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The Morelos district, located in the center of the Guerrero gold belt, Guerrero State, southern Mexico, has been the site of several recent gold ± copper skarn discoveries (total of ~8.5 Moz Au) associated with Upper Cretaceous-Paleocene granodiorite intrusions. The Limon, Guajes, and Media Luna skarn deposits developed along the contact of the El Limon granodiorite and the sedimentary host rocks. Native gold is associated with retrograde alteration of garnet-pyroxene skarn and is accompanied by native bismuth as well as telluride and sulfide minerals. The Media Luna deposit is characterized by a distinct magnesium-rich skarn mineralogy and contains an average of ~1 wt % Cu, in addition to gold and silver. This distinguishes it from the other gold-only skarn deposits in the Guerrero gold belt. This observation suggests that skarns in the Guerrero gold belt, which replaced magnesium-rich sedimentary protoliths, may host significant copper resources in addition to gold. Uranium-lead laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) zircon ages of the El Limon granodiorite range from 66.86 ± 0.40 to 65.88 ± 0.79 Ma. Preskarn molybdenite-quartz veins cut through the El Limon granodiorite and yield Re-Os molybdenite ages between 66.63 ± 0.22 and 65.55 ± 0.25 Ma. A series of pre-, syn-, and postskarn porphyritic dikes intruded the El Limon granodiorite, having zircon ages ranging from 65.9 ± 1.1 to 64.68 ± 0.38 Ma. The timing of skarn formation at Limon and Media Luna was constrained to 64.6 ± 1.2 to 63.2 ± 1.0 Ma, applying U-Pb LA-ICP-MS to garnet. The ubiquitous presence of garnet in skarn deposits and the time- and cost-efficiency of in situ geochronology are striking advantages of this method to constrain the timing of mineralization.

Objectives The United States has seen increasing shifts toward home- and community-based services (HCBS) in place of institutional care for long-term services and supports. However, research has neglected to assess whether these shifts have improved access to HCBS for persons with dementia. This paper identifies HCBS access barriers and facilitators, and discusses how barriers contribute to disparities for persons with dementia living in rural areas and exacerbate disparities for minoritized populations. Methods We analyzed qualitative data from 35 in-depth interviews. Interviews were held with stakeholders in the HCBS ecosystem, including Medicaid administrators, advocates for persons with dementia and caregivers, and HCBS providers. Results Barriers to HCBS access for persons with dementia range from community and infrastructure barriers (e.g., clinicians and cultural differences), to interpersonal and individual-level barriers (e.g., caregivers, awareness, and attitudes). These barriers affect the health and quality of life for persons with dementia and may affect whether individuals can remain in their home or community. Facilitators included a range of more comprehensive and dementia-attuned practices and services in health care, technology, recognition and support for family caregivers, and culturally competent and linguistically accessible education and services. Discussion System refinements, such as incentivizing cognitive screening, can improve detection and increase access to HCBS. Disparities in HCBS access experienced by minoritized persons with dementia may be addressed through culturally competent awareness campaigns and policies that recognize the necessity of familial caregivers in supporting persons with dementia. These findings can inform efforts to ensure more equitable access to HCBS, improve dementia competence, and reduce disparities.

Objectives The Behavioral Risk Factor Surveillance System sampled 54,076 caregivers between 2015 and 2017 providing an opportunity to evaluate risk factors for poor mental and physical health among a representative sample of U.S. adult caregivers. This study aimed to evaluate the impact of childcare, work status, and intensity of caregiving among men and women caring for older adults (n = 17,271). Methods Controlling for sociodemographic factors, separate logistic regression analysis for women and men were carried out to assess the main and interaction effects of childcare, work status, and intensity of caregiving on number of poor mental and physical health days in the last month. Results Intensive caregiving demands had adverse effects on both women and men, but being in the workforce was beneficial to both men and women. Women with children at home reported adverse mental health effects but better physical health, while men with children at home reported adverse physical health effects. For women, the combination of not working, children in the household, and high-intensity caregiving were most detrimental to their mental health. Among men, those not working with children in the household, regardless of caregiving intensity, were at highest risk of adverse mental health effects. Discussion Our findings identify caregivers at high risk of adverse outcomes but also point to the need for more fine-grained analyses of how families negotiate the allocation of childcare, work, and caregiving responsibilities over time.

