Disparate pathways for extrachromosomal DNA biogenesis and genomic DNA repair.

Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a CRISPR-based method for ecDNA induction with extensive characterization of newly formed ecDNA to examine their biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of 800kb, 1 Mb, and 1.8 Mb ecDNAs reaching or exceeding 15%. We show non-homologous end joining and microhomology-mediated end joining both contribute to ecDNA formation, while inhibition of DNA-PKcs and ATM have opposing impacts on ecDNA formation. EcDNA and the corresponding chromosomal excision scar can form at significantly different rates and respond differently to DNA-PKcs and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.

Hematopoietic stem cell transplantation for neuromyelitis optica spectrum disorder. Can immune tolerance be reestablished?

Neuromyelitis optica (NMO), which is also referred to as Devic's disease, was originally considered an aggressive subtype of multiple sclerosis (MS) presenting as optic neuritis and/or extensive transverse myelitis in which 50% of patients become blind or in a wheelchair within 5 years of onset. Subsequently, NMO was categorized as one of a spectrum of inflammatory and demyelinating autoimmune disorders that are distinct from multiple sclerosis and termed neuromyelitis optica spectrum disorder (NMOSD). NMOSD differs from multiple sclerosis by its clinical course, presentation, magnetic resonance imaging findings, clinical presentation, serum biomarker prognosis, and response to treatment. More recently, NMOSD has been subdivided according to auto-antibody status as aquaporin 4 (AQP4) seropositive NMO, myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD), and seronegative NMOSD. The only treatment to date that has resulted in treatment-free remissions, now lasting for more than 5-10 years with posttreatment disappearance of anti-AQP4 antibodies, is hematopoietic stem cell transplantation (HSCT) using either an allogeneic (matched sibling or unrelated) donor with a reduced toxicity conditioning regimen or an autologous stem cell source using a nonmyeloablative conditioning regimen of plasmapheresis (PLEX), cyclophosphamide (Cytoxan®), rabbit antithymocyte (ATG), and rituximab (Rituxan®). Post-HSCT long-term resolution of disease activity and disappearance of AQP4 antibodies is consistent with HSCT-induced immune tolerance.

Identification of glycosyltransferases mediating 2-O-arabinopyranosyl and 2-O-galactosyl substitutions of glucuronosyl side chains of xylan.

Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O-2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2-O-arabinopyranose (Arap) or 2-O-galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3-clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Arap from UDP-Arap onto O-2 of GlcA side chains of xylan, and thus it was named xylan 2-O-arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T-DNA knockout mutation showing a loss of the Arap substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus, lemon-scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2-O-Arap and 2-O-Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site-directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2-O-arabinopyranosylation and 2-O-galactosylation of GlcA side chains of xylan.

Early Adversity and Socioeconomic Factors in Pediatric Multiple Sclerosis: A Case-Control Study.

BACKGROUND AND OBJECTIVES: Psychosocial adversity and stress, known to predispose adults to neurodegenerative and inflammatory immune disorders, are widespread among children who experience socioeconomic disadvantage, and the associated neurotoxicity and proinflammatory profile may predispose these children to multiple sclerosis (MS). We sought to determine associations of socioeconomic disadvantage and psychosocial adversity with odds of pediatric-onset MS (POMS), age at POMS onset, and POMS disease activity. METHODS: This case-control study used data collected across 17 sites in the United States by the Environmental and Genetic Risk Factors for Pediatric Multiple Sclerosis Study. Cases (n = 381) were youth aged 3-21 years diagnosed with POMS or a clinically isolated demyelinating syndrome indicating high risk of MS. Frequency-matched controls (n = 611) aged 3-21 years were recruited from the same institutions. Prenatal and postnatal adversity and postnatal socioeconomic factors were assessed using retrospective questionnaires and zip code data. The primary outcome was MS diagnosis. Secondary outcomes were age at onset, relapse rate, and Expanded Disability Status Scale (EDSS). Predictors were maternal education, maternal prenatal stress events, child separation from caregivers during infancy and childhood, parental death during childhood, and childhood neighborhood disadvantage. RESULTS: MS cases (64% female, mean age 15.4 years, SD 2.8) were demographically similar to controls (60% female, mean age 14.9 years, SD 3.9). Cases were less likely to have a mother with a bachelor's degree or higher (OR 0.42, 95% CI 0.22-0.80, p = 0.009) and were more likely to experience childhood neighborhood disadvantage (OR 1.04 for each additional point on the neighborhood socioeconomic disadvantage score, 95% CI 1.00-1.07; p = 0.025). There were no associations of the socioeconomic variables with age at onset, relapse rate, or EDSS, or of prenatal or postnatal adverse events with risk of POMS, age at onset, relapse rate, or EDSS. DISCUSSION: Low socioeconomic status at the neighborhood level may increase the risk of POMS while high parental education may be protective against POMS. Although we did not find associations of other evaluated prenatal or postnatal adversities with POMS, future research should explore such associations further by assessing a broader range of stressful childhood experiences.

