Plant synthetic biology as a tool to help eliminate hidden hunger.

Agricultural systems are under increasing pressure from declining environmental conditions, a growing population, and changes in consumer preferences, resulting in widespread malnutrition-related illnesses. Improving plant nutritional content through biotechnology techniques such as synthetic biology is a promising strategy to help combat hidden hunger caused by the lack of affordable and healthy foods in human diets. Production of compounds usually found in animal-rich diets, such as vitamin D or omega-3 fatty acids, has been recently demonstrated in planta. Here, we review recent biotechnological approaches to biofortifying plants with vitamins, minerals, and other metabolites, and summarise synthetic biology advances that offer the opportunity to build on these early biofortification efforts.

Hemorrhagic transformation in acute ischemic stroke: hemorrhagic subtypes and symptomatic intracranial hemorrhage.

BACKGROUND: Few clinical studies perform detailed analyses of subtypes of intracranial hemorrhage (ICH) after mechanical thrombectomy (MT) used to treat acute ischemic stroke. Symptomatic intracranial hemorrhage (sICH) is a formidable complication of MT and is widely used in clinical trials as a safety outcome. However, variable definitions of sICH are used across clinical studies. OBJECTIVE: To radiographically subcategorize post-MT ICH development within this large cohort and examine overlap with sICH. Second, to examine the agreement of this definition of sICH with local site-reported occurrences of sICH to see how sICH rates change with modifications of the definitions used. METHODS: A large cohort of patients treated with MT for acute ischemic stroke (n=1395) was analyzed to (1) radiographically characterize hemorrhagic subtypes of intracranial hemorrhage (ICH) occurring after MT; (2) examine associations of hemorrhagic subtypes with sICH; and (3) compare core laboratory-adjudicated occurrences of sICH with site-reported sICH. RESULTS: The overall rate of ICH was 552/1395 patients (39.6%), and the overall rate of sICH was 47/1395 (3.4%). The most common type of ICH was hemorrhagic infarction type 1 (HI1), which represented 45.3% of all ICH cases- followed by HI2 (31.5%) and subarachnoid hemorrhage (SAH, 29.2%). Parenchymal hematoma 2 (PH2) represented only 3.3% of all ICH cases. Of the PH2 hemorrhages, only 33.3% were determined to be symptomatic. Of sICH cases, the most common ICH subtypes were HI2 (48.9%) and SAH (38.3%). Comparison of sICH rates as determined by core laboratory adjudication versus local site-reported results showed that only 14 patients were identified as having sICH with both definitions, with 47 patients total with sICH according to one definition, but not the other. CONCLUSIONS: Results of this analysis demonstrate the radiographic subtypes of ICH and also highlight the limitations of variable criteria used to define sICH, suggesting that it might be appropriate to revisit how sICH is defined post-MT. TRIAL REGISTRATION NUMBER: Clinical trial NCT03845491.

Instrumented Mouthguard Decoupling Affects Measured Head Kinematic Accuracy.

Many recent studies have used boil-and-bite style instrumented mouthguards to measure head kinematics during impact in sports. Instrumented mouthguards promise greater accuracy than their predecessors because of their superior ability to couple directly to the skull. These mouthguards have been validated in the lab and on the field, but little is known about the effects of decoupling during impact. Decoupling can occur for various reasons, such as poor initial fit, wear-and-tear, or excessive impact forces. To understand how decoupling influences measured kinematic error, we fit a boil-and-bite instrumented mouthguard to a 3D-printed dentition mounted to a National Operating Committee on Standards for Athletic Equipment (NOCSAE) headform. We also instrumented the headform with linear accelerometers and angular rate sensors at its center of gravity (CG). We performed a series of pendulum impact tests, varying impactor face and impact direction. We measured linear acceleration and angular velocity, and we calculated angular acceleration from the mouthguard and the headform CG. We created decoupling conditions by varying the gap between the lower jaw and the bottom face of the mouthguard. We tested three gap conditions: 0 mm (control), 1.6 mm, and 4.8 mm. Mouthguard measurements were transformed to the CG and compared to the reference measurements. We found that gap condition, impact duration, and impact direction significantly influenced mouthguard measurement error. Error was higher for larger gaps and in frontal (front and front boss) conditions. Higher errors were also found in padded conditions, but the mouthguards did not collect all rigid impacts due to inherent limitations. We present characteristic decoupling time history curves for each kinematic measurement. Exemplary frequency spectra indicating characteristic decoupling frequencies are also described. Researchers using boil-and-bite instrumented mouthguards should be aware of their limitations when interpreting results and should seek to address decoupling through advanced post-processing techniques when possible.

