Lipid Trolling to Optimize A3 Adenosine Receptor-Positive Allosteric Modulators (PAMs).

A3 adenosine receptor (A3AR) positive allosteric modulators (PAMs) (2,4-disubstituted-1H-imidazo[4,5-c]quinolin-4-amines) allosterically increase the Emax of A3AR agonists, but not potency, due to concurrent orthosteric antagonism. Following mutagenesis/homology modeling of the proposed lipid-exposed allosteric binding site on the cytosolic side, we functionalized the scaffold, including heteroatom substitutions and exocyclic phenylamine extensions, to increase allosteric binding. Strategically appended linear alkyl-alkynyl chains with terminal amino/guanidino groups improved allosteric effects at both human and mouse A3ARs. The chain length, functionality, and attachment position were varied to modulate A3AR PAM activity. For example, 26 (MRS8247, p-alkyne-linked 8 methylenes) and homologues increased agonist Cl-IB-MECA's Emax and potency ([35S]GTPγS binding). The putative mechanism involves a flexible, terminally cationic chain penetrating the lipid environment for stable electrostatic anchoring to cytosolic phospholipid head groups, suggesting "lipid trolling", supported by molecular dynamic simulation of the active-state model. Thus, we have improved A3AR PAM activity through rational design based on an extrahelical, lipidic binding site.

Evaluating current and future pedestrian mid-block crossing safety treatments using virtual reality simulation.

Virtual reality (VR) simulation offers a proactive, cost effective, immersive, and low risk platform for studying pedestrian safety. Within immersive virtual environments (IVEs), existing and alternative design conditions and intelligent transportation systems (ITS) technologies can be directly compared, prior to real-world implementation, to assess the impacts alternatives may have on pedestrian safety, perception, and behavior. Environmental factors can be controlled within IVEs so that test trials are replicable and directly comparable. Coupled with stated preference feedback, participants' observed preferences and behavior provide a comprehensive understanding of the impacts of proposed design alternatives. This research presents a case study of pedestrian behavior with three different mid-block crossing safety treatments modeled within a one-to-one scale IVE replication of a real-world location in Charlottesville, Virginia. The three safety treatments consider both passive and active collision avoidance designs and technologies, including (1) the existing painted crosswalk, (2) the addition of rectangular rapid flashing beacons (RRFBs), and (3) a pedestrian to everything (P2X) ITS phone application. Additionally, this paper demonstrates a VR simulation experimental design and framework for testing pedestrian safety treatments within naturalistic and replicable IVEs to assess both stated and observed preferences and behaviors of pedestrians. Repeated measures ANOVA indicated changes in both accepted gap size (p = 0.001) and crossing speed (p < 0.001) with alternative safety treatments. Generalized mixed models showed that pedestrians waited for statistically larger gap sizes (p = 0.02) without the assistance of alternative safety technologies (RRFBs and P2X application) and pedestrians crossed the street significantly faster (p = 0.001) without the alternative safety technologies, leading to unsafe dashing behavior. Through post-experiment surveys, it was found that participants perceived the As Built environment to be the least safe of the three treatments and that their sense of risk within the IVE was realistic. Considering both the observed crossing behavior and stated feedback, pedestrians exhibited intentionally unsafe darting behavior without assistive safety technology. This study demonstrates how VR simulation may be leveraged to study both stated preferences and observed behavior for understanding the safety implications of alternative roadway designs, providing a proactive approach for assessing and designing for pedestrian safety.

Percent Final Height Is a Novel Method That Identifies Differences Between the Rate of Development in American Children of Different Races.

