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RationaleCOPD can be assessed using multidimensional grading systems with components from three domains: pulmonary function tests, symptoms and systemic features. Clinically, measures may be used interchangeably, though it is not known if they share similar pathobiology.ObjectiveTo use RNA sequencing (RNA-seq) to determine if there is an overlap in the underlying biological mechanisms and consequences driving different components of the multidimensional grading systems.MethodsWhole blood was collected for RNA-seq from current and former smokers in the Genetic Epidemiology of COPD study. We tested the overlap in gene expression and biological pathways associated with case–control status and quantitative COPD phenotypes within and between the three domains.ResultsIn 2647 subjects, there were 3030 genes differentially expressed in any of the three domains or case–control status. There were five genes that overlapped between the three domains and case–control status, including G protein-coupled receptor 15(GPR15), sestrin 1 (SESN1) and interferon-induced guanylate-binding protein 1 (GBP1), which were associated with longitudinal decline in FEV1. The overlap between the three domains was enriched for pathways related to cellular components.ConclusionsWe identified gene sets and pathways that overlap between 12 COPD-related phenotypes and case–control status. There were no pathways represented in the overlap between the three domains and case–control status, but we identified multiple genes that demonstrated a consistent pattern of expression across several of the phenotypes. Patterns of gene expression correlation were generally similar to the correlation of clinical phenotypes in the PFT and symptom domains but not the systemic features.

Fetal perturbations in DNA methylation during lung development may reveal insights into the enduring impacts on adult lung health and disease during aging that have not been explored altogether before. We studied the association between genome-wide DNA-methylation and post-conception age in fetal-lung (n=78, 42 exposed to in-utero-smoke (IUS)) tissue and chronological age in adult-lung tissue (n=160, 114 with Chronic Obstructive Pulmonary Disease) using multi-variate linear regression models with covariate adjustment and tested for effect modification by phenotypes. Overlapping age-associations were evaluated for functional and tissue-specific enrichment using the Genotype-Tissue-Expression (GTEx) project. We identified 244 age-associated differentially methylated positions and 878 regions overlapping between fetal and adult-lung tissues. Hyper-methylated CpGs (96%) were enriched in transcription factor activity (FDR adjusted P=2x10-33) and implicated in developmental processes including embryonic organ morphogenesis, neurogenesis and growth delay. Hypo-methylated CpGs (2%) were enriched in oxido-reductase activity and VEGFA-VEGFR2 Signaling. Twenty-one age-by-sex and eleven age-by-pack-years interactions were statistically significant (FDR

Rationale: Smoking results in at least a decade lower life expectancy. Mortality among current smokers is two to three times as high as never smokers. DNA methylation is an epigenetic modification ...

A thorough knowledge of the anatomy of the deltoid and spring ligament complex is important for treatment of deformities that impact the foot and ankle. Both ligaments are interconnected, and the study of their anatomic characteristics is better performed together than in isolation. The deltoid ligament is a group of ligaments that derives its origin from the medial malleolus, and the spring ligament complex consist of a group of ligaments that connects the navicular and the sustentaculum tali of the calcaneus. They both play an important role in stabilization of the medial ankle and medial column of the foot.

Purpose This paper is a part of a series of papers seeking insight into a holistic perspective into the lives, experiences and vulnerabilities of male-to-female transgender persons (from here on referred to as “transgender persons”/“Ladyboys”) within the sex industry in Southeast Asia. “Ladyboy” in Thai context specifically refers to the cultural subgroup, rather than the person’s gender identity and is not seen as an offensive term. Among the minimal studies that have been conducted, the majority have focused on sexual health and the likelihood of contracting or spreading HIV/AIDS, while often ignoring the possibility of other vulnerabilities. The paper aims to discuss this issue. Design/methodology/approach The study interviews 60 transgender persons working within red light areas of Bangkok. The final research instrument was a questionnaire of 11 sub-themes, containing both multiple choice and open-ended questions. Findings This study found that 81 percent of participants had entered the sex industry due to financial necessity. There was also a high vulnerability among transgender sex workers to physical and sexual violence. This includes nearly a quarter (24 percent) who cite being forced to have sex and 26 percent who cite physical assault within the last 12 months. Social implications These findings can aid the development of programs and social services that address the needs of ladyboys, looking beyond gender expression and social identity to meet needs and vulnerabilities that often go overlooked. Originality/value This survey provides deeper understanding of the vulnerability of transgender sex workers, including their trajectory into sex work and potential alternatives.

