Erratum: iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells.

[This corrects the article DOI: 10.1016/j.isci.2023.107059.].

Effects of testosterone enanthate on aggression, risk-taking, competition, mood, and other cognitive domains during 28 days of severe energy deprivation.

RATIONALE: Behavioral effects of testosterone depend on dose, acute versus sustained formulation, duration of administration, personality, genetics, and endogenous levels of testosterone. There are also considerable differences between effects of endogenous and exogenous testosterone. OBJECTIVES: This study was the secondary behavioral arm of a registered clinical trial designed to determine if testosterone protects against loss of lean body mass and lower-body muscle function induced by a severe energy deficit typical of sustained military operations. METHODS: Behavioral effects of repeated doses of testosterone on healthy young men whose testosterone was reduced by severe energy deficit were examined. This was a double-blind, placebo-controlled, between-group study. Effects of four weekly intramuscular injections of testosterone enanthate (200 mg/week, N = 24) or matching placebo (N = 26) were evaluated. Determination of sample size was based on changes in lean body mass. Tasks assessing aggression, risk-taking, competition, social cognition, vigilance, memory, executive function, and mood were repeatedly administered. RESULTS: During a period of artificially induced, low testosterone levels, consistent behavioral effects of administration of exogenous testosterone were not observed. CONCLUSIONS: Exogeneous testosterone enanthate (200 mg/week) during severe energy restriction did not reliably alter the measures of cognition. Study limitations include the relatively small sample size compared to many studies of acute testosterone administration. The findings are specific to healthy males experiencing severe energy deficit and should not be generalized to effects of other doses, formulations, or acute administration of endogenous testosterone or studies conducted with larger samples using tests of cognitive function designed to detect specific effects of testosterone.

Determination of Ligand-Binding Affinity (Kd) Using Transverse Relaxation Rate (R2) in the Ligand-Observed 1H NMR Experiment and Applications to Fragment-Based Drug Discovery.

High hit rates from initial ligand-observed NMR screening can make it challenging to prioritize which hits to follow up, especially in cases where there are no available crystal structures of these hits bound to the target proteins or other strategies to provide affinity ranking. Here, we report a reproducible, accurate, and versatile quantitative ligand-observed NMR assay, which can determine Kd values of fragments in the affinity range of low µM to low mM using transverse relaxation rate R2 as the observable parameter. In this study, we examined the theory and proposed a mathematical formulation to obtain Kd values using non-linear regression analysis. We designed an assay format with automated sample preparation and simplified data analysis. Using tool compounds, we explored the assay reproducibility, accuracy, and detection limits. Finally, we used this assay to triage fragment hits, yielded from fragment screening against the CRBN/DDB1 complex.

A Degron Blocking Strategy Towards Improved CRL4CRBN Recruiting PROTAC Selectivity.

Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC-induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation.

iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells.

To address the limitation associated with degron based systems, we have developed iTAG, a synthetic tag based on IMiDs/CELMoDs mechanism of action that improves and addresses the limitations of both PROTAC and previous IMiDs/CeLMoDs based tags. Using structural and sequence analysis, we systematically explored native and chimeric degron containing domains (DCDs) and evaluated their ability to induce degradation. We identified the optimal chimeric iTAG(DCD23 60aa) that elicits robust degradation of targets across cell types and subcellular localizations without exhibiting the well documented "hook effect" of PROTAC-based systems. We showed that iTAG can also induce target degradation by murine CRBN and enabled the exploration of natural neo-substrates that can be degraded by murine CRBN. Hence, the iTAG system constitutes a versatile tool to degrade targets across the human and murine proteome.

Effect of exogenous testosterone in the context of energy deficit on risky choice: Behavioural and neural evidence from males.