Estimations of the reproducibility of U–Pb ages from SHRIMP (Sensitive High-Resolution Ion MicroProbe) instruments are based on data from studies that are nearly 2 decades old. Since that time, refinement of analytical procedures and operational improvements have reduced the historically identified uncertainties of SHRIMP U–Pb analysis. This paper investigates 36 SHRIMP thermal ionisation mass spectrometry (TIMS) double-dated “real-world” geologic samples from a variety of igneous rock types to better understand both geological and analytical sources of disagreement between the two dating methods. Geoscience Australia's (GA) use of high-precision chemical abrasion thermal ionisation mass spectrometry (CA-TIMS) for chronostratigraphy in Australian sedimentary basins has produced a substantial selection of precisely dated zircons, which we can use to cross-correlate the SHRIMP and CA-TIMS ages throughout the Phanerozoic. A total of 33 of the 36 ages were reported with external SHRIMP uncertainties less than 1 % (95 % confidence). Six of eight cases where the CA-TIMS age was outside the SHRIMP uncertainty envelope were in samples where the 95 % confidence interval of the reported SHRIMP age was below 0.66 % uncertainty, suggesting that SHRIMP analyses of untreated zircon with smaller uncertainties are probably overoptimistic. The mean age offset between SHRIMP and TIMS ages is 0.095 %, but the distribution appears bimodal. Geological explanations for age discrepancies between SHRIMP and CA-TIMS are suggested by considering intrusive and extrusive age results separately. All but one sample where the SHRIMP age is more than 0.25 % older are volcanic. This offset could be explained by the better single-grain age resolution of TIMS, allowing identification and exclusion of antecrysts from the eruptive population, while SHRIMP does not have a sufficient single-grain precision to deconvolve these populations – leading to an apparent older SHRIMP age. In contrast, SHRIMP ages from plutonic rocks – particularly plutonic rocks from the early Paleozoic – are typically younger than the CA-TIMS ages from the same samples, most likely reflecting Pb loss from non-chemically abraded SHRIMP zircons, while chemical abrasion of zircons prior to TIMS analysis destroyed or corrected these areas of Pb loss.

Running is an increasingly popular sport and form of exercise. Because of the importance of the hip in the biomechanics involved with running, forming the primary connection between the axial and appendicular skeleton of the lower extremities, accurate diagnosis and reporting of hip pathology are vital for appropriate management. This review provides an overview of the most common hip pathologies and injuries encountered in runners. Radiologic studies, primarily conventional radiography and magnetic resonance imaging (MRI) provide useful diagnostic information and should be used in combination with clinical findings to help guide therapeutic management.

This commentary honors Sharon Dunwoody’s scholarship and collaboration by focusing on two co-edited books on science communication. It explores the development of these two seminal publications that influenced the early development of the science communication field. In addition to placing the books in their respective time periods, the commentary highlights Dunwoody’s specific contributions as described by her two co-editors and long-time collaborators.