INTRODUCTION: The Greulich & Pyle (G&P) Radiographic Atlas of Skeletal Development uses hand x-rays obtained between 1926 and 1942 on children of Caucasian ancestry. Our study uses modern Caucasian, Black, Hispanic, and Asian children to investigate patterns of development as a function of percent final height (PFH). METHODS: A retrospective review, at a single institution, was conducted using children who received a hand x-ray, a height measurement taken within 60 days of that x-ray, and a final height. BA and CA were compared between races. PFH was calculated by dividing height at the time of the x-ray by their final height. To further evaluate differences between races in CA or BA, PFH was then modeled as a function of CA or BA using a fifth-degree polynomial regression, and mean ages at the 85th PFH were compared. Patients were then divided into Sanders stages 1, 2-4, and 5-8 and the mean PFH, CA, and BA of the Asian, Black, and Hispanic children were compared with the White children using Student t test. P values less than 0.05 were considered significant. RESULTS: We studied 498 patients, including 53 Asian, 83 Black, 190 Hispanic, and 172 White patients. Mean BA was significantly greater than CA in Black males (1.27 y) and females (1.36 y), Hispanic males (1.12 y) and females (1.29 y), and White females (0.74 y). Hispanic and Black patients were significantly more advanced in BA than White patients (P<0.001). At the 85th PFH, White and Hispanic males were older than Black males by at least 7 months (P<0.001), and White females were significantly older than Hispanic females by 6.4 months (P<0.001). At 85th PFH for males, Hispanic and Black males had greater BA than White males by at least 5 months (P<0.001), and Asian females had a greater BA than Black females by at least 5 months (P<0.001). Compared with White children, Hispanic children were significantly younger at Sanders 2-4 than White children, and Black children were skeletally older at Sanders 5-8. CONCLUSIONS: BA was greater than CA by ≥1 year in Black and Hispanic children, and that these children had a significantly greater BA than their White counterparts. Black males and Hispanic females reached their 85th PFH at younger ages, and Hispanic males and Asian females were the most skeletally mature at their 85th PFH. Our results suggest that BA and CA may vary as a function of race, and further studies evaluating growth via the 85th PFH may be necessary. LEVEL OF EVIDENCE: Therapeutic Study - Level IV.

Reinforcement expectation in the honeybee (Apis mellifera): Can downshifts in reinforcement show conditioned inhibition?

When animals learn the association of a conditioned stimulus (CS) with an unconditioned stimulus (US), later presentation of the CS invokes a representation of the US. When the expected US fails to occur, theoretical accounts predict that conditioned inhibition can accrue to any other stimuli that are associated with this change in the US. Empirical work with mammals has confirmed the existence of conditioned inhibition. But the way it is manifested, the conditions that produce it, and determining whether it is the opposite of excitatory conditioning are important considerations. Invertebrates can make valuable contributions to this literature because of the well-established conditioning protocols and access to the central nervous system (CNS) for studying neural underpinnings of behavior. Nevertheless, although conditioned inhibition has been reported, it has yet to be thoroughly investigated in invertebrates. Here, we evaluate the role of the US in producing conditioned inhibition by using proboscis extension response conditioning of the honeybee (Apis mellifera). Specifically, using variations of a "feature-negative" experimental design, we use downshifts in US intensity relative to US intensity used during initial excitatory conditioning to show that an odorant in an odor-odor mixture can become a conditioned inhibitor. We argue that some alternative interpretations to conditioned inhibition are unlikely. However, we show variation across individuals in how strongly they show conditioned inhibition, with some individuals possibly revealing a different means of learning about changes in reinforcement. We discuss how the resolution of these differences is needed to fully understand whether and how conditioned inhibition is manifested in the honeybee, and whether it can be extended to investigate how it is encoded in the CNS. It is also important for extension to other insect models. In particular, work like this will be important as more is revealed of the complexity of the insect brain from connectome projects.

Varenicline and Nicotine Replacement Therapy for Smokers Admitted to Hospitals: A Randomized Clinical Trial.