Purpose: This research investigated the presence and integrity of the opioid growth factor (OGF)—opioid growth factor receptor (OGFr) regulatory pathway in type 1 diabetic (T1D) rats, and i...

Mutations in Serpinf1 gene which encodes pigment epithelium-derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective matrix mineralization. We reported previously that PEDF reduced expression and synthesis of Sost/Sclerostin as well as other osteocytes genes encoding proteins that regulate matrix mineralization [1]. To determine whether PEDF had an effect on osteocyte gene expression in bone, we used bone explant cultures. First, osteocytes were isolated from surgical waste of bone fragments obtained from patients undergoing elective foot surgeries under approved IRB protocol by Penn State College of Medicine IRB committee. Primary osteocytes treated with PEDF reduced expression and synthesis of Sost/Sclerostin and matrix phosphoglycoprotein (MEPE) as well as dentin matrix protein (DMP-1). On the whole, PEDF reduced osteocyte protein synthesis by 50% and by 75% on mRNA levels. For bone explants, following collagenase digestion, bone fragments were incubated in alpha-MEM supplemented with 250 ng/ml of PEDF or BSA. After 7 days of incubation in a medium supplemented with PEDF, analysis of mRNA by PCR and protein by western blotting of encoded osteocyte proteins showed reduced Sclerostin synthesis by 39% and MEPE by 27% when compared to fragments incubated in medium supplemented with BSA. mRNA expression levels of osteocytes in bone fragments treated with PEDF were reduced by 50% for both SOST and MEPE when compared to BSA-treated bone fragments. Taken together, the data indicate that PEDF has an effect on osteocyte gene expression in bone and encourage further studies to examine effect of PEDF on bone formation indices in animal models and its effect on osteocyte gene expression in vivo following PEDF administration.

Background: Stroke alert systems provide rapid evaluation of suspected strokes to aid timely thrombolysis and mechanical thrombectomy. Given consequences of delayed or missed diagnosis, these systems are intentionally more sensitive than specific for confirmed stroke. However, substantial physician and hospital resources are involved. We sought to evaluate characteristics and predictors of stroke mimics and confirmed strokes in a large stroke alert program. Methods: The study is a retrospective investigation of the stroke alert system at the University of Colorado Hospital. Variables included patient, clinical, stroke and stroke alert characteristics, and outcome. Variables were compared for patients with confirmed stroke, and mimics. Results: Stroke alerts were evaluated in 125 patients over a one-month period in 2019. Median age was 60 years (IQR 47-72), 52% were female, 51% were white, and 24% black or African American. Median initial NIHSS score was 4 (IQR 1-11), and onset was outside the hospital in 70%. One third of stroke alerts were confirmed stroke or TIA, and 66% were mimics. Most stroke alerts were called by the departments of Emergency Medicine (78%), Medicine (10%) and Surgery (9%). The most common of more than 40 stroke alert symptoms and combinations were weakness (45%), aphasia (20%), altered mental status (16%) and facial droop or numbness (each 14%). Stroke mimics were more likely to be female (58% female vs. 41% male, OR 2.206, 95% CI 1.025-4.745, p=0.041), had better initial NIHSS scores [3 (IQR 1-9) mimics vs. 6 (IQR 2-15) stroke, p=0.015), and were more often discharged to home (77% mimics vs. 51% strokes, OR 2.051, 95% CI 1.269-3.316, p=0.004). Stroke onset location and service initiating stroke alert were not associated with confirmed stroke. Weakness was the only symptom associated with confirmed stroke (58% stroke vs. 37% mimics, OR 2.447, 95% CI 1.137-5.268, p=0.021). Conclusions: Of stroke alerts in our institution, two thirds were stroke mimics, and these were more likely to be female patients. Weakness as a stroke alert symptom, alone or with other symptoms, was associated with confirmed stroke. Additional study is warranted to improve specificity and optimize utilization of physician and hospital resources in stroke alert programs.