Evaluating ChatGPT's moral competence in health care-related ethical problems.

Objectives: Artificial intelligence tools such as Chat Generative Pre-trained Transformer (ChatGPT) have been used for many health care-related applications; however, there is a lack of research on their capabilities for evaluating morally and/or ethically complex medical decisions. The objective of this study was to assess the moral competence of ChatGPT. Materials and methods: This cross-sectional study was performed between May 2023 and July 2023 using scenarios from the Moral Competence Test (MCT). Numerical responses were collected from ChatGPT 3.5 and 4.0 to assess individual and overall stage scores, including C-index and overall moral stage preference. Descriptive analysis and 2-sided Student's t-test were used for all continuous data. Results: A total of 100 iterations of the MCT were performed and moral preference was found to be higher in the latter Kohlberg-derived arguments. ChatGPT 4.0 was found to have a higher overall moral stage preference (2.325 versus 1.755) when compared to ChatGPT 3.5. ChatGPT 4.0 was also found to have a statistically higher C-index score in comparison to ChatGPT 3.5 (29.03 ± 11.10 versus 19.32 ± 10.95, P =.0000275). Discussion: ChatGPT 3.5 and 4.0 trended towards higher moral preference for the latter stages of Kohlberg's theory for both dilemmas with C-indices suggesting medium moral competence. However, both models showed moderate variation in C-index scores indicating inconsistency and further training is recommended. Conclusion: ChatGPT demonstrates medium moral competence and can evaluate arguments based on Kohlberg's theory of moral development. These findings suggest that future revisions of ChatGPT and other large language models could assist physicians in the decision-making process when encountering complex ethical scenarios.

Optimizing Visualization in Shoulder Arthroscopy: An Evidence-Based Guide.

Advances in arthroscopy have contributed toward improved understanding and management of diverse pathological conditions in the shoulder. As a result, arthroscopy is often preferred by both patients and surgeons. However, surgery can be complicated by limited visualization. Techniques to improve visualization include patient and portal positioning, mechanical débridement, radiofrequency ablation, epinephrine added to irrigation fluid, tranexamic acid administration, and controlled hypotensive anesthesia. Despite published literature on each, a thorough understanding of the evidence supporting these techniques and adjuvants is essential to interpret the clinical utility of each.

Breast Cancer Cells Exhibit Mesenchymal-Epithelial Plasticity Following Dynamic Modulation of Matrix Stiffness.

Mesenchymal-epithelial transition (MET) is essential for tissue and organ development and is thought to contribute to cancer by enabling the establishment of metastatic lesions. Despite its importance in both health and disease, there is a lack of in vitro platforms to study MET and little is known about the regulation of MET by mechanical cues. Here, hyaluronic acid-based hydrogels with dynamic and tunable stiffnesses mimicking that of normal and tumorigenic mammary tissue are synthesized. The platform is then utilized to examine the response of mammary epithelial cells and breast cancer cells to dynamic modulation of matrix stiffness. Gradual softening of the hydrogels reduces proliferation and increases apoptosis of breast cancer cells. Moreover, breast cancer cells exhibit temporal changes in cell morphology, cytoskeletal organization, and gene expression that are consistent with mesenchymal-epithelial plasticity as the stiffness of the matrix is reduced. A reduction in matrix stiffness attenuates the expression of integrin-linked kinase, and inhibition of integrin-linked kinase impacts proliferation, apoptosis, and gene expression in cells cultured on stiff and dynamic hydrogels. Overall, these findings reveal intermediate epithelial/mesenchymal states as cells move along a matrix stiffness-mediated MET trajectory and suggest an important role for matrix mechanics in regulating mesenchymal-epithelial plasticity.

Clinical and economic evaluation of tralokinumab for atopic dermatitis.

Tralokinumab is the first selective interleukin 13 inhibitor approved for moderate to severe atopic dermatitis. This article reports the findings of a comprehensive literature review and extensive economic analysis to assess tralokinumab's safety, effectiveness, and cost. Evidence synthesis involved evaluating comparative effectiveness and conducting economic sensitivity analyses. This review was prepared by the University of Connecticut School of Pharmacy Academy of Managed Care Pharmacy (AMCP) Student Chapter. The student author group won the AMCP National Pharmacy and Therapeutics competition for their tralokinumab product review in March 2023.

Quantitative Measurement of Rate of Targeted Protein Degradation.