Importance: Varenicline is the most effective sole pharmacotherapy for smoking cessation. If used in combination with nicotine replacement therapy (NRT), cessation rates may be further improved, but the efficacy and safety of the combination need to be evaluated. Objective: To examine whether hospitalized smokers treated with varenicline and NRT lozenges achieve higher prolonged smoking abstinence rates compared with those treated with varenicline alone. Design, Setting, and Participants: A double-blind, placebo-controlled randomized clinical trial was conducted in adult medical or surgical inpatients of 5 Australian public hospitals with a history of smoking 10 cigarettes or more per day, interested in quitting, and available for 12-month follow-up between May 1, 2019, and May 1, 2021 (final 12-month data collection in May 2022). Data analysis was performed from June 1 to August 30, 2023. Interventions: A 12-week varenicline regimen was initiated during hospitalization at standard doses in all participants. Participants were randomized to additionally use NRT (2 mg) or placebo lozenges if there was an urge to smoke. Behavioral support (Quitline) was offered to all participants. Main Outcomes and Measures: The primary outcome was biochemically verified sustained abstinence at 6 months. Secondary outcomes included self-reported prolonged abstinence, 7-day point prevalence abstinence (3, 6, and 12 months), and medicine-related adverse events. Results: A total of 320 participants (mean [SD] age, 52.5 [12.1] years; 183 [57.2%] male) were randomized. The conduct of biochemical verification was affected by COVID-19 restrictions; consequently, the biochemically verified abstinence in the intervention vs control arms (18 [11.4%] vs 16 [10.1%]; odds ratio [OR], 1.14; 95% CI, 0.56-2.33) did not support the combination therapy. The secondary outcomes in the intervention vs control arms of 7-day point prevalence abstinence at 6 months (54 [34.2%] vs 37 [23.4%]; OR, 1.71; 95% CI, 1.04-2.80), prolonged abstinence at 12 months (47 [29.9%] vs 30 [19.1%]; OR, 1.77; 95% CI, 1.05-3.00), and 7-day point prevalence abstinence at 12-months (48 [30.6%] vs 31 [19.7%]; OR, 1.79; 95% CI, 1.07-2.99) significantly improved with the combination therapy. The self-reported 6-month prolonged abstinence (61 [38.6%] vs 47 [29.7%]; OR, 1.49; 95% CI, 0.93-2.39) favored the combination therapy but was not statistically significant. Medicine-related adverse events were similar in the 2 groups (102 [74.5%] in the intervention group vs 86 [68.3%] in the control group). Conclusions and Relevance: In this randomized clinical trial of the combination of varenicline and NRT lozenges in hospitalized adult daily smokers, the combination treatment improved self-reported abstinence compared with varenicline alone, without compromising safety, but it did not improve biochemically validated abstinence. Trial Registration: anzctr.org.au Identifier: ACTRN12618001792213.

Focal selective dorsal rhizotomy and concurrent deformity correction: a combined approach.

OBJECTIVE: Selective dorsal rhizotomy (SDR) is a neurosurgical procedure to reduce spasticity in children with cerebral palsy and spastic diplegia. The authors developed a procedure called focal SDR for children with spasticity predominantly in the L5 or S1 motor distribution, which can be combined with orthopedic correction of fixed soft-tissue or bony deformity. The authors describe in detail the technique of minimally invasive focal SDR and propose selection criteria. METHODS: The authors conducted a retrospective study of patients who underwent focal SDR at their institution and underwent baseline and 1-year postoperative 3D gait analysis. Modified Ashworth scale (MAS) and Gait Deviation Index (GDI) scores were the primary outcome measures. RESULTS: Ten patients met the study criteria, all with an underlying diagnosis of cerebral palsy. All underwent focal SDR at the unilateral or bilateral S1 level, and 4 additionally underwent focal SDR at the L5 level unilaterally or bilaterally. All but 1 patient underwent concurrent orthopedic surgery. The improvement in spasticity of the plantar flexors, as measured by the MAS score, was 2.2 (p < 0.001). In the patients who underwent L5 focal SDR, there was an improvement in the hamstring MAS score of 1.4 (p = 0.004). The mean improvement in the GDI score following focal SDR was 11 (range -6 to 29, p < 0.001). CONCLUSIONS: Focally impairing spasticity in the gastrocsoleus complex and/or hamstrings muscle group in the setting of less functionally impactful proximal tone is extremely common in cerebral palsy. The novel technique of focal SDR, combined with orthopedic intervention, improves spasticity scores and overall gait mechanics. Further investigation is warranted to define the ideal candidacy and outcomes.

Associations between Posttraumatic Stress Disorder and Diabetes in Vietnam-Era Women Veterans in the HealthViEWS Study.