Lack of training for school clinicians in evidence-based practices (EBPs) contributes to underutilization of such services for youth with attention-deficit/hyperactivity disorder (ADHD). Advances in web-based technology and videoconferencing have allowed for expanded access to and optimization of training. We describe the development and outcomes of a novel web-based platform for training school clinicians to gain skills in EBPs for school-age youth with ADHD. The training platform is adapted from an empirically supported, in-person training for a school–home behavioral intervention (Collaborative Life Skills program) and includes skill modules for working with teachers, parents, and students. Training methods include web-accessed manuals/handouts, skill example video clips, automated progress monitoring tools, and consultation/in-session coaching via videoconferencing. We gathered stakeholder qualitative and quantitative feedback during discovery and design phases of the iterative development. We then evaluated the usability, acceptability, fidelity and clinician and student outcomes of the remote training program. Focus group themes and qualitative feedback identified clinician preferences for remote training features (e.g., interactive, brief, role-plays/coaching methods), video tools (recorded samples of skills and therapy sessions), and progress monitoring tools (e.g., clear, easy to use). Clinician usability ratings of the platform were high with most components rated as moderately to very useful/easy to use. Clinician ratings of usability, fidelity implementing the treatment, and their EBP knowledge and confidence following training were favorable. Student’s outcomes were similar to those achieved in prior studies of clinician in-person training. Results support the promise of remote, web-based clinician training for the dissemination of evidence-based practices.

In eyes with diabetic macular edema, the relative efficacy of administering aflibercept monotherapy as compared with bevacizumab first with a switch to aflibercept if the eye condition does not improve sufficiently (a form of step therapy) is unclear.At 54 clinical sites, we randomly assigned eyes in adults who had diabetic macular edema involving the macular center and a visual-acuity letter score of 24 to 69 (on a scale from 0 to 100, with higher scores indicating better visual acuity; Snellen equivalent, 20/320 to 20/50) to receive either 2.0 mg of intravitreous aflibercept or 1.25 mg of intravitreous bevacizumab. The drug was administered at randomization and thereafter according to the prespecified retreatment protocol. Beginning at 12 weeks, eyes in the bevacizumab-first group were switched to aflibercept therapy if protocol-specified criteria were met. The primary outcome was the mean change in visual acuity over the 2-year trial period. Retinal central subfield thickness and visual acuity at 2 years and safety were also assessed.A total of 312 eyes (in 270 adults) underwent randomization; 158 eyes were assigned to receive aflibercept monotherapy and 154 to receive bevacizumab first. Over the 2-year period, 70% of the eyes in the bevacizumab-first group were switched to aflibercept therapy. The mean improvement in visual acuity was 15.0 letters in the aflibercept-monotherapy group and 14.0 letters in the bevacizumab-first group (adjusted difference, 0.8 letters; 95% confidence interval, -0.9 to 2.5; P = 0.37). At 2 years, the mean changes in visual acuity and retinal central subfield thickness were similar in the two groups. Serious adverse events (in 52% of the patients in the aflibercept-monotherapy group and in 36% of those in the bevacizumab-first group) and hospitalizations for adverse events (in 48% and 32%, respectively) were more common in the aflibercept-monotherapy group.In this trial of treatment of moderate vision loss due to diabetic macular edema involving the center of the macula, we found no evidence of a significant difference in visual outcomes over a 2-year period between aflibercept monotherapy and treatment with bevacizumab first with a switch to aflibercept in the case of suboptimal response. (Funded by the National Institutes of Health; Protocol AC ClinicalTrials.gov number, NCT03321513.).

Infections induce a set of pleiotropic responses in animals, including anorexia, adipsia, lethargy and changes in temperature, collectively termed sickness behaviours1. Although these responses have been shown to be adaptive, the underlying neural mechanisms have not been elucidated2–4. Here we use of a set of unbiased methodologies to show that a specific subpopulation of neurons in the brainstem can control the diverse responses to a bacterial endotoxin (lipopolysaccharide (LPS)) that potently induces sickness behaviour. Whole-brain activity mapping revealed that subsets of neurons in the nucleus of the solitary tract (NTS) and the area postrema (AP) acutely express FOS after LPS treatment, and we found that subsequent reactivation of these specific neurons in FOS2A-iCreERT2 (also known as TRAP2) mice replicates the behavioural and thermal component of sickness. In addition, inhibition of LPS-activated neurons diminished all of the behavioural responses to LPS. Single-nucleus RNA sequencing of the NTS–AP was used to identify LPS-activated neural populations, and we found that activation of ADCYAP1+ neurons in the NTS–AP fully recapitulates the responses elicited by LPS. Furthermore, inhibition of these neurons significantly diminished the anorexia, adipsia and locomotor cessation seen after LPS injection. Together these studies map the pleiotropic effects of LPS to a neural population that is both necessary and sufficient for canonical elements of the sickness response, thus establishing a critical link between the brain and the response to infection.