Previous research has shown greater risk aversion when people make choices about lives than cash. We tested the hypothesis that compared to placebo, exogenous testosterone administration would lead to riskier choices about cash than lives, given testosterone's association with financial risk-taking and reward sensitivity. A double-blind, placebo-controlled, randomized trial was conducted to test this hypothesis (Clinical Trials Registry: NCT02734238, www.clinicaltrials.gov). We collected functional magnetic resonance imaging (fMRI) data from 50 non-obese males before and shortly after 28 days of severe exercise-and-diet-induced energy deficit, during which testosterone (200 mg testosterone enanthate per week in sesame oil) or placebo (sesame seed oil only) was administered. Because we expected circulating testosterone levels to be reduced due to severe energy deficit, testosterone administration served a restorative function to mitigate the impact of energy deficit on testosterone levels. The fMRI task involved making choices under uncertainty for lives and cash. We also manipulated whether the outcomes were presented as gains or losses. Consistent with prospect theory, we observed the reflection effect such that participants were more risk averse when outcomes were presented as gains than losses. Brain activation in the thalamus covaried with individual differences in exhibiting the reflection effect. Testosterone did not impact choice, but it increased sensitivity to negative feedback following risky choices. These results suggest that exogenous testosterone administration in the context of energy deficit can impact some aspects of risky choice, and that individual differences in the reflection effect engage a brain structure involved in processing emotion, reward and risk.

Short sleep duration is associated with a wide variety of medical conditions among United States military service members.

OBJECTIVES: This cross-sectional study investigated self-reported sleep duration and its association with a comprehensive range of clinically-diagnosed medical condition categories (CDMCs), as well as the relationship between short sleep duration (≤6 h) and demographic/lifestyle factors, among United States military service members (SMs). METHODS: A stratified random sample of SMs (n = 20,819) completed an online questionnaire on usual daily hours of sleep and demographic/lifestyle characteristics. CDMCs for a six-month period prior to questionnaire completion were obtained from a comprehensive military electronic medical surveillance system and grouped into 33 CDMCs covering both broad and specific medical conditions. Prevalence of CDMCs was compared among three sleep duration categories (≤4, 5-6 and ≥7 h). RESULTS: SMs reported a mean ± standard deviation of 6.3 ± 1.4 h of sleep per day. After adjustment for demographic/lifestyle characteristics, shorter sleep duration was associated with higher odds of a medical condition in 25 of 33 CDMCs, with most (n = 20) demonstrating a dose-response relationship. The five CDMCs with the largest differences between ≤4 vs ≥ 7 h sleep were: diseases of the nervous system (odds ratio [OR] = 2.9, 95% confidence interval [95%CI] = 2.4-3.4), mental/behavioral diseases (OR = 2.7, 95%CI = 2.3-3.2), diseases of the musculoskeletal system (OR = 1.9, 95%CI = 1.6-2.1), diseases of the circulatory system (OR = 1.7, 95%CI = 1.3-2.2), and diseases of the digestive system (OR = 1.6, 95%CI = 1.2-2.0). Six hours of sleep or less was independently associated with older age, less formal education, race, Hispanic ethnicity, higher body mass index, smoking, and military service branch. CONCLUSIONS: In this young, physically active population, reporting shorter sleep duration was associated with a higher risk of multiple CDMCs.

Sleep and Injuries in Military Personnel With Suggestions for Improving Sleep and Mitigating Effects of Sleep Loss.

Sleep professionals suggest adults should sleep at least seven hours per night and define good sleep quality as 1) sleep onset =15 minutes, 2) one or fewer awakenings per night, 3) awake after sleep onset =20 minutes, and 4) sleep efficiency (ratio of sleep time to time in bed) =85%. This paper focuses on associations between injuries and sleep quality/duration among military personnel and strategies to optimize sleep and mitigate effects of sleep loss. Investigations among military personnel generally used convenience samples who self-reported their injury and sleep quality/quantity. Despite these limitations, data suggest that lower sleep quality or duration is associated with higher risk of musculoskeletal injury (MSI). Possible mechanisms whereby poor sleep quality/duration may influence MSI include hormonal changes increasing muscle catabolism, increases in inflammatory processes affecting post-exercise muscle damage, and effects on new bone formation. Sleep can be optimized by a slightly cool sleeping environment, bedding that maintains a stable thermal microclimate around the body, not using media devices near bedtime or in the sleeping environment, minimizing noise, and having regular bed and awaking times. Sleep loss mitigation strategies include napping (<30 to 90 minutes), sleep banking (extended time in bed), and judicious use of caffeine or modafinil.

SimPLIT: Simplified Sample Preparation for Large-Scale Isobaric Tagging Proteomics.