Background: Posttraumatic stress disorder (PTSD) is associated with incident diabetes. However, past studies have often included predominantly male samples, despite important sex and gender differences in diabetes. To address this limitation, this study examined the association between PTSD and diabetes in older Veteran women, a population with a high burden of PTSD. Materials and Methods: Data were collected from 4,105 women (Mage = 67.4 years), participating in the Health of Vietnam-Era Veteran Women's Study (HealthViEWS; Department of Veterans Affairs Cooperative Studies Program #579). Participants completed self-report measures of demographics, health conditions, and health behaviors. Information on military service was obtained through service records. A structured clinical interview was conducted by telephone to assess current and lifetime PTSD and other mental health disorders. Weighted descriptive and logistic regression analyses were used to examine associations between PTSD and diabetes. Results: The prevalence of diabetes was 28.4% among women with current full PTSD compared to 23.4%, 17.6%, and 17.5% for current subthreshold, remitted, and no PTSD. In unadjusted analyses, women with current full and subthreshold PTSD were 1.87 [1.49; 2.33] and 1.44 [1.11; 1.85] times more likely to have diabetes compared to women with no PTSD. Remitted PTSD was not associated with increased odds of diabetes. Effects were attenuated but remained significant after adjustment for relevant covariates. Conclusions: Vietnam-era women with current PTSD, including subthreshold symptoms, had a greater likelihood of diabetes compared to women without PTSD. These findings suggest that women with PTSD may benefit from increased diabetes prevention efforts.

Gartland classification concordance of supracondylar fractures among pediatric emergency medicine physicians, radiologists, and orthopedic surgeons.

BACKGROUND: The modified Gartland classification is the most widely accepted grading method of supracondylar humeral fractures among orthopedic surgeons and is relevant to identifying fractures that may require surgery. OBJECTIVE: To assess the interobserver reliability of the modified Gartland classification among pediatric radiologists, pediatric orthopedic surgeons, and pediatric emergency medicine physicians. MATERIALS AND METHODS: Elbow radiographs for 100 children with supracondylar humeral fractures were retrospectively independently graded by two pediatric radiologists, two pediatric orthopedic surgeons, and two pediatric emergency medicine physicians using the modified Gartland classification. A third grader of the same subspecialty served as a tie-breaker as needed to reach consensus. Readers were blinded to one another and to the medical record. The modified Gartland grade documented in the medical record by the treating orthopedic provider was used as the reference standard. Interobserver agreement was assessed using kappa statistics. RESULTS: There was substantial interobserver agreement (kappa = 0.77 [95% CI, 0.69-0.85]) on consensus fracture grade between the three subspecialties. Similarly, when discriminating between Gartland type I and higher fracture grades, there was substantial interobserver agreement between specialties (kappa = 0.77 [95% CI, 0.66-0.89]). The grade assigned by pediatric radiologists differed from the reference standard on 15 occasions, pediatric emergency medicine differed on 19 occasions, and pediatric orthopedics differed on 9 occasions. CONCLUSION: The modified Gartland classification for supracondylar humeral fractures is reproducible among pediatric emergency medicine physicians, radiologists, and orthopedic surgeons.

Key residues in the VDAC2-BAK complex can be targeted to modulate apoptosis.

BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial channel protein VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, while interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease or to promote BAK-mediated apoptosis for cancer therapy.

Venom-inspired somatostatin receptor 4 (SSTR4) agonists as new drug leads for peripheral pain conditions.

Persistent pain affects one in five people worldwide, often with severely debilitating consequences. Current treatment options, which can be effective for mild or acute pain, are ill-suited for moderate-to-severe persistent pain, resulting in an urgent need for new therapeutics. In recent years, the somatostatin receptor 4 (SSTR 4 ), which is expressed in sensory neurons of the peripheral nervous system, has emerged as a promising target for pain relief. However, the presence of several closely related receptors with similar ligand-binding surfaces complicates the design of receptor-specific agonists. In this study, we report the discovery of a potent and selective SSTR 4 peptide, consomatin Fj1, derived from extensive venom gene datasets from marine cone snails. Consomatin Fj1 is a mimetic of the endogenous hormone somatostatin and contains a minimized binding motif that provides stability and drives peptide selectivity. Peripheral administration of synthetic consomatin Fj1 provided analgesia in mouse models of postoperative and neuropathic pain. Using structure-activity studies, we designed and functionally evaluated several Fj1 analogs, resulting in compounds with improved potency and selectivity. Our findings present a novel avenue for addressing persistent pain through the design of venom-inspired SSTR 4 -selective pain therapeutics. One Sentence Summary: Venom peptides from predatory marine mollusks provide new leads for treating peripheral pain conditions through a non-opioid target.