BackgroundThe molecular basis of airway remodelling in chronic obstructive pulmonary disease (COPD) remains poorly understood. We identified gene expression signatures associated with chest computed tomography (CT) scan airway measures to understand molecular pathways associated with airway disease.MethodsIn 2396 subjects in the COPDGene Study, we examined the relationship between quantitative CT airway phenotypes and blood transcriptomes to identify airway disease-specific genes and to define an airway wall thickness (AWT) gene set score. Multivariable regression analyses were performed to identify associations of the AWT score with clinical phenotypes, bronchial gene expression and genetic variants.ResultsType 1 interferon (IFN)-induced genes were consistently associated with AWT, square root wall area of a hypothetical airway with 10 mm internal perimeter (Pi10) and wall area percentage, with the strongest enrichment in AWT. A score derived from 18 genes whose expression was associated with AWT was associated with COPD-related phenotypes including reduced lung function (forced expiratory volume in 1 s percentage predicted β= −3.4; pIKZF1, a gene that regulates IFN signalling and is associated with inflammatory diseases.ConclusionsA gene expression signature with IFN-stimulated genes from peripheral blood and bronchial brushings is associated with CT AWT, lung function and exacerbations. Shared genes and genetic associations suggest viral responses and/or autoimmune dysregulation as potential underlying mechanisms of airway disease in COPD.

Category: Bunion; Midfoot/Forefoot Introduction/Purpose: Hallux valgus (HV) is a tri-plane deformity of the foot corresponding to a medial deviation of the first metatarsal and a lateral deviation of the hallux. Understanding key angles between bones, as well as how these deformity changes in each plane, is critical to generating pre-operative insights into the most effective surgical correction of the deformity. While two- dimenional (2D) imaging can provide some information; utilizing three-dimensional (3D) imaging can include more precise and accurate measures of hallux valgus. The purpose of this study is to evaluate the metatarsal osteotomies for correction of hallux valgus deformity in axial, coronal and sagittal plane with 2D and 3D measurements with the hypothesis that 3D measurements will provide greater accuracy of pre and post surgical changes Methods: Ten cadaveric specimens were selected and weight-bearing CT scans were taken pre operatively with an applied an axial load of 80 pounds. Midshaft osteotomies were then performed on the cadaveric specimens followed by weight-bearing CT scans taken post-operatively.3D models of the pre-operative and post-operative specimens were created and differences in pre and post operative changes were analyzed using conventional 2D and 3D models of the first metatarsal (M1), second metatarsal (M2), fifth metatarsal (M5), and proximal phalanx of the hallux (PP1) using a paired student t-test. Quantitative examination of foot and ankle offset (FAO) alignment along with congruity of first metatarsal phalangeal and first tarsometatarsal joints Results: 3D measurements of the Inter-Metatarsal (IM) Angle, was 12.3 degrees pre-opeative and 10.4 degrees post-operative with no significant differences in all three planes while the M1M5 Angle, measured between the principle axes of the first metatarsal and fifth metatarsal was 25.3 degrees pre-operative and 22.0 degrees post-operative were statistically significant with the greatest change in the axial plane. 2D measurements of the hallux valgus angle, measured the longitudineal axes of the first metatarsal and proximal phalange of the hallux was 22.6 degrees pre opereative and 21.7 degrees post opereative with no statistical difference while the metatarsal parabola, measured in two dimensions projected onto the axial plane was 157.1 degrees before and 141.5 degrees after surgery was statistical significant. Conclusion: When comparing 2D and 3D pre and post surgical hallux valgus values, the only significant differences were identified in the absolute M1M5 values in the axial plane and a significant change was also observed in the metatarsal parabola angle. While the metatarsal parabola angle are sensitive measures, these results suggest that the M1 M5 angle allows for precise, efficient measurements in axial, coronal and sagittal planes that is previously unknown through two-dimensional radiographic measurements for quantifying the effect of metatarsal osteotomies on hallux valgus