Predictors of a relapsing course in myelin oligodendrocyte glycoprotein antibody-associated disease.

BACKGROUND: Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) is a recently described demyelinating disorder, and children represent about 50% of all cases. Almost half of the patients experience relapses, but very few studies have evaluated predictors of relapse risk, challenging clinical management. The study aimed to identify predictors at MOGAD onset that are associated with a relapsing course. METHODS: Prospectively collected data from paediatric patients with MOGAD seen by the US Network of Paediatric MS Centres were leveraged. Univariable and adjusted multivariable models were used to predict recurrent disease. RESULTS: We identified 326 MOGAD cases (mean age at first event 8.9 years [SD 4.3], 57% female, 77% white and 74% non-Hispanic) and 46% relapsed during a mean follow-up of 3.9 years (SD 4.1). In the adjusted multivariable model, female sex (HR 1.66, 95% CI 1.17 to 2.36, p=0.004) and Hispanic/Latino ethnicity (HR 1.77, 95% CI 1.19 to 2.64, p=0.005) were associated with a higher risk of relapsing MOGAD. Maintenance treatment initiated before a second event with rituximab (HR 0.25, 95% CI 0.07 to 0.92, p=0.037) or intravenous immunoglobulin (IVIG) (HR 0.35, 95% CI 0.14 to 0.88, p=0.026) was associated with lower risk of a second event in multivariable analyses. Conversely, maintenance steroids were associated with a higher estimated relapse risk (HR 1.76, 95% CI 0.90 to 3.45, p=0.097). CONCLUSION: Sex and ethnicity are associated with relapsing MOGAD. Use of rituximab or IVIG therapy shortly after onset is associated with a lower risk of the second event. Preventive treatment after a first event could be considered for those with a higher relapse risk.

Prediction of joint moments from kinematics using machine learning in children with congenital talipes equino varus and typically developing peers.

Background: Understanding joint loading and the crucial role of joint moments is essential for developing treatment strategies in gait analysis, which often requires the precise estimation of joint moments through an inverse dynamic approach. This process necessitates the use of a force plate synchronized with a motion capture system. However, effectively capturing ground reaction force in typically developing (TD) children and those with congenital talipes equino varus (CTEV) presents challenges, while the availability and high cost of additional force plates pose additional challenges. Therefore the study aimed to develop, train, and identify the most effective machine learning (ML) model to predict joint moments from kinematics for TD children and those with CTEV. Method: In a study at the Gait Lab, 13 children with bilateral CTEV and 17 TD children underwent gait analysis to measure kinematics and kinetics, using a 12-camera Qualisys Motion Capture System and an AMTI force plate. ML models were then trained to predict joint moments from kinematic data as input. Results: The random forest regressor and deep neural networks (DNN) proved most effective in predicting joint moments from kinematics for TD children, yielding better results. The Random Forest regressor achieved an average r of 0.75 and nRMSE of 23.03 % for TD children, and r of 0.74 and 23.82 % for CTEV. DNN achieved an average r of 0.75 and nRMSE of 22.83 % for TD children, and r of 0.76 and nRMSE of 23.9 % for CTEV. Conclusions: The findings suggest that using machine learning to predict joint moments from kinematics shows moderate potential as an alternative to traditional gait analysis methods for both TD children and those with CTEV. Despite its potential, the current prediction accuracy limitations hinder the immediate clinical application of these techniques for decision-making in a pediatric population.

