A strict requirement in proteasome substrates for spacing between ubiquitin tag and degradation initiation elements.

Proteins are typically targeted to the proteasome for degradation through the attachment of ubiquitin chains and the proteasome initiates degradation at a disordered region within the target protein. Yet some proteins with ubiquitin chains and disordered regions escape degradation. Here we investigate how the position of the ubiquitin chain on the target protein relative to the disordered region modulates degradation and show that the distance between the two determines whether a protein is degraded efficiently. This distance depends on the type of the degradation tag and is likely a result of the separation on the proteasome between the receptor that binds the tag and the site that engages the disordered region.

Matrix Effects in GC-MS Profiling of Common Metabolites after Trimethylsilyl Derivatization.

Metabolite profiling using gas chromatography coupled to mass spectrometry (GC-MS) is one of the most frequently applied and standardized methods in research projects using metabolomics to analyze complex samples. However, more than 20 years after the introduction of non-targeted approaches using GC-MS, there are still unsolved challenges to accurate quantification in such investigations. One particularly difficult aspect in this respect is the occurrence of sample-dependent matrix effects. In this project, we used model compound mixtures of different compositions to simplify the study of the complex interactions between common constituents of biological samples in more detail and subjected those to a frequently applied derivatization protocol for GC-MS analysis, namely trimethylsilylation. We found matrix effects as signal suppression and enhancement of carbohydrates and organic acids not to exceed a factor of ~2, while amino acids can be more affected. Our results suggest that the main reason for our observations may be an incomplete transfer of carbohydrate and organic acid derivatives during the injection process and compound interaction at the start of the separation process. The observed effects were reduced at higher target compound concentrations and by using a more suitable injection-liner geometry.

Postdeployment Respiratory Health: The Roles of the Airborne Hazards and Open Burn Pit Registry and the Post-Deployment Cardiopulmonary Evaluation Network.

Background: Following deployment to the Southwest Asia theater of operations and Afghanistan, many service members and veterans report respiratory symptoms and concerns about their military and environmental exposures. The US Department of Veterans Affairs (VA) established the national Airborne Hazards and Open Burn Pit Registry (AHOBPR) in 2014 to help better understand long-term health conditions that may be related to these exposures. Observations: The AHOBPR provides an online questionnaire and optional health evaluation performed by a primary care or environmental health clinician. The clinical evaluation provides an opportunity for the service member or veteran to talk with a health care professional about their symptoms, exposures, and potential treatment. Data derived from questionnaire responses and health evaluations facilitate medical surveillance and research. The VA also established a network of specialists, referred to as the Post-Deployment Cardiopulmonary Evaluation Network (PDCEN). The PDCEN identifies veterans within the AHOBPR who self-report certain conditions or have unexplained dyspnea and conducts comprehensive diagnostic evaluations. Primary objectives of PDCEN evaluations are to define respiratory and related conditions that are present, determine whether conditions are related to deployment, and work with the veteran's clinician to identify treatments and/or follow-up care to improve their health. We utilize a case example to illustrate the role of the primary care practitioner in connecting veterans to PDCEN clinical evaluations. Conclusions: AHOBPR clinical evaluations represent an initial step to better understand postdeployment health conditions. The PDCEN clinical evaluation extends the AHOBPR evaluation by providing specialty care for certain veterans requiring more comprehensive evaluation while systematically collecting and analyzing clinical data to advance the field.

Associations Between Food Addiction and Substance-Use Disorders: A Critical Overview of their Overlapping Patterns of Consumption.

Purpose of Review: Research on patterns of overconsumption in individuals with food addiction (FA) has focused largely on binge eating. However, compulsive overeating can be varied and dimensional. This review focuses on the similarities between the patterns of consumption in FA and in other clinically established substance-use disorders, such as alcohol and nicotine dependence. It also highlights features that make FA unique to other addiction disorders. Recent Findings: Overall, there is substantial evidence that binge-like overconsumption is a characteristic of various substance-use and eating disorders. Likewise, it appears that different overeating patterns can reflect addictive-like eating. One pattern may be compulsive grazing - defined as the repetitive inability to resist consumption of small amounts of food. Summary: This review adds to the increasingly compelling picture that FA and binge-eating disorder are unique conditions, and that FA resembles other substance-use disorders. We conclude that a variety of overeating patterns can reflect addictive eating behaviours in vulnerable individuals, one of which may be compulsive grazing.