Targeted protein degradation (TPD) is a therapeutic approach that leverages the cell's natural machinery to degrade targets instead of inhibiting them. This is accomplished by using mono- or bifunctional small molecules designed to induce the proximity of target proteins and E3 ubiquitin ligases, leading to ubiquitination and subsequent proteasome-dependent degradation of the target. One of the most significant attributes of the TPD approach is its proposed catalytic mechanism of action, which permits substoichiometric exposure to achieve the desired pharmacological effects. However, apart from one in vitro study, studies supporting the catalytic mechanism of degraders are largely inferred based on potency. A more comprehensive understanding of the degrader catalytic mechanism of action can help aspects of compound development. To address this knowledge gap, we developed a workflow for the quantitative measurement of the catalytic rate of degraders in cells. Comparing a selective and promiscuous BTK degrader, we demonstrate that both compounds function as efficient catalysts of BTK degradation, with the promiscuous degrader exhibiting faster rates due to its ability to induce more favorable ternary complexes. By leveraging computational modeling, we show that the catalytic rate is highly dynamic as the target is depleted from cells. Further investigation of the promiscuous kinase degrader revealed that the catalytic rate is a better predictor of optimal degrader activity toward a specific target compared to degradation magnitude alone. In summary, we present a versatile method for mapping the catalytic activity of any degrader for TPD in cells.

ß-Amino Acids Reduce Ternary Complex Stability and Alter the Translation Elongation Mechanism.

Templated synthesis of proteins containing non-natural amino acids (nnAAs) promises to expand the chemical space available to biological therapeutics and materials, but existing technologies are still limiting. Addressing these limitations requires a deeper understanding of the mechanism of protein synthesis and how it is perturbed by nnAAs. Here we examine the impact of nnAAs on the formation and ribosome utilization of the central elongation substrate: the ternary complex of native, aminoacylated tRNA, thermally unstable elongation factor, and GTP. By performing ensemble and single-molecule fluorescence resonance energy transfer measurements, we reveal that both the (R)- and (S)-ß2 isomers of phenylalanine (Phe) disrupt ternary complex formation to levels below in vitro detection limits, while (R)- and (S)-ß3-Phe reduce ternary complex stability by 1 order of magnitude. Consistent with these findings, (R)- and (S)-ß2-Phe-charged tRNAs were not utilized by the ribosome, while (R)- and (S)-ß3-Phe stereoisomers were utilized inefficiently. (R)-ß3-Phe but not (S)-ß3-Phe also exhibited order of magnitude defects in the rate of translocation after mRNA decoding. We conclude from these findings that non-natural amino acids can negatively impact the translation mechanism on multiple fronts and that the bottlenecks for improvement must include the consideration of the efficiency and stability of ternary complex formation.

Skeletal muscle BMAL1 is necessary for transcriptional adaptation of local and peripheral tissues in response to endurance exercise training.

OBJECTIVE: In this investigation, we addressed the contribution of the core circadian clock factor, BMAL1, in skeletal muscle to both acute transcriptional responses to exercise and transcriptional remodeling in response to exercise training. Additionally, we adopted a systems biology approach to investigate how loss of skeletal muscle BMAL1 altered peripheral tissue homeostasis as well as exercise training adaptations in iWAT, liver, heart, and lung of male mice. METHODS: Combining inducible skeletal muscle specific BMAL1 knockout mice, physiological testing and standardized exercise protocols, we performed a multi-omic analysis (transcriptomics, chromatin accessibility and metabolomics) to explore loss of muscle BMAL1 on muscle and peripheral tissue responses to exercise. RESULTS: Muscle-specific BMAL1 knockout mice demonstrated a blunted transcriptional response to acute exercise, characterized by the lack of upregulation of well-established exercise responsive transcription factors including Nr4a3 and Ppargc1a. Six weeks of exercise training in muscle-specific BMAL1 knockout mice induced significantly greater and divergent transcriptomic and metabolomic changes in muscle. Surprisingly, liver, lung, inguinal white adipose and heart showed divergent exercise training transcriptomes with less than 5% of 'exercise-training' responsive genes shared for each tissue between genotypes. CONCLUSIONS: Our investigation has uncovered the critical role that BMAL1 plays in skeletal muscle as a key regulator of gene expression programs for both acute exercise and training adaptations. In addition, our work has uncovered the significant impact that altered exercise response in muscle and its likely impact on the system plays in the peripheral tissue adaptations to exercise training. Our work also demonstrates that if the muscle adaptations diverge to a more maladaptive state this is linked to increased gene expression signatures of inflammation across many tissues. Understanding the molecular targets and pathways contributing to health vs. maladaptive exercise adaptations will be critical for the next stage of therapeutic design for exercise mimetics.