Large scale proteomic profiling of cell lines can reveal molecular signatures attributed to variable genotypes or induced perturbations, enabling proteogenomic associations and elucidation of pharmacological mechanisms of action. Although isobaric labeling has increased the throughput of proteomic analysis, the commonly used sample preparation workflows often require time-consuming steps and costly consumables, limiting their suitability for large scale studies. Here, we present a simplified and cost-effective one-pot reaction workflow in a 96-well plate format (SimPLIT) that minimizes processing steps and demonstrates improved reproducibility compared to alternative approaches. The workflow is based on a sodium deoxycholate lysis buffer and a single detergent cleanup step after peptide labeling, followed by quick off-line fractionation and MS2 analysis. We showcase the applicability of the workflow in a panel of colorectal cancer cell lines and by performing target discovery for a set of molecular glue degraders in different cell lines, in a 96-sample assay. Using this workflow, we report frequently dysregulated proteins in colorectal cancer cells and uncover cell-dependent protein degradation profiles of seven cereblon E3 ligase modulators (CRL4CRBN). Overall, SimPLIT is a robust method that can be easily implemented in any proteomics laboratory for medium-to-large scale TMT-based studies for deep profiling of cell lines.

Membrane excitability: Ankyrins keep neuromuscular junctions firing.

Voltage-gated sodium channels are clustered and immobilized at high densities in electrically excitable cells. A new study shows that ankyrins are essential to tether sodium channels and prevent synaptic fatigue at the neuromuscular junction.

Effects of testosterone administration on fMRI responses to executive function, aggressive behavior, and emotion processing tasks during severe exercise- and diet-induced energy deficit.

BACKGROUND: Clinical administration of testosterone is widely used due to a variety of claimed physical and cognitive benefits. Testosterone administration is associated with enhanced brain and cognitive function, as well as mood, in energy-balanced males, although such relationships are controversial. However, the effects of testosterone administration on the brains of energy-deficient males, whose testosterone concentrations are likely to be well below normal, have not been investigated. METHODS: This study collected functional magnetic resonance imaging (fMRI) data from 50 non-obese young men before (PRE) and shortly after (POST) 28 days of severe exercise-and-diet-induced energy deficit during which testosterone (200 mg testosterone enanthate per week in sesame oil, TEST) or placebo (sesame seed oil only, PLA) were administered. Scans were also collected after a post-energy-deficit weight regain period (REC). Participants completed five fMRI tasks that assessed aspects of: 1) executive function (Attention Network Task or ANT; Multi-Source Interference Task or MSIT; AXE Continuous Processing Task or AXCPT); 2) aggressive behavior (Provoked Aggression Task or AGG); and 3) latent emotion processing (Emotional Face Processing or EMO). RESULTS: Changes over time in task-related fMRI activation in a priori defined task-critical brain regions during performance of 2 out of 5 tasks were significantly different between TEST and PLA, with TEST showing greater levels of activation during ANT in the right anterior cingulate gyrus at POST and during MSIT in several brain regions at REC. Changes over time in objective task performance were not statistically significant; testosterone-treated volunteers had greater self-reported anger during AGG at POST. CONCLUSIONS: Testosterone administration can alter some aspects of brain function during severe energy deficit and increase levels of anger.

Dietary Supplement Use in US Army Personnel: A Mixed-Methods, Survey and Focus-Group Study Examining Decision Making and Factors Associated With Use.

BACKGROUND: Dietary supplement (DS) use by Army personnel is high and is a safety and readiness issue. OBJECTIVE: Our aim was to examine factors motivating use of DSs among US Army personnel and preferred safety education strategies. DESIGN: This mixed-method study used a validated DS questionnaire and subsequent focus groups that were formed based on questionnaire-identified demographic characteristics. An embedded qualitative dominant design was used. PARTICIPANTS/SETTING: Data were collected from April to July 2015 from active duty soldiers at 3 military installations in the United States. MAIN OUTCOME MEASURES: A self-report questionnaire (n = 289) provided data on demographic characteristics, health, exercise, detailed use, and attitudes regarding DS safety and efficacy. Fourteen focus-group sessions (n = 129) examined factors motivating DS use, education strategies, and identified themes and DS-related behaviors. STATISTICAL ANALYSIS PERFORMED: Descriptive statistics and χ2 analyses were conducted. RESULTS: Of the soldiers who completed questionnaires, 83% were male, 60% were enlisted, and 40% were officers; mean age ± standard deviation was 27.6 ± 0.36 years and 75% used at least 1 type of DS per week: 52% used protein/amino acids, 47% used multivitamins/minerals, and 35% used a combination of products. Focus groups indicated reasons for use included physical appearance, fitness, peer endorsement, ease of access, limited availability of healthy food, occupational demands, and health. Participants requested education from an expert on safe use that was not focused on dangerous products. CONCLUSIONS: Soldiers are high DS users, especially products marked for purported performance enhancement. Motivating factors for DS use are fitness/appearance and occupational demands, but soldiers lack knowledge of DS regulatory requirements and safety/efficacy. Soldiers wished to receive education on DSs from trusted health care professionals, such as registered dietitian nutritionists, that was not focused on dangerous products. Study findings suggest guidance and education should occur before periods of high DS use, such as deployment.