The uptake of topical formulations can be enhanced through laser pretreatments. Newer technologies, such as non-ablative fractional lasers (NAFLs), can target dermal tissues and spare the stratum corneum (SC), which can mitigate common side effects associated with ablative laser treatment.To discuss the parameters of 2 NAFL pretreatment systems (1440-nm/1927-nm diode system or 1550-nm erbium-doped glass/1927-nm thulium system), which can be paired with topicals to improve their cutaneous uptake.The parameters of the 1440-nm/1927-nm diode system and 1550-nm erbium-doped glass/1927-nm thulium system were reviewed, as well as in vivo experiments using both laser systems.Multiple parameters should be optimized to enhance the uptake of various topical formulations and minimize side effects, including wavelength, spot size, density, pulse duration, thermal properties, and topical formulation. Chosen therapeutic parameters must account for patient-specific factors, such as treatment indication, therapeutic area, and Fitzpatrick skin type. In vivo experiments have demonstrated that both laser systems are effective in enhancing the uptake of various topical therapies relative to untreated controls.Individualization of laser system parameters increases efficiency and minimizes risk.

Background Providing continuous health insurance coverage during the perinatal period may increase access to and utilization of labor neuraxial analgesia. This study tested the hypothesis that implementation of the 2010 Dependent Coverage Provision of the Patient Protection and Affordable Care Act, requiring private health insurers to allow young adults to remain on their parent’s plan until age 26, was associated with increased labor neuraxial analgesia use. Methods This study used a natural experiment design and birth certificate data for spontaneous vaginal deliveries in 28 US states between 2009 and 2013. The intervention was the Dependent Coverage Provision, categorized into pre- and post-intervention periods (January 2009-August 2010 and September 2010-December 2013, respectively). The exposure was women age, categorized as exposed (21 to 25 years) and unexposed (27 to 31 years). The outcome was the labor neuraxial analgesia utilization rate. Results Of the 4,515,667 birth certificates analyzed, 3,033,129 (67.2%) indicated labor neuraxial analgesia use. For women aged 21 to 25 years, labor neuraxial analgesia utilization rates were 64.9% during the pre-intervention period and 68.9% during the post-intervention period (difference: 4.0%; 95% CI: 3.9, 4.2). For women aged 27 to 31 years, labor neuraxial analgesia utilization rates were 64.9% during the pre-intervention period and 67.7% during the post-intervention period (difference: 2.8%; 95% CI: 2.7, 2.9). After adjustment, implementation of the Dependent Coverage Provision was associated with a 1.0% (95% CI: 0.8, 1.2) absolute increase in labor neuraxial analgesia utilization rate among women aged 21 to 25 years compared with women aged 27 to 31 years. The increase was statistically significant for White and Hispanic women but not for Black and Other race and ethnicity women. Conclusions Implementation of the Dependent Coverage Provision was associated with a statistically significant increase in labor neuraxial analgesia use, but the small effect size unlikely of clinical significance.

Venous thromboembolism (VTE) is a significant cause of morbidity and mortality among patients with inflammatory bowel disease (IBD). However, data on national trends remain limited.To assess national trends in VTE-associated hospitalisations among patients with IBD as well as risk factors for, and mortality associated with, these events METHODS: Using the U.S. Nationwide Inpatient Sample from 2000-2018, temporal trends in VTE were assessed using the National Cancer Institute's Joinpoint Regression Program with estimates presented as the average annual percent change (AAPC) with 95% confidence intervals (CIs).Between 2000 and 2018, there were 4,859,728 hospitalisations among patients with IBD, with 128,236 (2.6%) having a VTE, and 6352 associated deaths. The rate of VTE among hospitalised patients with IBD increased from 192 to 295 cases per 10,000 hospitalisations (AAPC 2.4%, 95%CI 1.4%, 3.4%, p 0.001), and remained significant when stratified by ulcerative colitis (UC) and Crohn's disease as well as by deep vein thrombosis and pulmonary embolism. On multivariable analysis, increasing age, male sex, UC (aOR: 1.30, 95%CI 1.26, 1.33), identifying as non-Hispanic Black, and chronic corticosteroid use (aOR: 1.22, 95%CI 1.16, 1.29) were associated with an increased risk of a VTE-associated hospitalisation.Rates of VTE-associated hospitalisations are increasing among patients with IBD. Continued efforts need to be placed on education and risk reduction.