Trends in reported outcomes and patient reported outcome measures (PROMs) in humeral shaft fracture literature: a systematic review.

PURPOSE: With a lack of standardization among outcome measures in fracture literature, cross-study comparisons remain limited. This systematic review aimed to identify trends in outcome measures reported by studies of the treatment of humeral shaft fractures. METHODS: A systematic review was performed of studies reporting clinical outcomes of humeral shaft fractures indexed in PubMed. Extracted data included demographics, fracture characteristics, treatment modalities, outcomes, patient reported outcome measures (PROMs), and journal characteristics. Cochran-Armitage tests and linear regressions were used to identify data trends. Pearson chi-square and Kruskal-Wallis tests were used for comparisons between studies. RESULTS: This review included 197 studies with outcomes of 15,445 humeral shaft fractures. 126 studies reported PROMs and 37 different PROMs were used. The Constant Score was most commonly reported (34% of studies), followed by ASES Score (21%), MEPS (21%), and DASH Score (20%). There was a significant increase in PROM usage over time (p = 0.016) and in articles using three or more PROMs (p = 0.005). The number of PROMs were significantly greater in prospective cohort studies and RCTs (p = 0.012) compared to retrospective cohort studies and case series (p = 0.044 for both). Post-treatment shoulder motion was reported in 43% of studies and 34% reported elbow motion. 86% of studies reported complications as an outcome parameter. Time to union and nonunion rate were published in 69% and 88% of studies, respectively. CONCLUSION: This study identified increasing PROM usage over time and disparities in the reporting of outcomes in humeral shaft fracture literature requiring further validation and standardization of available outcome measures.

Fluorescence labeling strategies for the study of ion channel and receptor cell surface expression: A comprehensive toolkit for extracellular labeling of TRPV1.

Regulation of ion channel expression on the plasma membrane is a major determinant of neuronal excitability, and identifying the underlying mechanisms of this expression is critical to our understanding of neurons. A critical aspect of measuring changes in ion channel expression is uniquely identifying ion channels located on the cell surface. To accomplish this goal we demonstrate two orthogonal strategies to label extracellular sites of the ion channel TRPV1 that minimally perturb the function of the channel: 1) We use the amber codon suppression technique to introduce a non-canonical amino acid (ncAA) with tetrazine click chemistry compatible with a trans-cyclooctene coupled fluorescent dye. 2) By inserting the circularly permutated HaloTag (cpHaloTag) in an extracellular loop of TRPV1, we incorporate a click-chemistry site for a chloroalkane-linked fluorescent dye of our choosing. Optimization of ncAA insertion sites was accomplished by screening residue positions between the S1 and S2 transmembrane domains with elevated missense variants in the human population, and we identified T468 as a rapid labeling site (∼5 minutes) based on functional as well as biochemical assays in HEK293T/17 cells. After several rounds of adapting the linker lengths and backbone placement of cpHaloTag on the extracellular side of TRPV1, our efforts led to a channel construct that robustly expressed as a fully functional TRPV1exCellHalo fusion with intact wild-type gating properties. The TRPV1exCellHalo construct was used in a single molecule experiment to track TRPV1 on the cell surface and validate studies that show decreased mobility of the channel upon activation. The success of these extracellular label TRPV1 (exCellTRPV1) constructs as tools to track surface expression of the channel will shed significant light on the mechanisms regulating expression and provide a general scheme to introduce similar modifications to other cell surface receptors.

Symptomatology in Unilateral Versus Bilateral Superior Canal Dehiscence Patients Undergoing Unilateral Surgery.