Optical vagus nerve modulation of heart and respiration via heart-injected retrograde AAV.

Vagus nerve stimulation has shown many benefits for disease therapies but current approaches involve imprecise electrical stimulation that gives rise to off-target effects, while the functionally relevant pathways remain poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate targeted neural communication with light-sensitive actuators (opsins) and can be targeted to organs of interest based on the location of viral delivery. Here, we tested whether retrograde adeno-associated virus (rAAV2-retro) injected in the heart can be used to selectively express opsins in vagus nerve fibers controlling cardiac function. Furthermore, we investigated whether perturbations in cardiac function could be achieved with photostimulation at the cervical vagus nerve. Viral injection in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Photostimulation using both one-photon stimulation and two-photon holography with a GRIN-lens incorporated nerve cuff, was tested on the pilot-cohort of injected mice. Changes in heart rate, surface electrocardiogram, and respiratory responses were observed in response to both one- and two-photon photostimulation. The results demonstrate feasibility of retrograde labeling for organ targeted optical neuromodulation.

Optogenetic stimulation of cholinergic fibers for the modulation of insulin and glycemia.

Previous studies have demonstrated stimulation of endocrine pancreas function by vagal nerve electrical stimulation. While this increases insulin secretion, expected concomitant reductions in circulating glucose do not occur. A complicating factor is the non-specific nature of electrical nerve stimulation. Optogenetic tools, however, provide the potential for cell-type specific neural stimulation using genetic targeting and/or spatially shaped excitation light. Here, we demonstrate light-activated stimulation of the endocrine pancreas by targeting parasympathetic (cholinergic) axons. In a mouse model expressing ChannelRhodopsin2 (ChR2) in cholinergic cells, serum insulin and glucose were measured in response to (1) ultrasound image-guided optical stimulation of axon terminals in the pancreas or (2) optical stimulation of axons of the cervical vagus nerve. Measurements were made in basal-glucose and glucose-stimulated conditions. Significant increases in plasma insulin occurred relative to controls under both pancreas and cervical vagal stimulation, while a rapid reduction in glycemic levels were observed under pancreatic stimulation. Additionally, ultrasound-based measurements of blood flow in the pancreas were increased under pancreatic stimulation. Together, these results demonstrate the utility of in-vivo optogenetics for studying the neural regulation of endocrine pancreas function and suggest its therapeutic potential for the control of insulin secretion and glucose homeostasis.

The effect of sleep restriction on cognitive performance in elite cognitive performers: a systematic review.

STUDY OBJECTIVES: To synthesize original articles exploring the effects of sleep restriction on cognitive performance specifically for Elite Cognitive Performers, i.e. those who engage in cognitively demanding tasks with critical or safety-critical outcomes in their occupation or area of expertise. METHODS: Backward snowballing techniques, gray literature searches, and traditional database searches (Embase, MEDLINE, Web of Science, Google Scholar, PSYCinfo, and SportDiscus) were used to obtain relevant articles. A quality assessment was performed, and the risk of training effects was considered. Results were narratively synthesized. Fourteen articles fit the criteria. Cognitive outcomes were divided into three categories defined by whether cognitive demands were "low-salience," "high-salience stable," or "high-salience flexible." RESULTS: Low-salience tests (i.e. psychomotor vigilance tasks & serial reaction tests), mainly requiring vigilance and rudimentary attentional capacities, were sensitive to sleep restriction, however, this did not necessarily translate to significant performance deficits on low-salience occupation-specific task performance. High-salience cognitive outcomes were typically unaffected unless when cognitive flexibility was required. CONCLUSIONS: Sleep restriction is of particular concern to occupations whereby individuals perform (1) simple, low-salience tasks or (2) high-salience tasks with demands on the flexible allocation of attention and working memory, with critical or safety-critical outcomes.

Generation of Leads for γ-Secretase Modulation.