The Aging Surgeon: Considerations for Navigating a Successful and Satisfying Career.

Our careers as surgeons are some of the busiest and perhaps most sought after in existence. We have all put in countless years of tenacious effort, at times blood, frequent sweat, and occasional tears, to have the privilege to care for others and correct their ailments. Many of us are like freight trains rolling down the tracks indefinitely. But all too often we finish our training and head down those tracks without considering what stops we should make along the way, which forks in the tracks we should consider taking, and perhaps most often, we do not consider how we are going to eventually stop the train. Most of us have been witness to colleagues who keep working beyond their prime, be it for lack of alternative opportunities, lack of hobbies to retire to, or for lack of insight into their own decline. From these observations was born this presidential panel. As you can see, it is a collection of past presidents of So Cal ACS, with the exception for Dr Freischlag (who we all know would have served as president at some point had she never relocated away from Southern California). Each of these speakers has unique experience from their own careers that they will share with us so we can take pause and consider their insights and wisdom for how to navigate a successful and satisfying career.

Evaluating Chat Generative Pre-trained Transformer Responses to Common Pediatric In-toeing Questions.

OBJECTIVE: Chat generative pre-trained transformer (ChatGPT) has garnered attention in health care for its potential to reshape patient interactions. As patients increasingly rely on artificial intelligence platforms, concerns about information accuracy arise. In-toeing, a common lower extremity variation, often leads to pediatric orthopaedic referrals despite observation being the primary treatment. Our study aims to assess ChatGPT's responses to pediatric in-toeing questions, contributing to discussions on health care innovation and technology in patient education. METHODS: We compiled a list of 34 common in-toeing questions from the "Frequently Asked Questions" sections of 9 health care-affiliated websites, identifying 25 as the most encountered. On January 17, 2024, we queried ChatGPT 3.5 in separate sessions and recorded the responses. These 25 questions were posed again on January 21, 2024, to assess its reproducibility. Two pediatric orthopaedic surgeons evaluated responses using a scale of "excellent (no clarification)" to "unsatisfactory (substantial clarification)." Average ratings were used when evaluators' grades were within one level of each other. In discordant cases, the senior author provided a decisive rating. RESULTS: We found 46% of ChatGPT responses were "excellent" and 44% "satisfactory (minimal clarification)." In addition, 8% of cases were "satisfactory (moderate clarification)" and 2% were "unsatisfactory." Questions had appropriate readability, with an average Flesch-Kincaid Grade Level of 4.9 (±2.1). However, ChatGPT's responses were at a collegiate level, averaging 12.7 (±1.4). No significant differences in ratings were observed between question topics. Furthermore, ChatGPT exhibited moderate consistency after repeated queries, evidenced by a Spearman rho coefficient of 0.55 ( P = 0.005). The chatbot appropriately described in-toeing as normal or spontaneously resolving in 62% of responses and consistently recommended evaluation by a health care provider in 100%. CONCLUSION: The chatbot presented a serviceable, though not perfect, representation of the diagnosis and management of pediatric in-toeing while demonstrating a moderate level of reproducibility in its responses. ChatGPT's utility could be enhanced by improving readability and consistency and incorporating evidence-based guidelines. LEVEL OF EVIDENCE: Level IV-diagnostic.

Infection with mpox virus via the genital mucosae increases shedding and transmission in the multimammate rat (Mastomys natalensis).

The 2022 mpox virus (MPXV) outbreak was sustained by human-to-human transmission; however, it is currently unclear which factors lead to sustained transmission of MPXV. Here we present Mastomys natalensis as a model for MPXV transmission after intraperitoneal, rectal, vaginal, aerosol and transdermal inoculation with an early 2022 human outbreak isolate (Clade IIb). Virus shedding and tissue replication were route dependent and occurred in the presence of self-resolving localized skin, lung, reproductive tract or rectal lesions. Mucosal inoculation via the rectal, vaginal and aerosol routes led to increased shedding, replication and a pro-inflammatory T cell profile compared with skin inoculation. Contact transmission was higher from rectally inoculated animals. This suggests that transmission might be sustained by increased susceptibility of the anal and genital mucosae for infection and subsequent virus release.