Cigarette smoking impacts DNA methylation, but the investigation of sex-specific features of lung tissue DNA methylation in smokers has been limited. Women appear more susceptible to cigarette smoke, and often develop more severe lung disease at an earlier age with less smoke exposure. We aimed to analyse whether there are sex differences in DNA methylation in lung tissue and whether these DNA methylation marks interact with smoking. We collected lung tissue samples from former smokers who underwent lung tissue resection. One hundred thirty samples from white subjects were included for this analysis. Regression models for sex as a predictor of methylation were adjusted for age, presence of COPD, smoking variables and technical batch variables revealed 710 associated sites. 294 sites demonstrated robust sex-specific methylation associations in foetal lung tissue. Pathway analysis identified 6 nominally significant pathways including the mitophagy pathway. Three CpG sites demonstrated a suggested interaction between sex and pack-years of smoking: GPR132, ANKRD44 and C19orf60. All of them were nominally significant in both male- and female-specific models, and the effect estimates were in opposite directions for male and female; GPR132 demonstrated significant association between DNA methylation and gene expression in lung tissue (P

Rationale: Genetic association studies have identified rs2076295 in association with idiopathic pulmonary fibrosis (IPF). We hypothesized that rs2076295 is the functional variant regulating DSP (de...

BackgroundThe human microbiome has a role in the development of human diseases. Individual microbiome profiles are highly personalized, though many species are shared. Understanding the relationship between the human microbiome and disease may inform future individualized treatments. Specifically, the blood microbiome, once believed sterile, may be a surrogate for some lung and gut microbial characteristics. We sought associations between the blood microbiome and lung-relevant host factors.MethodsBased on reads not mapped to the human genome, we detected microbial nucleic acid signatures in peripheral blood RNA-sequencing for 2,590 current and former smokers with and without chronic obstructive pulmonary disease (COPD) from the COPDGene study. We used the GATK microbial pipeline PathSeq to infer microbial profiles. We tested associations between the inferred profiles and lung disease relevant phenotypes and examined links to host gene expression pathways.ResultsThe four phyla with highest abundance across all subjects were Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. We observed associations between exacerbation phenotypes and the relative abundance ofStaphylococcus, AcidovoraxandCupriavidus. The genusFlavobacteriumwas associated with emphysema and change in emphysema. Our host-microbiome interaction analysis revealed clustering of genera associated with emphysema, systemic inflammation, airway remodeling and exacerbations, through links to lung-relevant host pathways.ConclusionsThis study is the first to identify a bacterial microbiome signature in the peripheral blood of current and former smokers. Understanding the relationships between the systemic microbial populations and lung disease severity may inform novel interventions and aid in the understanding of exacerbation phenotypes.