Exploring Disparities in Pancreatic Ductal Adenocarcinoma Outcomes among Asian and Pacific Islander Subgroups.

Pancreatic ductal adenocarcinoma (PDAC) is a challenging malignancy with known disparities in outcomes across ethnicities. Studies specifically investigating PDAC in Asian populations are sparse, overlooking the rich diversity within this group. This research seeks to fill that gap by examining survival differences across the broad spectrum of Asian ethnicities, acknowledging the complexity and varied experiences within these communities. Utilizing the National Cancer Database from 2004 to 2019, we categorized patients into East Asian, Southeast Asian, South Asian, and Pacific Islander groups. Non-Asians or Pacific Islanders were excluded. Overall survival was analyzed using a Cox hazards model. The study consisted of 13,254 patients. Most patients were East Asian (59.4%, n = 7,866). Southeast Asians exhibited the poorest survival in unadjusted analysis (HR, 1.32; 95% confidence interval, 1.23-1.42; P < 0.001) compared with South Asians who exhibited the best survival. Multivariable analysis revealed significantly worse survival for East Asians and Pacific Islanders relative to South Asians, whereas Southeast Asians' results were not significantly different. Asian subgroup differences notably affect PDAC outcomes. Research on genetic and cultural aspects, especially in Southeast Asians, and tackling health disparities are crucial for enhancing survival in this diverse disease. SIGNIFICANCE: This study highlights the significant survival disparities among Asian subgroups with pancreatic cancer, utilizing a large national database. By differentiating among East Asian, Southeast Asian, South Asian, and Pacific Islander groups, it underscores the need for tailored research and healthcare approaches. Addressing these differences is essential for developing culturally sensitive interventions and potentially improving outcomes in a disease that uniquely affects these diverse populations.

Clinical and magnetic resonance imaging outcomes in pediatric-onset MS patients on fingolimod and ocrelizumab.

BACKGROUND: Observational studies looking at clinical a++nd MRI outcomes of treatments in pediatric MS, could assess current treatment algorithms, and provide insights for designing future clinical trials. OBJECTIVE: To describe baseline characteristics and clinical and MRI outcomes in MS patients initiating ocrelizumab and fingolimod under 18 years of age. METHODS: MS patients seen at 12 centers of US Network of Pediatric MS were included in this study if they had clinical and MRI follow-up and started treatment with either ocrelizumab or fingolimod prior to the age of 18. RESULTS: Eighty-seven patients initiating fingolimod and 52 initiating ocrelizumab met the inclusion criteria. Before starting fingolimod, mean annualized relapse rate was 0.43 (95 % CI: 0.29 - 0.65) and 78 % developed new T2 lesions while during treatment it was 0.12 (95 % CI: 0.08 - 1.9) and 47 % developed new T2 lesions. In the ocrelizumab group, the mean annualized relapse rate prior to initiation of treatment was 0.64 (95 % CI: 0.38-1.09) and a total of 83 % of patients developed new T2 lesions while during treatment it was 0.09 (95 % CI: 0.04-0.21) and none developed new T2 lesions. CONCLUSION: This study highlights the importance of evaluating current treatment methods and provides insights about the agents in the ongoing phase III trial comparing fingolimod and ocrelizumab.

A new subjective well-being index using anchored best-worst scaling.

Subjective well-being (SWB) describes an individual's life evaluation. Direct elicitation methods for SWB via rating scales do not force individuals to trade-off among life domains, whilst best-worst scaling (BWS) approaches only provide relative measures. This paper instead offers a dual-response BWS task, where respondents nominate areas of most and least importance and satisfaction with respect to 11 SWB domains, whilst also eliciting anchoring points to obtain an absolute measure of domain satisfaction. Combining domain satisfaction and importance produces a robust measure of individual SWB, but statistically unique relative to other life satisfaction measures utilizing single- and multi-item ratings, including global satisfaction and those aggregated over SWB domains, as well as eudemonia. Surveying 2500 Australians reveals anchored-BWS improves discrimination amongst domains in terms of importance and satisfaction, illustrating its value as a diagnostic tool for SWB measurement to focus services, policy, and initiatives in areas to most impact wellbeing. This includes highlighting a major discrepancy between health satisfaction and importance, whilst also reporting that SWB is significantly lower for Indigenous, unemployed, middle-aged, males and lower income groups.

