) + the STAIRS-program or a control group receiving CAU + some basic psychoeducation. Quantitative efficacy data on functional and personal recovery and associated aspects will be collected using self-report questionnaires at the start of the intervention, immediately following the intervention, and at the six-month follow-up. Insights into patients' experiences on perceived effects and the way in which different program elements contribute to this effect, as well as the usability and acceptability of the program, will be gained by conducting qualitative interviews with patients from the experimental group, who are selected using maximum variation sampling. Finally, data on healthcare resource use, productivity loss and quality of life will be collected and analysed to assess the cost-effectiveness and cost-utility of the STAIRS-program. DISCUSSION: Well-designed recovery-oriented programs for patients suffering from MDD are scarce. If efficacy and cost-effectiveness are demonstrated with this study and patients experience the STAIRS program as usable and acceptable, this program can be a valuable addition to CAU. The qualitative interviews may give insights into what works for whom, which can be used to promote implementation. TRIAL REGISTRATION: This trial was registered at ClinicalTrials.gov on 1 July 2021, registration number NCT05440812.

Statistical Mechanics Metrics in Pairing and Parsing In Silico and Phenotypic Data of a Novel Genetic NFκB1 (c.T638A) Variant.

(1) Background: Mutations in NFκB1, a transcriptional regulator of immunomodulating proteins, are a known cause of inborn errors of immunity. Our proband is a 22-year-old male with a diagnosis of common variable immunodeficiency (CVID), cytopenias with massive splenomegaly, and nodular regenerative hyperplasia of the liver. Genetic studies identified a novel, single-point mutation variant in NFκB1, c. T638A p. V213E. (2) Methods: Next-generation panel sequencing of the patient uncovered a novel single-point mutation in the NFκB1 gene that was modeled using the I-TASSER homology-modeling software, and molecular dynamics were assessed using the YASARA2 software (version 20.14.24). (3) Results: This variant replaces valine with glutamic acid at position 213 in the NFκB1 sequence. Molecular modeling and molecular dynamic studies showed altered dynamics in and around the rel homology domain, ankyrin regions, and death domain of the protein. We postulate that these changes alter overall protein function. (4) Conclusions: This case suggests the pathogenicity of a novel variant using protein-modeling techniques and molecular dynamic simulations.

Mapping the Arabidopsis thaliana proteome in PeptideAtlas and the nature of the unobserved (dark) proteome; strategies towards a complete proteome.

This study describes a new release of the Arabidopsis thaliana PeptideAtlas proteomics resource providing protein sequence coverage, matched mass spectrometry (MS) spectra, selected PTMs, and metadata. 70 million MS/MS spectra were matched to the Araport11 annotation, identifying ∼0.6 million unique peptides and 18267 proteins at the highest confidence level and 3396 lower confidence proteins, together representing 78.6% of the predicted proteome. Additional identified proteins not predicted in Araport11 should be considered for building the next Arabidopsis genome annotation. This release identified 5198 phosphorylated proteins, 668 ubiquitinated proteins, 3050 N-terminally acetylated proteins and 864 lysine-acetylated proteins and mapped their PTM sites. MS support was lacking for 21.4% (5896 proteins) of the predicted Araport11 proteome - the 'dark' proteome. This dark proteome is highly enriched for certain ( e.g. CLE, CEP, IDA, PSY) but not other ( e.g. THIONIN, CAP,) signaling peptides families, E3 ligases, TFs, and other proteins with unfavorable physicochemical properties. A machine learning model trained on RNA expression data and protein properties predicts the probability for proteins to be detected. The model aids in discovery of proteins with short-half life ( e.g. SIG1,3 and ERF-VII TFs) and completing the proteome. PeptideAtlas is linked to TAIR, JBrowse, PPDB, SUBA, UniProtKB and Plant PTM Viewer.

Implementation of genomic medicine for rare disease in a tertiary healthcare system: Mayo Clinic Program for Rare and Undiagnosed Diseases (PRaUD).

BACKGROUND: In the United States, rare disease (RD) is defined as a condition that affects fewer than 200,000 individuals. Collectively, RD affects an estimated 30 million Americans. A significant portion of RD has an underlying genetic cause; however, this may go undiagnosed. To better serve these patients, the Mayo Clinic Program for Rare and Undiagnosed Diseases (PRaUD) was created under the auspices of the Center for Individualized Medicine (CIM) aiming to integrate genomics into subspecialty practice including targeted genetic testing, research, and education. METHODS: Patients were identified by subspecialty healthcare providers from 11 clinical divisions/departments. Targeted multi-gene panels or custom exome/genome-based panels were utilized. To support the goals of PRaUD, a new clinical service model, the Genetic Testing and Counseling (GTAC) unit, was established to improve access and increase efficiency for genetic test facilitation. The GTAC unit includes genetic counselors, genetic counseling assistants, genetic nurses, and a medical geneticist. Patients receive abbreviated point-of-care genetic counseling and testing through a partnership with subspecialty providers. RESULTS: Implementation of PRaUD began in 2018 and GTAC unit launched in 2020 to support program expansion. Currently, 29 RD clinical indications are included in 11 specialty divisions/departments with over 142 referring providers. To date, 1152 patients have been evaluated with an overall solved or likely solved rate of 17.5% and as high as 66.7% depending on the phenotype. Noteworthy, 42.7% of the solved or likely solved patients underwent changes in medical management and outcome based on genetic test results. CONCLUSION: Implementation of PRaUD and GTAC have enabled subspecialty practices advance expertise in RD where genetic counselors have not historically been embedded in practice. Democratizing access to genetic testing and counseling can broaden the reach of patients with RD and increase the diagnostic yield of such indications leading to better medical management as well as expanding research opportunities.

Serous endometrial intra-epithelial carcinoma: an observational study.

BACKGROUND: Serous endometrial intra-epithelial carcinoma is described as a malignant, superficial spreading lesion with risk of extra-uterine spread at time of diagnosis, and poor outcome. OBJECTIVE: To evaluate the surgical management of patients with serous endometrial intra-epithelial carcinoma and its impact on oncologic outcomes and complications. METHODS: This Dutch observational retrospective cohort study evaluated all patients diagnosed with pure serous endometrial intra-epithelial carcinoma in the Netherlands, between January 2012 and July 2020. The pathological examination was reviewed by two pathologists with expertise in gynecological oncology. Clinical data were obtained when the diagnosis was confirmed. Primary outcome is progression-free survival, secondary outcomes are duration of follow-up, adverse events related to surgery, and overall survival. RESULTS: A total of 23 patients from 13 medical centers were included, of whom 15 (65.2%) presented with post-menopausal blood loss. In 17 patients (73.9%) the intra-epithelial lesion was present in an endometrial polyp. All patients underwent hysterectomy, of whom 12 patients (52.2%) were surgically staged. None of the staged patients showed extra-uterine disease. Two patients received adjuvant brachytherapy. There were no recurrences of disease (median follow-up duration of 35.6 months (range 1.0-108.6) and no disease-related deaths in this cohort. CONCLUSION: In patients with serous endometrial intra-epithelial carcinoma, median progression-free survival reached nearly 3 years and no recurrences have been reported. Our results do not endorse World Health Organization 2014 advice to treat serous endometrial intra-epithelial carcinoma as high-grade, high-risk endometrial carcinoma. Full surgical staging might possibly lead to overtreatment.