Introduction Cachexia contributes to increased mortality and reduced quality of life in Chronic Obstructive Pulmonary Disease (COPD) and may be associated with underlying gene expression changes. Our goal was to identify differential gene expression signatures associated with COPD cachexia in current and former smokers. Methods We analyzed whole-blood gene expression data from participants with COPD in a discovery cohort (COPDGene, N = 400) and assessed replication (ECLIPSE, N = 114). To approximate the consensus definition using available criteria, cachexia was defined as weight-loss > 5% in the past 12 months or low body mass index (BMI) (2) and 1/3 criteria: decreased muscle strength (six-minute walk distance 2 among women and 2 among men) in COPDGene. In ECLIPSE, cachexia was defined as weight-loss > 5% in the past 12 months or low BMI and 3/5 criteria: decreased muscle strength, anorexia, abnormal biochemistry (anemia or high c-reactive protein (> 5 mg/l)), fatigue, and low FFMI. Differential gene expression was assessed between cachectic and non-cachectic subjects, adjusting for age, sex, white blood cell counts, and technical covariates. Gene set enrichment analysis was performed using MSigDB. Results The prevalence of COPD cachexia was 13.7% in COPDGene and 7.9% in ECLIPSE. Fourteen genes were differentially downregulated in cachectic versus non-cachectic COPD patients in COPDGene (FDR Discussion Several replicated genes regulating heme metabolism were downregulated among participants with COPD cachexia. Impaired heme biosynthesis may contribute to cachexia development through free-iron buildup and oxidative tissue damage.

COPD likely has developmental origins; however, the underlying molecular mechanisms are not fully identified. Investigation of lung tissue-specific epigenetic modifications such as DNA methylation using network approaches might facilitate insights linking in utero smoke (IUS) exposure and risk for COPD in adulthood.We performed genome-wide methylation profiling for adult lung DNA from 160 surgical samples and 78 fetal lung DNA samples isolated from discarded tissue at 8–18 weeks of gestation. Co-methylation networks were constructed to identify preserved modules that shared methylation patterns in fetal and adult lung tissues and associations with fetal IUS exposure, gestational age and COPD.Weighted correlation networks highlighted preserved and co-methylated modules for both fetal and adult lung data associated with fetal IUS exposure, COPD and lower adult lung function. These modules were significantly enriched for genes involved in embryonic organ development and specific inflammation-related pathways, including Hippo, phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), Wnt, mitogen-activated protein kinase and transforming growth factor-β signalling. Gestational age-associated modules were remarkably preserved for COPD and lung function, and were also annotated to genes enriched for the Wnt and PI3K/AKT pathways.Epigenetic network perturbations in fetal lung tissue exposed to IUS and of early lung development recapitulated in adult lung tissue from ex-smokers with COPD. Overlapping fetal and adult lung tissue network modules highlighted putative disease pathways supportive of exposure-related and age-associated developmental origins of COPD.

Objective: Diabetes affects more than 29 million individuals in the United States, resulting in healthcare costs approaching $245 billion. Approximately 15% of these individuals will develop a chronic, non-healing foot ulcer (diabetic foot ulcer [DFU]) that, if untreated, may lead to amputation. The current treatments for DFU are expensive, have significant side-effects, and often result in non-compliance. A new topical treatment is described that accelerates cutaneous wound repair and is disease modifying by targeting underlying aberrant diabetic pathways. Approach: The efficacy of naltrexone (NTX), an opioid receptor antagonist, and Regranex® was compared in preclinical studies using type 1 diabetic rats. Dorsal cutaneous wounds were treated topically with 0.03% NTX, Regranex, or moisturizing cream alone. Wound closure, DNA synthesis, and cytokine production were monitored. Results: Wound closure rates with topical NTX in type 1 diabetic rats were comparable to Regranex. Topical NTX accelerated DNA synt...