The understanding of the neural control of appetite sheds light on the pathogenesis of eating disorders such as anorexia nervosa and obesity. Both diseases are a result of maladaptive eating behaviors (overeating or undereating) and are associated with life-threatening health problems. The fine regulation of appetite involves genetic, physiological, and environmental factors, which are detected and integrated in the brain by specific neuronal populations. For centuries, the hypothalamus has been the center of attention in the scientific community as a key regulator of appetite. The hypothalamus receives and sends axonal projections to several other brain regions that are important for the integration of sensory and emotional information. These connections ensure that appropriate behavioral decisions are made depending on the individual’s emotional state and environment. Thus, the mechanisms by which higher-order brain regions integrate exteroceptive information to coordinate feeding is of great importance. In this review, we will focus on the functional and anatomical projections connecting the hypothalamus to the limbic system and higher-order brain centers in the cortex. We will also address the mechanisms by which specific neuronal populations located in higher-order centers regulate appetite and how maladaptive eating behaviors might arise from altered connections among cortical and subcortical areas with the hypothalamus.

Objective To determine whether infertility diagnoses differ between Black ethnic subgroups. Design Retrospective review Setting An urban safety net hospital. Patients Women seeking infertility care between 2005-2015. Interventions Charts of women with infertility and PCOS ICD-9 diagnoses were reviewed to confirm diagnoses. Data was stratified by race and subsequently by ethnicity to evaluate differences in infertility diagnoses between Black American, Black Haitian, and Black African women. White American women were used as the comparison group. Main Outcome Measures Infertility diagnoses between Black ethnic subgroups and White women. Results A total of 358 women met inclusion criteria including 99 Black American, 110 Black Haitian, 61 Black African, and 88 White American women. Anovulation/polycystic ovarian syndrome (PCOS) was the most common diagnosis in each ethnic group, accounting for 40% of infertility among White American, 57% among Black American, 25% among Black Haitian, and 21% among Black African women. There were no significant differences in individual infertility diagnoses between Black and White women. Between ethnic subgroups, multivariate analysis showed significantly higher odds of infertility due to anovulation/PCOS in Black American women compared to Black African women (odds ratio [OR]=4.9; 95% CI=1.4-17.0). Compared to Black African women, higher odds of tubal factor infertility were observed in Black American (OR=4.7; 95% CI=1.16-18.7) and Black Haitian women (OR=4.0; 95% CI=1.1-14.0). Conclusions Infertility diagnoses were not homogeneous across Black ethnic groups. Studies examining infertility should specify ethnic subgroups within race as this may affect results.

The rapid development of 2 nucleoside-modified mRNA vaccines that are safe and highly effective against coronavirus disease 2019 has transformed the vaccine field. The mRNA technology has the advantage of accelerated immunogen discovery, induction of robust immune responses, and rapid scale up of manufacturing. Efforts to develop genital herpes vaccines have been ongoing for 8 decades without success. The advent of mRNA technology has the potential to change that narrative. Developing a genital herpes vaccine is a high public health priority. A prophylactic genital herpes vaccine should prevent HSV-1 and HSV-2 genital lesions and infection of dorsal root ganglia, the site of latency. Vaccine immunity should be durable for decades, perhaps with the assistance of booster doses. While these goals have been elusive, new efforts with nucleoside-modified mRNA-lipid nanoparticle vaccines show great promise. We review past approaches to vaccine development that were unsuccessful or partially successful in large phase 3 trials, and describe lessons learned from these trials. We discuss our trivalent mRNA-lipid nanoparticle approach for a prophylactic genital herpes vaccine and the ability of the vaccine to induce higher titers of neutralizing antibodies and more durable CD4+ T follicular helper cell and memory B cell responses than protein-adjuvanted vaccines.