Timing of High-glucose Diet in the C. elegans Lifecycle Impacts Fertility Phenotypes.

Human metabolic diseases and high-sugar diets have been associated with infertility. Previous studies show that high-glucose diet also affects fertility in C. elegans, leading to decreased offspring production and delayed reproductive timing. We tested whether the timing of glucose exposure affects these fertility defects or the embryo to larval transition. We found that decreased offspring production was strictly a response to high-glucose exposure in adulthood, whereas the delayed reproductive profile was influenced by both developmental and adult diets. We found no effect of high-glucose diet on the number of embryos that develop to the first larval stage. Together, these results suggest that the decreased offspring production and delayed reproductive profile may be separable phenotypes, and that a high-glucose diet reduces the number of offspring by interfering with processes regulated during adulthood.

BRAFV600E-Driven Lung Adenocarcinoma Requires Copper to Sustain Autophagic Signaling and Processing.

The transition metal copper (Cu) is an essential micronutrient required for development and proliferation, but the molecular mechanisms by which Cu contributes to these processes is not fully understood. Although traditionally studied as a static cofactor critical for the function of Cu-dependent enzymes, an expanding role for Cu is emerging to include its novel function as a dynamic mediator of signaling processes through the direct control of protein kinase activity. We now appreciate that Cu directly binds to and influences MEK1/2 and ULK1/2 kinase activity, and show here that reductions in MAPK and autophagic signaling are associated with dampened growth and survival of oncogenic BRAF-driven lung adenocarcinoma cells upon loss of Ctr1. Efficient autophagy, clonogenic survival, and tumorigenesis of BRAF-mutant cells required ULK1 Cu-binding. Although treatment with canonical MAPK inhibitors resulted in the upregulation of protective autophagy, mechanistically, the Cu chelator tetrathiomolybdate (TTM) was sufficient to target both autophagic and MAPK signaling as a means to blunt BRAF-driven tumorigenic properties. These findings support leveraging Cu chelation with TTM as an alternative therapeutic strategy to impair autophagy and MAPK signaling. As traditional MAPK monotherapies initiate autophagy signaling and promote cancer cell survival. IMPLICATIONS: We establish that copper chelation therapy inhibits both autophagy and MAPK signaling in BRAFV600E-driven lung adenocarcinoma, thus overcoming the upregulation of protective autophagy elicited by canonical MAPK pathway inhibitors.

gmxapi: A GROMACS-native Python interface for molecular dynamics with ensemble and plugin support.

Gmxapi provides an integrated, native Python API for both standard and advanced molecular dynamics simulations in GROMACS. The Python interface permits multiple levels of integration with the core GROMACS libraries, and legacy support is provided via an interface that mimics the command-line syntax, so that all GROMACS commands are fully available. Gmxapi has been officially supported since the GROMACS 2019 release and is enabled by default in current versions of the software. Here we describe gmxapi 0.3 and later. Beyond simply wrapping GROMACS library operations, the API permits several advanced operations that are not feasible using the prior command-line interface. First, the API allows custom user plugin code within the molecular dynamics force calculations, so users can execute custom algorithms without modifying the GROMACS source. Second, the Python interface allows tasks to be dynamically defined, so high-level algorithms for molecular dynamics simulation and analysis can be coordinated with loop and conditional operations. Gmxapi makes GROMACS more accessible to custom Python scripting while also providing support for high-level data-flow simulation algorithms that were previously feasible only in external packages.

ER-GCaMP6f: An Endoplasmic Reticulum-Targeted Genetic Probe to Measure Calcium Activity in Astrocytic Processes.