Although cigarette smoke (CS) is the primary risk factor for chronic obstructive pulmonary disease (COPD), the underlying molecular mechanisms for the significant variability in developing COPD in response to CS are incompletely understood. We performed lung gene expression profiling of two different wild-type murine strains (C57BL/6 and NZW/LacJ) and two genetic models with mutations in COPD genome-wide association study genes (HHIP and FAM13A) after 6 months of chronic CS exposure and compared the results to human COPD lung tissues. We identified gene expression patterns that correlate with severity of emphysema in murine and human lungs. Xenobiotic metabolism and nuclear erythroid 2-related factor 2-mediated oxidative stress response were commonly regulated molecular response patterns in C57BL/6, Hhip+/−, and Fam13a−/− murine strains exposed chronically to CS. The CS-resistant Fam13a−/− mouse and NZW/LacJ strain revealed gene expression response pattern differences. The Fam13a−/− strain diverged in gene expression compared with C57BL/6 control only after CS exposure. However, the NZW/LacJ strain had a unique baseline expression pattern, enriched for nuclear erythroid 2-related factor 2-mediated oxidative stress response and xenobiotic metabolism, and converged to a gene expression pattern similar to the more susceptible wild-type C57BL/6 after CS exposure. These results suggest that distinct molecular pathways may account for resistance to emphysema. Surprisingly, there were few genes commonly modulated in mice and humans. Our study suggests that gene expression responses to CS may be largely species and model dependent, yet shared pathways could provide biologically significant insights underlying individual susceptibility to CS.

Medial displacement calcaneal osteotomies have been shown to be successful in the surgical management of adult acquired flatfoot, in particular, stage 2 deformity. Classically, the medial displacement calcaneal osteotomy technique has been performed. However, a calcaneal Z osteotomy has been more recently described and applied in the surgical management of flatfoot deformity. Although the potential advantages of the calcaneal Z technique have been reported, data on its effect on the subtalar joint are lacking. A validated flatfoot model was induced in 8 cadaveric feet that had been randomly assigned to either medial displacement calcaneal osteotomy (n = 4) or calcaneal Z osteotomy (n = 4). The feet were loaded through the tibia with a constant ground reaction force of 400 N, with a simultaneous increase in the Achilles tendon force to 300 or 500 N. The subtalar joint pressures were recorded before and after osteotomy. We did not detect any statistically significant differences between the 2 techniques in terms of their effects on subtalar joint pressure.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous and complex syndrome. Network-based analysis implemented by SWIM software can be exploited to identify key molecular switches - called “switch genes” - for disease. Genes contributing to common biological processes or define given cell types are frequently co-regulated and co-expressed, giving rise to expression network modules. Consistently, we found that the COPD correlation network built by SWIM consists of three well-characterized modules: one populated by switch genes, all up-regulated in COPD cases and related to the regulation of immune response, inflammatory response, and hypoxia (like TIMP1, HIF1A, SYK, LY96, BLNK and PRDX4); one populated by well-recognized immune signature genes, all up-regulated in COPD cases; one where the GWAS genes AGER and CAVIN1 are the most representative module genes, both down-regulated in COPD cases. Interestingly, 70% of AGER negative interactors are switch genes including PRDX4, whose activation strongly correlates with the activation of known COPD GWAS interactors SERPINE2, CD79A, and POUF2AF1. These results suggest that SWIM analysis can identify key network modules related to complex diseases like COPD.

Chronic obstructive pulmonary disease (COPD) is the leading cause of respiratory mortality worldwide. Genetic risk loci provide novel insights into disease pathogenesis. We performed a genome-wide association study in 35,735 cases and 222,076 controls from the UK Biobank and additional studies from the International COPD Genetics Consortium. We identified 82 loci with P-value < 5 × 10−8; 47 were previously described in association with either COPD or population-based lung function. Of the remaining 35 novel loci, 13 were associated with lung function in 79,055 individuals from the SpiroMeta consortium. Using gene expression and regulation data, we identified enrichment for loci in lung tissue, smooth muscle and several lung cell types. We found 14 COPD loci shared with either asthma or pulmonary fibrosis. COPD genetic risk loci clustered into groups of quantitative imaging features and comorbidity associations. Our analyses provide further support to the genetic susceptibility and heterogeneity of COPD., Editorial summary Genome-wide analysis of chronic obstructive pulmonary disease identifies 82 loci, 35 of which are new. Integration of gene expression and genomic annotation data shows enrichment of signals in lung tissue, smooth muscle and several lung cell types.