A rice GT61 glycosyltransferase possesses dual activities mediating 2-O-xylosyl and 2-O-arabinosyl substitutions of xylan.

MAIN CONCLUSION: A member of the rice GT61 clade B is capable of transferring both 2-O-xylosyl and 2-O-arabinosyl residues onto xylan and another member specifically catalyses addition of 2-O-xylosyl residue onto xylan. Grass xylan is substituted predominantly with 3-O-arabinofuranose (Araf) as well as with some minor side chains, such as 2-O-Araf and 2-O-(methyl)glucuronic acid [(Me)GlcA]. 3-O-Arabinosylation of grass xylan has been shown to be catalysed by grass-expanded clade A members of the glycosyltransferase family 61. However, glycosyltransferases mediating 2-O-arabinosylation of grass xylan remain elusive. Here, we performed biochemical studies of two rice GT61 clade B members and found that one of them was capable of transferring both xylosyl (Xyl) and Araf residues from UDP-Xyl and UDP-Araf, respectively, onto xylooligomer acceptors, whereas the other specifically catalysed Xyl transfer onto xylooligomers, indicating that the former is a xylan xylosyl/arabinosyl transferase (named OsXXAT1 herein) and the latter is a xylan xylosyltransferase (named OsXYXT2). Structural analysis of the OsXXAT1- and OsXYXT2-catalysed reaction products revealed that the Xyl and Araf residues were transferred onto O-2 positions of xylooligomers. Furthermore, we demonstrated that OsXXAT1 and OsXYXT2 were able to substitute acetylated xylooligomers, but only OsXXAT1 could xylosylate GlcA-substituted xylooligomers. OsXXAT1 and OsXYXT2 were predicted to adopt a GT-B fold structure and molecular docking revealed candidate amino acid residues at the predicted active site involved in binding of the nucleotide sugar donor and the xylohexaose acceptor substrates. Together, our results establish that OsXXAT1 is a xylan 2-O-xylosyl/2-O-arabinosyl transferase and OsXYXT2 is a xylan 2-O-xylosyltransferase, which expands our knowledge of roles of the GT61 family in grass xylan synthesis.

The smallest worthwhile effect on pain intensity of exercise therapy for people with chronic low back pain: a discrete choice experiment study.

OBJECTIVE: To identify the smallest worthwhile effect (SWE) of exercise therapy for people with non-specific chronic low back pain (CLBP). DESIGN: Discrete choice experiment. METHODS: The SWE was estimated as the lowest reduction in pain that participants would consider exercising worthwhile, compared to not exercising i.e., effects due to natural history and other components (e.g., regression to the mean). We recruited English-speaking adults in Australia with non-specific CLBP to our online survey via email obtained from a registry of previous participants and advertisements on social media. We used discrete choice experiment to estimate the SWE of exercise compared to no exercise for pain intensity. We analysed the discrete choice experiment using a mixed logit model, and mitigated hypothetical bias through certainty calibration, with sensitivity analyses performed with different certainty calibration thresholds. RESULTS: Two-hundred and thirteen participants completed the survey. The mean age (±SD) was 50.7±16.5, median (IQR) pain duration 10 years (5-20), and mean pain intensity (±SD) was 5.8±2.3 on a 0-10 numerical rating scale. For people with CLBP the SWE of exercise was a between-group reduction in pain of 20%, compared to no exercise. In the sensitivity analyses, the SWE varied with different levels of certainty calibration; from 0% without certainty calibration to 60% with more extreme certainty calibration. CONCLUSION: This patient-informed threshold of clinical importance could guide the interpretation of findings from randomised trials and meta-analyses of exercise therapy compared to no exercise.

Targeting IL-6 trans-signalling by sgp130Fc attenuates severity in SARS-CoV-2 -infected mice and reduces endotheliopathy.