Ca2+ is a major second messenger involved in cellular and subcellular signaling in a wide range of cells, including astrocytes, which use calcium ions to communicate with other cells in the brain. Even though a variety of genetically encoded Ca2+ indicators have been developed to study astrocyte calcium signaling, understanding the dynamics of endoplasmic reticulum calcium signaling is greatly limited by the currently available tools. To address this, we developed an endoplasmic reticulum-targeted calcium indicator, ER-GCaMP6f, which is anchored to the cytosolic side of the organelle and measures signaling that occurs in close proximity to the endoplasmic reticulum of astrocytes. Using a combination of confocal and super-resolution microscopy techniques, we demonstrate the localization of the indicator in the endoplasmic reticulum in both cell soma and processes of astrocytes. Combining ER-GCaMP6f with total internal reflection fluorescence microscopy, we show that Ca2+ fluctuations in small astrocytic processes can be detected, which are otherwise not observable with existing indicators and standard wide-field and confocal techniques. We also compared the ER-GCaMP6f indicator against currently used plasma membrane-tethered and cytosolic GCaMP6f indicators. ER-GCaMP6f identifies dynamics in calcium signaling of endoplasmic reticulum resident receptors that are missed by plasma membrane-anchored indicators. We also generated an adeno-associated virus (AAV5) and demonstrate that ER-GCaMP6f can be expressed in vivo and by measured calcium activity in brain slices. ER-GCaMP6f provides a powerful tool to study calcium signaling in close proximity to the endoplasmic reticulum in astrocyte cell soma and processes both in culture and in brain slices.

Exploring women's experiences of treatment for binge eating disorder: Methylphenidate vs. cognitive behavioural therapy.

OBJECTIVE: The current qualitative study explored the personal experiences of a sample of women with binge eating disorder (BED). The women were previously enrolled in a 12-week randomized controlled trial comparing pharmacotherapy (methylphenidate [MP]) and cognitive behavioural therapy (CBT). METHODS: Semi-structured interviews were conducted with 15 women who completed the trial (8 MP, 7 CBT) to obtain their narrative accounts. Key themes were then identified from transcribed tape recordings, using thematic analysis. RESULTS: Participants described self-awareness as bringing greater attention to their binge eating, and to their thoughts and emotions. Furthermore, both groups valued the interpersonal relationships with the clinicians and their ability to create a safe and comforting environment. In the MP group, many participants described the medication as reducing their preoccupation with food, and hence, binge frequency. In CBT, there was a focus on psychoeducation and obtaining a "toolbox" of long-term binge-management skills that could also be used following treatment. In both groups, stress was described as a primary trigger for a binge and/or a cause of relapse. DISCUSSION: Although patients reported having a positive experience in the therapies, it is suggested that broader stress regulation skills training would be useful to evaluate further, to bolster relapse prevention skills. These qualitative findings add a much-needed lived-experience perspective on clinical treatments for binge eating. This is especially significant considering that a psychostimulant similar to MP is the only approved pharmacotherapy for BED, and to date, little is known about the patient's subjective experiences when taking this medication.

The Role of School Connectedness in Supporting the Health and Well-Being of Youth: Recommendations for School Nurses.

The importance of students feeling connected in school cannot be overstated, as this perception is crucial to support their health and well-being. A lack of school connectedness can lead to adverse physical and mental health outcomes, including bully victimization. Numerous factors, including individual, social, and environmental, influence students' perceived sense of school connectedness. School nurses are well positioned to establish and maintain school connectedness due to their knowledge, accessibility to students, and familiarity with the school environment. This article details the importance of school connectedness and describes the associations between school connectedness, bullying, and mental health. In addition, we offer recommendations geared toward school nurses regarding strengthening school connectedness and promoting a culture of care and inclusivity within school environments, especially salient in the context of the COVID-19 pandemic.

The Efficacy of eHealth Interventions for the Treatment of Adults Diagnosed With Full or Subthreshold Binge Eating Disorder: Systematic Review and Meta-analysis.