Mass spectrometry-based protein quantitation is currently used to measure therapeutically relevant protein biomarkers in CAP/CLIA setting to predict likely responses of known therapies. Selected reaction monitoring (SRM) is the method of choice due to its outstanding analytical performance. However, data-independent acquisition (DIA) is now emerging as a proteome-scale clinical assay. We evaluated the ability of DIA to profile the patient-specific proteomes of sample-limited tumor biopsies and to quantify proteins of interest in a targeted fashion using formalin-fixed, paraffin-embedded (FFPE) tumor biopsies ( n = 12) selected from our clinical laboratory. DIA analysis on the tumor biopsies provided 3713 quantifiable proteins including actionable biomarkers currently in clinical use, successfully separated two gastric cancers from colorectal cancer specimen solely on the basis of global proteomic profiles, and identified subtype-specific proteins with prognostic or diagnostic value. We demonstrate the potential use of DIA-based quantitation to inform therapeutic decision-making using TUBB3, for which clinical cutoff expression levels have been established by SRM. Comparative analysis of DIA-based proteomic profiles and mRNA expression levels found positively and negatively correlated protein-gene pairs, a finding consistent with previously reported results from fresh-frozen tumor tissues.

Chronic obstructive pulmonary disease (COPD), one of the leading causes of death worldwide, is substantially influenced by genetic factors. Alpha-1 antitrypsin deficiency demonstrates that rare coding variants of large effect can influence COPD susceptibility. To identify additional rare coding variants in patients with severe COPD, we conducted whole exome sequencing analysis in 2543 subjects from two family-based studies (Boston Early-Onset COPD Study and International COPD Genetics Network) and one case-control study (COPDGene). Applying a gene-based segregation test in the family-based data, we identified significant segregation of rare loss of function variants in TBC1D10A and RFPL1 (P-value

Although the butterflies of North America have received considerable taxonomic attention, overlooked species and instances of hybridization continue to be revealed. The present study assembles a DNA barcode reference library for this fauna to identify groups whose patterns of sequence variation suggest the need for further taxonomic study. Based on 14,626 records from 814 species, DNA barcodes were obtained for 96% of the fauna. The maximum intraspecific distance averaged 1/4 the minimum distance to the nearest neighbor, producing a barcode gap in 76% of the species. Most species (80%) were monophyletic, the others were para- or polyphyletic. Although 15% of currently recognized species shared barcodes, the incidence of such taxa was far higher in regions exposed to Pleistocene glaciations than in those that were ice-free. Nearly 10% of species displayed high intraspecific variation (>2.5%), suggesting the need for further investigation to assess potential cryptic diversity. Aside from aiding the identification of all life stages of North American butterflies, the reference library has provided new perspectives on the incidence of both cryptic and potentially over-split species, setting the stage for future studies that can further explore the evolutionary dynamics of this group.

Genomic regions identified by genome-wide association studies explain only a small fraction of heritability for chronic obstructive pulmonary disease (COPD). Alpha-1 antitrypsin deficiency shows that rare coding variants of large effect also influence COPD susceptibility. We hypothesized that exome sequencing in families identified through a proband with severe, early-onset COPD would identify additional rare genetic determinants of large effect.To identify rare genetic determinants of severe COPD.We applied filtering approaches to identify potential causal variants for COPD in whole exomes from 347 subjects in 49 extended pedigrees from the Boston Early-Onset COPD Study. We assessed the power of this approach under different levels of genetic heterogeneity using simulations. We tested genes identified in these families using gene-based association tests in exomes of 204 cases with severe COPD and 195 resistant smokers from the COPDGene study. In addition, we examined previously described loci associated with COPD using these datasets.We identified 69 genes with predicted deleterious nonsynonymous, stop, or splice variants that segregated with severe COPD in at least two pedigrees. Four genes (DNAH8, ALCAM, RARS, and GBF1) also demonstrated an increase in rare nonsynonymous, stop, and/or splice mutations in cases compared with resistant smokers from the COPDGene study; however, these results were not statistically significant. We demonstrate the limitations of the power of this approach under genetic heterogeneity through simulation.Rare deleterious coding variants may increase risk for COPD, but multiple genes likely contribute to COPD susceptibility.