BACKGROUND: SARS-CoV-2 infection is considered as a relapsing inflammatory process with a dysregulation of IL-6 signalling. Classic IL-6 signalling is thought to represent a defence mechanism against pathogens. In contrast, IL-6 trans-signalling has pro-inflammatory effects. In severe COVID-19, therapeutic strategies have focused on global inhibition of IL-6, with controversial results. We hypothesized that specific blockade of IL-6 trans-signalling could inhibit inflammatory response preserving the host defence activity inherent to IL-6 classic signalling. METHODS: To test the role of the specific IL-6 trans-signalling inhibition by sgp130Fc in short- and long-term consequences of COVID-19, we used the established K18-hACE2 transgenic mouse model. Histological as well as immunohistochemical analysis, and pro-inflammatory marker profiling were performed. To investigate IL-6 trans-signalling in human cells we used primary lung microvascular endothelial cells and fibroblasts in the presence/absence of sgp130Fc. FINDINGS: We report that targeting IL-6 trans-signalling by sgp130Fc attenuated SARS-CoV-2-related clinical symptoms and mortality. In surviving mice, the treatment caused a significant decrease in lung damage. In vitro, IL-6 trans-signalling induced strong and persisting JAK1/STAT3 activation in endothelial cells and lung fibroblasts with proinflammatory effects, which were attenuated by sgp130Fc. Our data also suggest that in those cells with scant amounts of IL-6R, the induction of gp130 and IL-6 by IL-6:sIL-6R complex sustains IL-6 trans-signalling. INTERPRETATION: IL-6 trans-signalling fosters progression of COVID-19, and suggests that specific blockade of this signalling mode could offer a promising alternative to mitigate both short- and long-term consequences without affecting the beneficial effects of IL-6 classic signalling. These results have implications for the development of new therapies of lung injury and endotheliopathy in COVID-19. FUNDING: The project was supported by ISCIII, Spain (COV-20/00792 to MB, PI23/01351 to MARH) and the European Commission-Next generation EU (European Union) (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global, SGL2103029 to MB). PID2019-110587RB-I00 (MB) supported by MICIN/AEI/10.13039/501100011033/and PID2022-143034OB-I00 (MB) by MICIN/AEI/10.13039/501100011033/FEDER. MAR-H acknowledges support from ISCIII, Spain and the European Commission-Next generation EU (European Union), through CSIC's Global Health PTI.

Is it resectable? A survey regarding tumor classification and vascular involvement.

INTRODUCTION: The anatomic location of the pancreas can result in involvement of major vasculature, which may act as a contraindication to resection. Several classification systems have been developed. We sought to discover the variations in the HPB community determining PDAC resectability. METHODS: The multiple-choice survey was distributed to all full members of the IHPBA. Questions were asked regarding demographics and clinical scenarios regarding tumor resectability. RESULTS: 164 responses were submitted. Most of the respondents were male and had been in practice for over 10 years. The median age range was 40-50 years old. Most practiced in either Asia (n = 57,35.9%), North America (n = 52,32.7%), or Europe (n = 32,20.1%). Classification systems used to determine resectability were: NCCN (n = 42,26.3%), JPS (n = 35,21.9%), International consensus (n = 33,20.6%), AHPBA/SSO (n = 23,14.4%), Alliance (n = 3,1.9%), and other/no-classification (n = 23,14.5%). There was significant variation in the frequency of the most common answer within the scenarios (84.7%-33.5%). Participant concordance with their stated classification system found a median rate of 62.5%. Participant decision of tumor resectability was not dependent on their adopted classification system. CONCLUSION: When classifying PDAC resectability, there is significant variation between surgeons as to how they would classify a specific tumour, independent of the classification system they use. In addition, surgeons do not show high concordance with the definitions within that classification system.

Activation of gp130 signaling in T cells drives TH17-mediated multi-organ autoimmunity.

The IL-6-gp130-STAT3 signaling axis is a major regulator of inflammation. Activating mutations in the gene encoding gp130 and germline gain-of-function mutations in STAT3 (STAT3GOF) are associated with multi-organ autoimmunity, severe morbidity, and adverse prognosis. To dissect crucial cellular subsets and disease biology involved in activated gp130 signaling, the gp130-JAK-STAT3 axis was constitutively activated using a transgene, L-gp130, specifically targeted to T cells. Activating gp130 signaling in T cells in vivo resulted in fatal, early onset, multi-organ autoimmunity in mice that resembled human STAT3GOF disease. Female mice had more rapid disease progression than male mice. On a cellular level, gp130 signaling induced the activation and effector cell differentiation of T cells, promoted the expansion of T helper type 17 (TH17) cells, and impaired the activity of regulatory T cells. Transcriptomic profiling of CD4+ and CD8+ T cells from these mice revealed commonly dysregulated genes and a gene signature that, when applied to human transcriptomic data, improved the segregation of patients with transcriptionally diverse STAT3GOF mutations from healthy controls. The findings demonstrate that increased gp130-STAT3 signaling leads to TH17-driven autoimmunity that phenotypically resembles human STAT3GOF disease.