BACKGROUND: There has been a recent rise in the use of eHealth treatments for a variety of psychological disorders, including eating disorders. OBJECTIVE: This meta-analysis of randomized controlled trials is the first to evaluate the efficacy of eHealth interventions specifically for the treatment of binge eating disorder (characterized by compulsive overconsumption of food, in a relatively short period, and without compensatory behaviors such as purging or fasting). METHODS: A search on the electronic databases PubMed, Web of Science, Embase, MEDLINE, and CINAHL was conducted for randomized controlled trials that compared the efficacy of eHealth treatment interventions with waitlist controls. RESULTS: From the databases searched, 3 studies (298 participants in total) met the inclusion criteria. All interventions were forms of internet-based guided cognitive behavioral therapy. The results of the analysis demonstrated that when compared with waitlist controls, individuals enrolled in eHealth interventions experienced a reduction in objective binge episodes (standardized mean difference [SMD] -0.77, 95% CI -1.38 to -0.16) and eating disorder psychopathology (SMD -0.71, 95% CI -1.20 to -0.22), which included shape (SMD -0.61, 95% CI -1.01 to -0.22) and weight concerns (SMD -0.91, 95% CI -1.33 to -0.48). There was no significant difference in BMI between the eHealth interventions and controls (SMD -0.01, 95% CI -0.40 to 0.39). CONCLUSIONS: These findings provide promising results for the use of internet-based cognitive behavioral therapy for binge eating disorder treatment and support the need for future research to explore the efficacy of these eHealth interventions.

Copper biology.

Metals are vital for life as they are necessary for essential biological processes. Traditionally, metals are categorized as either dynamic signals or static cofactors. Redox-inactive metals such as calcium (Ca), potassium (K), sodium (Na), and zinc (Zn) signal through large fluctuations in their metal-ion pools. In contrast, redox-active transition metals such as copper (Cu) and iron (Fe) drive catalysis and are largely characterized as static cofactors that must be buried and protected within the active sites of proteins, due to their ability to generate damaging reactive-oxygen species through Fenton chemistry. Cu has largely been studied as a static cofactor in fundamental processes from cellular respiration to pigmentation, working through cytochrome c oxidase and tyrosinase, respectively. However, within the last decade, a new paradigm in nutrient sensing and protein regulation - termed 'metalloallostery' - has emerged, expanding the repertoire of Cu beyond the catalytic proteins to dynamic signaling molecules essential for cellular processes that impact normal physiology and disease states. In this Primer we introduce both the 'traditional' and emerging roles for Cu in biology and the many ways in which Cu intersects with human health.

Mechanisms of substrate recognition by the 26S proteasome.

The majority of regulated protein degradation in eukaryotes is accomplished by the 26S proteasome, the large proteolytic complex responsible for removing regulatory proteins and damaged proteins. Proteins are targeted to the proteasome by ubiquitination, and degradation is initiated at a disordered region within the protein. The ability of the proteasome to precisely select which proteins to break down is necessary for cellular functioning. Recent studies reveal the subtle mechanisms of substrate recognition by the proteasome - diverse ubiquitin chains can act as potent proteasome targeting signals, ubiquitin receptors function uniquely and cooperatively, and modification of initiation regions modulate degradation. Here, we summarize recent findings illuminating the nature of substrate recognition by the proteasome.

The feasibility of a personality targeted intervention for addictive overeating: FoodFix.

Recent reviews have identified potential treatment targets for addictive overeating. These include: motivational interviewing, development of specific coping strategies for emotional regulation and the use of harm minimisation strategies based on interventions for substance use disorders. However, there is very little experiential evidence. The aim of this study was to determine the feasibility of a personality-targeted motivational interviewing intervention in adults above the healthy-weight range with symptoms of addictive eating, to reduce symptoms of addictive overeating and improve dietary profiles. Individuals with overweight and obesity (BMI >25 kg/m2) with addictive eating as defined by the modified Yale Food Addiction Scale (mYFAS) were recruited to a three-session intervention held over 3 months. Sessions were conducted by telehealth and facilitated by dietitians. Fifty-two individuals were randomised to either intervention or control (mean age 43.6 ± 12.2yrs, mean BMI 36.7 ± 6.8 kg/m2, 96% female). At three month follow up, there were significant reductions from baseline (BL) for both groups in total YFAS 2.0 symptoms, however, these changes were not significantly different between groups (intervention BL 8.0 ± 2.7; 3-months 6.5 ± 3.8, control BL 8.1 ± 2.5; 3-months 6.9 ± 3.9, p > 0.05). At 3 months the intervention group significantly reduced their energy from non-core foods compared with control (intervention BL 48% energy/day; 3-months 38%, control BL 41% energy/day; 3-months 38%, p < 0.01). The FoodFix intervention provides insight to the development of future management interventions for addictive eating.