It is imperative for health care organizations to foster leadership skills in their workforce. Leadership development programs offer a potential mechanism to achieve this goal. These development programs are likely not equally effective for all participants. This study evaluates the efficacy of one such program and determines personality predictors of its efficacy. Before and after a 12-month leadership development program, 28 physicians from various disciplines completed self-reported measures of leadership knowledge across 3 domains. At baseline, participants also provided personality data across the Big-5 factors of personality as well as 2 narrow facets (learning-goal orientation and preference for collaboration). Results suggest that leadership development programs can increase knowledge across leadership domains. Extraversion and conscientiousness predict changes in knowledge. Learning-goal-orientation and preference for collaboration personality facets provide incremental predictive power. Leadership development programs can improve self-rated knowledge across a range of leadership domains and is differentially effective for people based on their personalities.

Objectives Recent national organ distribution rule changes could have implications on distance between donor and recipient hospitals and cold ischemia time. With cold ischemia time being an unavoidable detriment to organ quality, any strategies that minimize it should maximize organ quality. This study evaluated the significance of the kidney allocation system and the Share 35 rule changes on kidney and liver transplant outcomes. Materials and methods This retrospective study included deceased liver and kidney donor and their recipient data from the Organ Procurement and Transplantation Network. Variables were analyzed using propensity score matching and Cox hazards model distance (from donor hospital to organ recovery center), and effects on survival outcomes of trans-planted livers and kidneys in the context of the recent rule changes were analyzed. Results Transplanted organs have significantly better outcomes when the distance is 0 miles versus median distances for locally transported organs of 18 and 22 miles for kidney and liver, respectively. Cold ischemia time, when corrected, accounts for this finding, thus suggesting that cold ischemia time is the factor most responsible for viability of a transplanted organ. This significance remains evident for liver transplants even after the Share 35 rule change but not for kidney transplants following the December 2014 kidney allocation system change. Conclusions Liver transplants showed a higher risk of lower viability with travel, and the Share 35 rule did not appear to change this result. Kidney transplant outcomes appear to have improved after the kidney allocation system change. Potential strategies for minimizing cold ischemia time and improving outcomes include more free-standing organ recovery centers in centralized locations.

To characterise inflammatory bowel disease (IBD) trends and associated risk during delivery hospitalisations.Cross-sectional.US delivery hospitalisations.Delivery hospitalisations in the 2000-2018 National Inpatient Sample.This study analysed a nationally representative hospital discharge database based on the presence of IBD. Temporal trends in IBD were analysed using joinpoint regression to estimate the average annual percent change (AAPC). IBD severity was characterised by the presence of diagnoses such as penetrating and stricturing disease and history of bowel resection. Risks for adverse outcomes were analysed based on presence of IBD. Poisson regression models were performed with unadjusted and adjusted risk ratios (aRR) as measures of effect.Prevalence of IBD and associated adverse outcomes.Of 73 109 790 delivery hospitalisations, 89 965 had a diagnosis of IBD. IBD rose from 0.06% in 2000 to 0.21% in 2018 (AAPC 7.3%, 95% CI 6.7-7.9%). Among deliveries with IBD, IBD severity diagnoses increased from 4.1% to 8.1% from 2000 to 2018. In adjusted analysis, IBD was associated with increased risk for preterm delivery (aRR 1.50, 95% CI 1.47-1.53), severe maternal morbidity (aRR 1.93, 95% CI 1.83-2.04), venous thrombo-embolism (aRR 2.76, 95% CI 2.39-3.18) and surgical injury during caesarean delivery hospitalisation (aRR 5.03, 95% CI 4.76-5.31). In the presence of a severe IBD diagnosis, risk was further increased for all adverse outcomes.IBD is increasing in the obstetric population and is associated with adverse outcomes. Risk is increased in the presence of a severe IBD diagnosis.Deliveries among women with inflammatory bowel disease are increasing. Disease severity is associated with adverse outcomes.