OBJECTIVE: To compare symptomatology in patients with unilateral versus bilateral superior semicircular canal dehiscence who underwent unilateral surgical repair. STUDY DESIGN: Retrospective cohort study. SETTING: Single surgeon series at tertiary academic medical center from 2002 to 2021. METHODS: Patients were administered a standardized questionnaire regarding the presence or absence of 16 symptoms (11 auditory and 8 vestibular) pre- and postoperatively. Symptom rates were compared between patients with unilateral and bilateral dehiscence, and paired statistical testing was used to analyze symptom improvement with surgery. RESULTS: Our final cohort included 125 patients, 93 (74%) with unilateral superior canal dehiscence syndrome (SCDS) and 32 (26%) with bilateral SCDS. Bilateral patients had an increased burden of auditory and vestibular symptoms compared to unilateral patients before surgery (7.6 vs 6.2, P = .03) and after surgery (3.1 vs 1.9, P = .02). Both groups experienced a significant reduction of symptoms following repair (P < .01 for both). CONCLUSION: Our study has 2 key findings: First, patients with bilateral dehiscence seem to be more symptomatic, reporting more auditory and vestibular symptoms both before and after surgery. Second, bilateral patients still seem to benefit from unilateral repair, demonstrating a significant reduction in the number of symptoms with surgery. Our findings may help inform the management of the sizable proportion of SCDS patients with bilateral defects.

Reduced B0/B1 + sensitivity in velocity-selective inversion arterial spin labeling using adiabatic refocusing pulses.

PURPOSE: To mitigate the B0/B1 + sensitivity of velocity-selective inversion (VSI) pulse trains for velocity-selective arterial spin labeling (VSASL) by implementing adiabatic refocusing. This approach aims to achieve artifact-free VSI-based perfusion imaging through single-pair label-control subtractions, reducing the need for the currently required four-pair dynamic phase-cycling (DPC) technique when using a velocity-insensitive control. METHODS: We introduce a Fourier-transform VSI (FT-VSI) train that incorporates sinc-modulated hard excitation pulses with MLEV-8-modulated adiabatic hyperbolic secant refocusing pairs. We compare performance between this train and the standard composite refocusing train, including with and without DPC, for dual-module VSI VSASL. We evaluate (1) simulated velocity-selective profiles and subtraction fidelity across a broad B0/B1 + range, (2) subtraction fidelity in phantoms, and (3) image quality, artifact presence, and gray-matter perfusion heterogeneity (as measured by the spatial coefficient of variation) in healthy human subjects. RESULTS: Adiabatic refocusing significantly improves FT-VSI robustness to B0/B1 + inhomogeneity for a single label-control subtraction. Subtraction fidelity is dramatically improved in both simulation and phantoms compared with composite refocusing without DPC, and is similar compared with DPC methods. In humans, marked artifacts seen with the non-DPC composite refocusing approach are eliminated, corroborated by significantly reduced gray-matter heterogeneity (via lower spatial coefficient of variation values). CONCLUSION: A novel VSASL labeling train using adiabatic refocusing pulses for VSI was found to reduce artifacts related to B0/B1 + inhomogeneity, thereby providing an alternative to DPC and its associated limitations, which include increased vulnerability to physiological noise and motion, reduced functional MRI applicability, and suboptimal data censoring.

Spaceflight-associated pain.

PURPOSE OF REVIEW: Consequences of the expanding commercial spaceflight industry include an increase in total number of spaceflight participants and an accompanying surge in the average number of medical comorbidities compared with government-based astronaut corps. A sequela of these developments is an anticipated rise in acute and chronic pain concerns associated with spaceflight. This review will summarize diagnostic and therapeutic areas of interest that can support the comfort of humans in spaceflight. RECENT FINDINGS: Painful conditions that occur in space may be due to exposure to numerous stressors such as acceleration and vibration during launch, trauma associated with extravehicular activities, and morbidity resulting directly from weightlessness. Without normal gravitational forces and biomechanical stress, the hostile environment of space causes muscle atrophy, bone demineralization, joint stiffness, and spinal disc dysfunction, resulting in a myriad of pain generators. Repeated insults from abnormal environmental exposures are thought to contribute to the development of painful musculoskeletal and neuropathic conditions. SUMMARY: As humanity invests in Lunar and Martian exploration, understanding the painful conditions that will impede crew productivity and mission outcomes is critical. Preexisting pain and new-onset acute or chronic pain resulting from spaceflight will require countermeasures and treatments to mitigate long-term health effects.

Partial wrapping of single-stranded DNA by replication protein A and modulation through phosphorylation.

Single-stranded DNA (ssDNA) intermediates which emerge during DNA metabolic processes are shielded by replication protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper to direct the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent functional specificity requires knowledge of the structural conformation of ssDNA when RPA-bound. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and double electron-electron resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

Tropism for ciliated cells is the dominant driver of influenza viral burst size in the human airway.