Altered copper homeostasis underlies sensitivity of hepatocellular carcinoma to copper chelation.

Hepatocellular carcinoma (HCC), the most common primary liver cancer, of which ∼800 000 new cases will be diagnosed worldwide this year, portends a five-year survival rate of merely 17% in patients with unresectable disease. This dismal prognosis is due, at least in part, from the late stage of diagnosis and the limited efficacy of systemic therapies. As a result, there is an urgent need to identify risk factors that contribute to HCC initiation and provide targetable vulnerabilities to improve patient survival. While myriad risk factors are known, elevated copper (Cu) levels in HCC patients and the incidence of hepatobiliary malignancies in Wilson disease patients, which exhibit hereditary liver Cu overload, suggests the possibility that metal accumulation promotes malignant transformation. Here we found that expression of the Cu transporter genes ATP7A, ATP7B, SLC31A1, and SLC31A2 was significantly altered in liver cancer samples and were associated with elevated Cu levels in liver cancer tissue and cells. Further analysis of genomic copy number data revealed that alterations in Cu transporter gene loci correlate with poorer survival in HCC patients. Genetic loss of the Cu importer SLC31A1 (CTR1) or pharmacologic suppression of Cu decreased the viability, clonogenic survival, and anchorage-independent growth of human HCC cell lines. Mechanistically, CTR1 knockdown or Cu chelation decreased glycolytic gene expression and downstream metabolite utilization and as a result forestalled tumor cell survival after exposure to hypoxia, which mimics oxygen deprivation elicited by transarterial embolization, a standard-of-care therapy used for patients with unresectable HCC. Taken together, these findings established an association between altered Cu homeostasis and HCC and suggest that limiting Cu bioavailability may provide a new treatment strategy for HCC by restricting the metabolic reprogramming necessary for cancer cell survival.

Use of Multiple Ion Fragmentation Methods to Identify Protein Cross-Links and Facilitate Comparison of Data Interpretation Algorithms.

Multiple ion fragmentation methods involving collision-induced dissociation (CID), higher-energy collisional dissociation (HCD) with regular and very high energy settings, and electron-transfer dissociation with supplementary HCD (EThcD) are implemented to improve the confidence of cross-link identifications. Three different S. cerevisiae proteasome samples cross-linked by diethyl suberthioimidate (DEST) or bis(sulfosuccinimidyl)suberate (BS3) are analyzed. Two approaches are introduced to combine interpretations from the above four methods. Working with cleavable cross-linkers such as DEST, the first approach searches for cross-link diagnostic ions and consistency among the best interpretations derived from all four MS2 spectra associated with each precursor ion. Better agreement leads to a more definitive identification. Compatible with both cleavable and noncleavable cross-linkers such as BS3, the second approach multiplies scoring metrics from a number of fragmentation experiments to derive an overall best match. This significantly increases the scoring gap between the target and decoy matches. The validity of cross-links fragmented by HCD alone and identified by Kojak, MeroX, pLink, and Xi was evaluated using multiple fragmentation data. Possible ways to improve the identification credibility are discussed. Data are available via ProteomeXchange with identifier PXD018310.

The proteasome 19S cap and its ubiquitin receptors provide a versatile recognition platform for substrates.

Proteins are targeted to the proteasome by the attachment of ubiquitin chains, which are markedly varied in structure. Three proteasome subunits-Rpn10, Rpn13, and Rpn1-can recognize ubiquitin chains. Here we report that proteins with single chains of K48-linked ubiquitin are targeted for degradation almost exclusively through binding to Rpn10. Rpn1 can act as a co-receptor with Rpn10 for K63 chains and for certain other chain types. Differences in targeting do not correlate with chain affinity to receptors. Surprisingly, in steady-state assays Rpn13 retarded degradation of various single-chain substrates. Substrates with multiple short ubiquitin chains can be presented for degradation by any of the known receptors, whereas those targeted to the proteasome through a ubiquitin-like domain are degraded most efficiently when bound by Rpn13 or Rpn1. Thus, the proteasome provides an unexpectedly versatile binding platform that can recognize substrates targeted for degradation by ubiquitin chains differing greatly in length and topology.