Functional donor-site morbidity following reconstruction with pectoralis major flaps: A systematic review.

Background: Pectoralis major muscle/myocutaneous flaps (PMMFs) are commonly used in reconstructive surgery, but may result in shoulder disability on the donor side. A systematic review evaluating this morbidity could be beneficial for guiding patients and providers considering this procedure. Methods: In October 2022, a systematic review of studies evaluating quantitative/qualitative measures of functional morbidity after PMMF was conducted. The results were categorized into PMMF's effect on range of motion (ROM), strength, and ability to complete shoulder-related activities/quality of life. Results: Eleven studies were included for analysis, which analyzed standard PMMF and two PMMF variants that spared portions of the muscle. Three of five studies demonstrated reduced shoulder ROM for standard PMMF versus controls lasting at least 4 months after head and neck reconstruction. Two of five studies, including two prospective studies demonstrated reduced shoulder strength for standard PMMF versus controls lasting at least 3 months after surgery. Five of nine studies found significant impairment in the ability to conduct shoulder-related activities, including work, up to one year postoperatively for standard PMMF versus controls. Muscle-sparing PMMF variants exhibited more promising outcomes in some categories. Conclusion: Standard PMMF results in prolonged reductions in shoulder ROM and strength, which may impair patients in shoulder-related activities. Other reconstructive options should be considered in patients who frequently participate in such activities. For patients requiring PMMF, muscle-sparing PMMF variants should be considered as alternatives to the standard PMMF.

Interaction between Permeation Enhancers and Lipid Bilayers.

Permeation enhancers (PEs) are a class of molecules that interact with the epithelial membrane and transiently increase its transcellular permeability. Although there have been few clinical trials of PE coformulated drugs, the mechanism of action of PEs remains elusive. In this paper, the interaction between two archetypes of PEs [salcaprozate sodium (SNAC) and sodium caprate (C10)] and membranes is investigated with extensive all-atom molecular dynamics simulations. The simulations show that (1) the association between the neutral PEs and membranes is favored in free energy, (2) the propensity of neutral PE aggregation is larger in aqueous solution than in lipid bilayers, (3) the equilibrium distribution of neutral PEs in membranes is fast, e.g., accessible with unbiased MD simulations, and (4) the micelle of neutral PEs formed in aqueous solution does not rupture the membranes (e.g., not forming pores or breaking up the membrane) under simulation conditions. All results combined, this study indicates that PEs insert into the membranes in an equilibrium or near equilibrium process. This study lays the foundation for future investigations of how PEs impact the free energy of permeation for small molecules.

Computationally designed sensors detect endogenous Ras activity and signaling effectors at subcellular resolution.

The utility of genetically encoded biosensors for sensing the activity of signaling proteins has been hampered by a lack of strategies for matching sensor sensitivity to the physiological concentration range of the target. Here we used computational protein design to generate intracellular sensors of Ras activity (LOCKR-based Sensor for Ras activity (Ras-LOCKR-S)) and proximity labelers of the Ras signaling environment (LOCKR-based, Ras activity-dependent Proximity Labeler (Ras-LOCKR-PL)). These tools allow the detection of endogenous Ras activity and labeling of the surrounding environment at subcellular resolution. Using these sensors in human cancer cell lines, we identified Ras-interacting proteins in oncogenic EML4-Alk granules and found that Src-Associated in Mitosis 68-kDa (SAM68) protein specifically enhances Ras activity in the granules. The ability to subcellularly localize endogenous Ras activity should deepen our understanding of Ras function in health and disease and may suggest potential therapeutic strategies.

Measuring the Effect of Off-Balancing Vectors on the Delivery of High-Quality CPR during Ambulance Transport: A Proof of Concept Study.