Influenza viruses pose a significant burden on global human health. Influenza has a broad cellular tropism in the airway, but how infection of different epithelial cell types impacts replication kinetics and burden in the airways is not fully understood. Using primary human airway cultures, which recapitulate the diverse epithelial cell landscape of the human airways, we investigated the impact of cell type composition on virus tropism and replication kinetics. Cultures were highly diverse across multiple donors and 30 independent differentiation conditions and supported a range of influenza replication. Although many cell types were susceptible to influenza, ciliated and secretory cells were predominantly infected. Despite the strong tropism preference for secretory and ciliated cells, which consistently make up 75% or more of infected cells, only ciliated cells were associated with increased virus production. Surprisingly, infected secretory cells were associated with overall reduced virus output. The disparate response and contribution to influenza virus production could be due to different pro- and antiviral interferon-stimulated gene signatures between ciliated and secretory populations, which were interrogated with single-cell RNA sequencing. These data highlight the heterogeneous outcomes of influenza virus infections in the complex cellular environment of the human airway and the disparate impacts of infected cell identity on multiround burst size, even among preferentially infected cell types.

Virtual Reality for Sub-Acute Pain After Orthopedic Traumatic Musculoskeletal Injuries: A Mixed Methods Pilot Study.

OBJECTIVES: Acute orthopedic traumatic musculoskeletal injuries are prevalent, costly, and often lead to persistent pain and functional limitations. Psychological risk factors (pain catastrophizing and anxiety) exacerbate these outcomes but are often overlooked in acute orthopedic care. Addressing gaps in current treatment approaches, this mixed methods pilot study explored the use of a therapeutic virtual reality (VR; RelieVRx), integrating mindfulness and cognitive behavioral therapy, for pain self-management at home following orthopedic injury. METHODS: We enrolled 10 adults with recent orthopedic injuries and elevated pain catastrophizing or pain anxiety from Level 1 Trauma Clinics within the Mass General Brigham healthcare system. Participants completed daily RelieVRx sessions at home for 8 weeks, which included pain education, relaxation, mindfulness, games, and dynamic breathing biofeedback. Primary outcomes were a-priori feasibility, appropriateness, acceptability, satisfaction, and safety. Secondary outcomes were pre-post measures of pain, physical function, sleep, depression, and mechanisms (pain self-efficacy, mindfulness, and coping). RESULTS: The VR and study procedures met or exceeded all benchmarks. We observed preliminary improvements in pain, physical functioning, sleep, depression, and mechanisms. Qualitative exit interviews confirmed high satisfaction with RelieVRx and yielded recommendations for promoting VR-based trials with orthopedic patients. DISCUSSION: The results support a larger randomized clinical trial of RelieVRx versus a sham placebo control to replicate the findings and explore mechanisms. There is potential for self-guided VR to promote evidence-based pain management strategies and address the critical mental health care gap for patients following acute orthopedic injuries.

Methionine Restriction Reduces Lung Cancer Progression and Increases Chemotherapy Response.

Targeting tumor metabolism through dietary interventions is an area of growing interest, and may help to improve the significant mortality of aggressive cancers, including non-small cell lung cancer (NSCLC). Here we show that the restriction of methionine in the aggressive KRAS/Lkb1-mutant NSCLC autochthonous mouse model drives decreased tumor progression and increased carboplatin treatment efficacy. Importantly, methionine restriction during early stages of tumorigenesis prevents the lineage switching known to occur in the model, and alters the tumor immune microenvironment (TIME) to have fewer tumor-infiltrating neutrophils. Mechanistically, mutations in LKB1 are linked to anti-oxidant production through changes to cystathionine-ß-synthase (CBS) expression. Human cell lines with rescued LKB1 show increased CBS levels and resistance to carboplatin, which can be partially rescued by methionine restriction. Furthermore, LKB1 rescued cells, but not mutant cells, show less G2-M arrest and apoptosis in high methionine conditions. Knock-down of CBS sensitized both LKB1 mutant and non-mutated lines to carboplatin, again rescuing the carboplatin resistance of the LKB1 rescued lines. Given that immunotherapy is commonly combined with chemotherapy for NSCLC, we next wanted to understand if T cells are impaired by MR. Therefore, we examined the ability of T cells from MR and control tumor bearing mice to proliferate in culture and found that T cells from MR treated mice had no defects in proliferation, even though we continued the MR conditions ex vivo. We also identified that CBS is most highly correlated with smoking, adenocarcinomas with alveolar and bronchiolar features, and adenosquamous cell carcinomas, implicating its roles in oxidative stress response and lineage fate in human tumors. Taken together, we have shown the importance of MR as a dietary intervention to slow tumor growth and improve treatment outcomes for NSCLC.