Zinc-chelating BET bromodomain inhibitors equally target islet endocrine cell types.

Inhibition of the bromodomain and extraterminal domain (BET) protein family is a potential strategy to prevent and treat diabetes; however, the clinical use of BET bromodomain inhibitors (BETis) is associated with adverse effects. Here, we explore a strategy for targeting BETis to ß cells by exploiting the high-zinc (Zn2+) concentration in ß cells relative to other cell types. We report the synthesis of a novel, Zn2+-chelating derivative of the pan-BETi (+)-JQ1, (+)-JQ1-DPA, in which (+)-JQ1 was conjugated to dipicolyl amine (DPA). As controls, we synthesized (+)-JQ1-DBA, a non-Zn2+-chelating derivative, and (-)-JQ1-DPA, an inactive enantiomer that chelates Zn2+. Molecular modeling and biophysical assays showed that (+)-JQ1-DPA and (+)-JQ1-DBA retain potent binding to BET bromodomains in vitro. Cellular assays demonstrated (+)-JQ1-DPA attenuated NF-ĸB target gene expression in ß cells stimulated with the proinflammatory cytokine interleukin 1ß. To assess ß-cell selectivity, we isolated islets from a mouse model that expresses green fluorescent protein in insulin-positive ß cells and mTomato in insulin-negative cells (non-ß cells). Surprisingly, Zn2+ chelation did not confer ß-cell selectivity as (+)-JQ1-DPA was equally effective in both ß and α cells; however, (+)-JQ1-DPA was less effective in macrophages, a nonendocrine islet cell type. Intriguingly, the non-Zn2+-chelating derivative (+)-JQ1-DBA displayed the opposite selectivity, with greater effect in macrophages compared with (+)-JQ1-DPA, suggesting potential as a macrophage-targeting molecule. These findings suggest that Zn2+-chelating small molecules confer endocrine cell selectivity rather than ß-cell selectivity in pancreatic islets and provide valuable insights and techniques to assess Zn2+ chelation as an approach to selectively target small molecules to pancreatic ß cells.NEW & NOTEWORTHY Inhibition of BET bromodomains is a novel potential strategy to prevent and treat diabetes mellitus. However, BET inhibitors have negative side effects. We synthesized a BET inhibitor expected to exploit the high zinc concentration in ß cells to accumulate in ß cells. We show our inhibitor targeted pancreatic endocrine cells; however, it was less effective in immune cells. A control inhibitor showed the opposite effect. These findings help us understand how to target specific cells in diabetes treatment.

Clinical, electrophysiologic and serologic evidence of cancer associated retinopathy preceding a diagnosis of breast cancer.

We report the case of a woman in her 50s who presented headaches, blurred vision, diplopia and loss of peripheral vision. She was treated for normal tension glaucoma based on optic nerve cupping prior to the development of diplopia. Records demonstrated visual field constriction over 4 months despite well-controlled intraocular pressures. Examination revealed decreased visual acuity and visual field constriction. The multifocal electroretinogram was abnormal. After a thorough review of her medical and family history, a concern for cancer-associated retinopathy developed. Blood samples were positive for antiretinal antibodies against 23 kDA and 46 kDA proteins. Cancer screening was recommended as the aetiology for retinopathy was unknown and revealed a left breast lump. Following lumpectomy with adjuvant chemoradiation, her visual acuity normalised and visual field defects completely resolved. This case serves to provide an example that distant systemic symptoms may be a manifestation of the underlying malignancy and the importance of clinical suspicion with prompt evaluation.

High-Throughput Cell-Based Screening of Small Molecule KRAS Signaling Inhibitors Using a Homogeneous Time-Resolved Fluorescence (HTRF) Assay.