Herein, we disclose three structurally differentiated γ-secretase modulators (GSMs) based on an oxadiazine scaffold. The analogues from series I potently inhibit the generation of Aß42 in vitro when the substituents at 3 and 4 positions of the oxadiazine moiety adopt an α orientation (cf. 11). To address the concern around potential reactivity of the exocyclic double bond present in series I toward nucleophilic attack, compounds containing either an endocyclic double bond, such as 20 (series II), or devoid of an olefinic moiety, such as 27 (series III), were designed and validated as novel GSMs. Compound 11 and azepine 20 exhibit robust lowering of CSF Aß42 in rats treated with a 30 mg/kg oral dose.

Caffeine, Energy Beverage Consumption, Fitness, and Sleep in U.S. Army Aviation Personnel.

BACKGROUND: Caffeine-containing products and dietary supplements are widely used by military populations, but little is known about their use by aviation personnel. This study assessed self-reported sleep, fitness, work-schedules, and caffeine/energy drink use.METHODS: A standardized survey was conducted in person by study personnel using tablet computers. A total of 188 aircrew members from the Combat Aviation Brigade at Fort Campbell, KY, participated in the survey. Focus groups were conducted with a subset of 47 subjects.RESULTS: The majority of subjects reported their physical fitness, health, and diets were good. They reported sleeping about 6 h per day and stated they needed additional sleep to feel fully rested. Their caffeine consumption averaged 346 ± 23 mg · d-1 with most derived from coffee (139 ± 12 mg · d-1) and energy drinks (110 ± 13 mg · d-1). About half (55%) of participants used energy drinks at least once per week and they consumed greater amounts of caffeine than nonusers. Focus group data indicated crewmembers primarily consumed energy drinks to enhance performance degraded by variations in work schedules and lack of sufficient sleep. Participants expressed a desire for additional education on diets and energy drinks as well as on aeromedical policies governing energy drink and supplement use.CONCLUSIONS: Caffeinated products, including coffee and energy drinks, are routinely used by Army aircrews to increase alertness. Aircrew personnel consider them generally safe, but would like to receive education about these beverages, other dietary issues, and Army policies governing their use in aircrew.Bukhari AS, Caldwell JA, DiChiara AJ, Merrill EP, Wright AO, Cole RE, Hatch-McChesney A, McGraw SM, Lieberman HR. Caffeine, energy beverage consumption, fitness, and sleep in U.S. Army aviation personnel. Aerosp Med Hum Perform. 2020; 91(8):641-650.

Cyp1 Inhibition Prevents Doxorubicin-Induced Cardiomyopathy in a Zebrafish Heart-Failure Model.

Doxorubicin is a highly effective chemotherapy agent used to treat many common malignancies. However, its use is limited by cardiotoxicity, and cumulative doses exponentially increase the risk of heart failure. To identify novel heart failure treatment targets, a zebrafish model of doxorubicin-induced cardiomyopathy was previously established for small-molecule screening. Using this model, several small molecules that prevent doxorubicin-induced cardiotoxicity both in zebrafish and in mouse models have previously been identified. In this study, exploration of doxorubicin cardiotoxicity is expanded by screening 2271 small molecules from a proprietary, target-annotated tool compound collection. It is found that 120 small molecules can prevent doxorubicin-induced cardiotoxicity, including 7 highly effective compounds. Of these, all seven exhibited inhibitory activity towards cytochrome P450 family 1 (CYP1). These results are consistent with previous findings, in which visnagin, a CYP1 inhibitor, also prevents doxorubicin-induced cardiotoxicity. Importantly, genetic mutation of cyp1a protected zebrafish against doxorubicin-induced cardiotoxicity phenotypes. Together, these results provide strong evidence that CYP1 is an important contributor to doxorubicin-induced cardiotoxicity and highlight the CYP1 pathway as a candidate therapeutic target for clinical cardioprotection.

Primary Presentation of Pulmonary Hypertension in the Peripartum: Preparing for Patients With Eisenmenger Physiology.

A Bosnian woman at 20 weeks' gestation presented with dyspnea and hypoxia. She was diagnosed with Eisenmenger physiology with severe pulmonary hypertension, ventricular septal defect, and patent ductus arteriosus. Given high maternal mortality, coordination of care with a multidisciplinary team approach may allow for best possible outcomes. (Level of Difficulty: Intermediate.).