AIM: This study aims to demonstrate the feasibility of quantifying the off-balancing vectors experienced during ambulance transport and comparing them to high-quality cardiopulmonary resuscitation (HQ-CPR) metrics. METHODS: Ten participants completed a total of 20 evolutions of compression-only HQ-CPR in an ambulance driven in a manner that minimized or increased linear and angular off-balancing vectors. Linear and angular velocity, linear and angular acceleration, and linear jerk were recorded. HQ-CPR variables measured were compression fraction and proportion of compressions with depth >5 cm (depth%), rate 100-120 (rate%), full chest recoil (recoil%), and hand position (hand%). A composite score was calculated: [(depth% + rate% + recoil% + hand%)/4) * compression fraction]. Difficulty of HQ-CPR performance was measured with the Borg rating of perceived exertion (RPE) Scale. A series of mixed effects models were fitted regressing each HQ-CPR metric on each off-balancing vector. RESULTS: HQ-CPR data and vector quantity data were successfully recorded in all evolutions. Rate% was negatively associated with increasing linear velocity (slope = -3.82, standard error [SE] 1.12, p = 0.005), linear acceleration (slope = -5.52, SE 1.93, p = 0.013), linear jerk (slope = -17.60, SE 5.78, p = 0.007), angular velocity (slope = -75.74, SE 22.72, p = 0.004), and angular acceleration (slope = -152.53, SE 59.60, p = 0.022). Compression fraction was negatively associated with increasing linear velocity (slope = -1.35, SE 0.37, p = 0.004), linear acceleration (slope = -1.67, SE 0.48, p = 0.003), linear jerk (slope = -4.90, SE 1.86, p = 0.018), angular velocity (slope = -25.66, SE 6.49, p = 0.001), and angular acceleration (slope = -45.35, SE 18.91, p = 0.031). Recoil% was negatively associated with increasing linear velocity (slope = -5.80, SE 2.21, p = 0.023) and angular velocity (slope = -116.96, SE 44.24, p = 0.019)). Composite score was negatively associated with increasing linear velocity (slope = -4.49, SE 1.45, p = 0.009) and angular velocity (slope = -86.13, SE 31.24, p = 0.014) and approached a negative association with increasing magnitudes of linear acceleration (slope -5.54, SE 2.93, p = 0.075), linear jerk (slope = -17.43, SE 8.80, p = 0.064), and angular acceleration (slope = -170.43, SE 80.73, p = 0.051). Borg RPE scale was positively associated with all off-balancing vectors. Depth%, hand%, mean compression depth, and mean compression rate were not correlated with any off-balancing vector. CONCLUSION: Off-balancing vector data can be successfully quantified during ambulance transport and compared with HQ-CPR performance parameters. Increasing off-balancing vectors experienced during ambulance transport are associated with worse HQ-CPR metrics and increased perceived physical exertion. These data may help guide future drive styles, ambulance design, or use of mechanical CPR devices to improve HQ-CPR delivery during selected patient transport scenarios.

Gene-environment interactions and risk of pediatric-onset multiple sclerosis associated with time spent outdoors.

BACKGROUND: Our previous study identified a significant association between lower time spent outdoors, as a proxy of sun exposure, and a higher risk of pediatric-onset multiple sclerosis (POMS). UV radiation modulates the expression of several genes, but it is unknown whether these genes modify the effect of sun exposure on POMS risk. METHODS: In an age- and sex-matched case-control study, we evaluated the additive and multiplicative interactions between time spent outdoors and genetic non-HLA risk variants for developing POMS within the metabolic pathways of UV radiation, including CD28(rs6435203), CD86(rs9282641), and NFkB1(rs7665090) and the top two HLA risk factors (presence of DRB1×15 and absence of A*02). RESULTS: In an adjusted model (332 POMS cases, 534 healthy controls), greater time compared to <30 min/day spent outdoors during the prior summer and higher UV radiation dose were associated with decreased odds of POMS (OR 0.66, 95% CI 0.56-0.78, p < 0.001; OR 0.78, 95 % CI 0.62-0.98, p = 0.04, respectively). No significant additive or multiplicative interactions were found between risk factors. CONCLUSIONS: The exploration of gene-environment interactions in the risk of developing MS can unravel the underlying mechanisms involved. Although we do not have evidence that our candidate genes contribute to interactions, other genes may.