Genome-wide (exome or genome) sequencing (GWS) has the potential to detect incidental findings (IFs): variants unrelated to the primary indication for testing that may be of medical or personal utility. As GWS becomes increasingly common in clinical practice, it is important to understand the impact of IFs on the individuals and their families. Our goal was to explore the immediate and long-term lived experience of individuals who received IFs as part of diagnostic GWS. We interviewed parents who received an IF as part of the CAUSES translational research study at Children's and Women's Health Centre of British Columbia. Five hundred families were offered trio-based GWS for their child with a suspected, undiagnosed genetic condition. Nine hundred and one of the 1000 parents chose to find out about IFs and 21 parents received an IF for themselves. Twelve of these parents participated in this study. They were interviewed an average of 2.3 years after the IFs were returned. Thematic analysis of transcribed interviews revealed that the participants' decisions and motivations to receive IFs were influenced by personal values and beliefs and by having a child with a suspected genetic condition. Participants' experiences were also influenced by the type of IF received, having a personal or family history of a related condition, their personal interpretation and perceived utility of the information, and the impact of the IF on other family members. Participants expressed either no regret or mild decisional regret on the Decisional Regret Scale. Two years post results, most participants reported little negative impact from receiving the IF. The utility of the information varied: some reported lifestyle changes and proactive screening, while others felt the information may be more relevant in the future. Understanding the immediate and longer term impact of receiving IFs from GWS can inform both pre- and post-test genetic counseling.

Obesity is associated with increased morbidity and mortality from influenza and SARS-CoV-2 infection. While vaccination is the most effective strategy for preventing influenza virus infection, our previous studies showed that influenza vaccines fail to provide optimal protection in obese individuals despite reaching canonical correlates of protection.

We investigate some concrete independence results for systems of reverse mathematics which emerge from monotonicity properties of number-theoretic functions. Natural properties of the less than or equal to relation with respect to sums of natural numbers lead to independence results for first-order Peano arithmetic. Natural properties of the less than or equal to relation with respect to sums and products of natural numbers lead to independence results for arithmetical transfinite recursion. By considering number-theoretic functions of arbitrary arities, we obtain independence results for systems beyond arithmetical transfinite recursion. We discuss how these embeddability relations are related to tree embeddability relations and we consider variants where the tree embeddability relation is not assumed to preserve infima. The findings of this paper are complementary to results on Kruskal-like theorems proved earlier by the first author. This article is part of the theme issue ‘Modern perspectives in Proof Theory’.

Supplementary Figure 7: Schematic representation that summarizes the proposed mechanism of action of CBS9106.

Supplementary Figure 8: MLN4924 attenuates tumor growth inhibition by CBS9106 in human xenograft model. (A) SCID mice bearing HCT116 tumor xenografts are treated with vehicle (5% GA, p.o. + 10% HBC, s.c.), CBS9106 (250 mg/kg p.o. once per week), MLN4924 (10 or 60 mg/kg s.c. once per week), or combination of CBS9106 and MLN4924 (n = 6-8 mice/group) for two weeks. Data are expressed as the mean {plus minus} SEM. (B) Body weight change is measured during the treatment (means {plus minus} SEM). (C) Tumors are excised at 4 or 24 hours and the levels of Neddylated cullins and CRM1 are measured in the tumor lysates by western blot analysis (n = 3). Data are expressed as the mean {plus minus} SD. V:Vehicle.