With recent advances proving that effective inhibition of KRAS is possible, there have been significant efforts made to develop inhibitors of specific mutant alleles. Here we describe a detailed protocol that employs homogeneous time-resolved fluorescence (HTRF) to identify compounds acting on KRAS signaling in malignant cell lines. This method allows for high-throughput, cell-based screens of large compound libraries for the development of RAS-targeted therapeutics.

FLAG-KRAS4B as a Model System for KRAS4B Proteoform and PTM Evaluation by Mass Spectrometry.

Prior analysis of intact and modified protein forms (proteoforms) of KRAS4B isolated from cell lines and tumor samples by top-down mass spectrometry revealed the presence of novel posttranslational modifications (PTMs) and potential evidence of context-specific KRAS4B modifications. However, low endogenous proteoform signal resulted in ineffective characterization, making it difficult to visualize less abundant PTMs or perform follow-up PTM validation using standard proteomic workflows. The NCI RAS Initiative has developed a model system, whereby KRAS4B bearing an N-terminal FLAG tag can be stably expressed within a panel of cancer cell lines. Herein, we present a method for combining immunoprecipitation with complementary proteomic methods to directly analyze N-terminally FLAG-tagged KRAS4B proteoforms and PTMs. We provide detailed protocols for FLAG-KRAS4B purification, proteoform analysis by targeted top-down LC-MS/MS, and validation of abundant PTMs by bottom-up LC-MS/MS with example results.

Identifying candidate mechanisms of comorbidity in disordered eating and posttraumatic stress disorder symptoms among U.S. veterans: A network analytic approach.

OBJECTIVE: Comorbidity between posttraumatic stress disorder (PTSD) and disordered eating (DE) symptoms is common, reflecting a possible reciprocal relationship between these disorders. Network analysis may reveal candidate mechanisms underlying their comorbidity and highlight important treatment targets. METHOD: Two national samples of U.S. veterans endorsing trauma exposure self-reported PTSD and DE symptoms. The discovery sample included veterans from all service eras (n = 434). The validation sample included recently separated post-9/11 veterans (n = 507). We fit graphical lasso models to evaluate the network structure of PTSD factors based on the seven-factor "hybrid" model and DE symptoms within each sample. We used strength scores to identify the most central symptoms within the networks and identified bridge symptoms connecting PTSD and DE features. We tested for network invariance between self-identified men and women within each sample and across the studies. RESULTS: PTSD and DE symptoms clustered as expected within networks for each sample. The strongest nodes in the networks included both PTSD and DE features. The strongest bridge symptoms in both studies included overevaluation of shape and weight, negative affect, and avoidance. Networks were invariant across men and women in each sample and largely invariant across samples. CONCLUSIONS: Cross-sectional network models of PTSD and DE symptoms largely replicated across national samples of U.S. veterans and between men and women within samples. Cognitive features of both disorders, along with avoidance, may partially underlie comorbidity and represent potential treatment targets. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

From posttraumatic stress symptoms to suicidal ideation among military veterans: Pathways founded on meaning in life and gratitude.

Military veterans experience higher rates of suicidal ideation compared to nonveteran populations. Importantly, suicidal ideation often precedes and predicts fatal and nonfatal suicide attempts, and thus it is critical to better understand factors that increase risk for suicidal ideation to inform suicide prevention efforts in this population. One key predictor of suicidal ideation is exposure to traumatic experiences and their sequelae, particularly posttraumatic stress symptoms (PTSS). However, little work has explored how deficits in well-being contribute to this association among veterans. We tested two aspects of well-being-meaning in life and gratitude-as potential mechanisms underlying the pathway from PTSS to suicidal ideation among 7,388 men and women veterans who recently separated from service. A parallel mediation analysis revealed significant paths from more severe PTSS to more frequent suicidal ideation through diminished meaning in life, B = 0.005, SE = 0.001, 95% CI [0.004, 0.007], and gratitude, B = 0.001, SE = 0.001, 95% CI [< 0.001, 0.002]. Gender differences were also observed. Although the results related to meaning in life appeared to replicate across gender, pathways involving gratitude differed among men and women. Overall, our findings suggest that helping veterans build meaning and appreciation in everyday life may be a proactive and holistic approach to suicide prevention.