Supplementary Figure 1: CBS9106 reduces CRM1 protein levels through neddylation -mediated degradation in MM.1S cells. (A) MM.1S cells are treated with CBS9106 (50 nM) and/or MLN4924 (100 nM) for indicated period (0-8 hours). (B) Cells are treated with increasing concentrations of MLN4924 (0, 10, 20, 50, or 100 nM) and/or CBS9106 (50 nM) for 24 hours. (A, B) Protein levels of CRM1, CyclinB1, CulNEDD8, p53, PARP, p27, CDT1, and beta-actin are determined by western blot analysis. The measured band density was normalized relative to that of the control sample, with the control value set to 1. MM.1S cells was kindly provided by Dr. H. Yasui (University of tokyo, Tokyo, Japan). Cleaved PARP:clPARP.

Supplementary Figure 6: Inhibitors of autophagy do not interfere with the efficacy of low dose MLN4924. HCT116 cells are treated with CBS9106 (100 nM), MLN4924 (50 nM or 1 microM) or 3-MA (5 mM) alone or in combination for 24 hours. Protein levels of CRM1, CulNEDD8, p53, HIF-1alpha, p70S6 Kinase (S6K), phospho-Thr389 p70S6 Kinase (p-S6K), LC3-I/II, p62, and beta-actin are determined by western blot analysis. The measured band density was normalized relative to that of the control sample, with the control value set to 1. Antibodies against HIF-1alpha (3716), S6K (2708), p-S6K (9234), or p62 (8025) were obtained from Cell Signaling Technology.

Supplementary Figure 3: The cell growth inhibition and apoptosis by CBS9106 is suppressed by MLN4924 in MM.1S cells. (A) MM.1S cells are treated with the indicated concentration of CBS9106 and/or MLN4924 for 72 hours (n = 6). Cell viability is determined by cell counting kit-8. (B) Cells are treated with CBS9106 (50 nM) and/or MLN4924 (10 or 100 nM) for indicated period (48-96 hours, n = 6). Cell growth is determined by cell counting kit-8. (C) The apoptotic population (indicated by sub-G1) is measured by flow cytometry after staining with propidium iodide. MM.1S cells are treated with CBS9106 (50 or 100 nM), LMB (1 nM), or MLN4924 (1, 2, 5, 10, or 50 nM) either alone or in combination for 24 hours (n = 3). Data are expressed as the mean {plus minus} SD. *P

Chromosome region maintenance 1 (CRM1) mediates the nuclear export of proteins and mRNAs, and is overexpressed in various cancers. Recent studies have also reported that CRM1 protein expression is a negative prognostic factor in patients with cancer. Therefore, CRM1 is considered a potential target for anticancer therapy. Our previous study demonstrated that CBS9106, a synthetic small-molecular inhibitor of CRM1, decreases CRM1 protein through proteasomal degradation without affecting CRM1 mRNA levels. However, the mechanism by which CRM1 is degraded is not well understood. Here, we demonstrate a novel signaling pathway that plays an important role in CBS9106-induced CRM1 degradation. We found that MLN4924, a selective inhibitor of NEDD8-activating enzyme (NAE), effectively inhibits cullin neddylation and attenuates CBS9106-induced CRM1 degradation in a time- and dose-dependent manner. MLN4924 also attenuated CBS9106-induced nuclear accumulation of Ran-binding protein 1 (RanBP1), cell growth inhibition, and apoptosis. Furthermore, RNAi-mediated knockdown of neddylation pathway proteins (NEDD8 and UBA3) or cullin ring ligase (CRL) component protein (Rbx1) attenuated CRM1 protein degradation and G1 phase cell-cycle arrest by CBS9106. Knockdown of CSN5 or CAND1 also partially inhibited CBS9106-induced CRM1 degradation. These findings demonstrate that CBS9106-induced CRM1 degradation is conferred by CRL activity involving the neddylation pathway, and that this response to CBS9106 leads to cell growth inhibition and apoptosis. Mol Cancer Ther; 13(12); 3013–23. ©2014 AACR.

Supplementary materials and methods and Supplementary figure legends