The landscape of basic science in the United States and around the world is changing, and the field of orthopaedic research is positioned to lead by embracing a culture of collaborative, team science that reflects our field's interdisciplinary nature. In this article we hope to address some of the cultural challenges and programmatic barriers that impede a team science approach in the US and suggest opportunities for change. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1673-1679, 2016.

Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3 mg/kg/day sS-31. Mitochondrial function was assessed in vivo using (31)P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased in vivo mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly.

Category: Diabetes Introduction/Purpose: Complications associated with the diabetes include increased incidence of fracture healing, delayed fracture healing, delayed osteoblasts cell replication, decreased angiogenesis, migration and/or osteoblast cell differentiation. The cellular events involved in bone healing are adversely affected by diabetes; however, can be modulated by the Opioid Growth Factor (OGF)–OGF receptor (OGFr) is an inhibitory peptide that downregulates DNA synthesis in a tissue nonspecific manner. Diabetes is associated with elevated serum levels of OGF and dysregulation of the OGFr leading to multiple complications related to healing, sensitivity, and regeneration. This study explores the presence and function of the OGF-OGFr axis in bone tissue from type 1 diabetic rats examining intact and fractured femurs during early phases of the repair process Methods: Seven-week-old Sprague Dawley rats were injected with streptozotocin (40mg/kg i.p.) to induce T1D; other rats received buffer only and served as controls. After one month, hyperglycemia rats underwent surgery to produce a fracture at the distal third of the femur. Four diabetic rats received opioid antagoinist (naltrexone) and calcium sulfate and all remaining rats received calcium sulfate with water only. X-rays were taken immediately after surgery and after rats were euthanized on post-surgery; femur and tibia were collected for protein isolation, western blot analysis along with frozen or paraffin-embedded for histological analysis Results: Immunofluorescence indicated approximately 90% increase in opioid growth factor receptor expression in diabetic femurs compared to age-matched normal femurs. Western Blotting also suggested an increase in the receptor protein in diabetic bones relative to normal bone. TRAP staining for osteoclasts was greater in control and opioid antagonist-treated diabetic fractures when compared to the number of osteoclasts in vehicle-treated diabetic fractured femurs. Safranin O stained sections revealed approximately more bone in opioid growth receptor antagonist-treated diabetic bone fractures than in vehicle-treated bone fractures Conclusion: These data support our hypothesis that expression levels of OGFr are dysregulated in the bone of diabetic patients leading to complications in bone healing. Moreover, modulation of the OGF-OGFr pathway with receptor antagonists restored some aspects of bone healing. With further study, these preliminary results support the role of the OGF-OGFr axis in treatment of diabetic bone healing. New therapies to target dysregulation of the OGF-OGFr regulatory pathway in diabetes would provide a safe and effective disease-modifying treatment for delayed bone healing.

SummaryChronic obstructive pulmonary disease (COPD) is the leading cause of respiratory mortality worldwide. Genetic risk loci provide novel insights into disease pathogenesis. To broaden COPD genetic risk loci discovery and identify cell type and phenotype associations we performed a genome-wide association study in 35,735 cases and 222,076 controls from the UK Biobank and additional studies from the International COPD Genetics Consortium. We identified 82 loci with P value < 5×10−8; 47 were previously described in association with either COPD or population-based lung function. Of the remaining 35 novel loci, 13 were associated with lung function in 79,055 individuals from the SpiroMeta consortium. Using gene expression and regulation data, we identified enrichment for loci in lung tissue, smooth muscle and alveolar type II cells. We found 9 shared genomic regions between COPD and asthma and 5 between COPD and pulmonary fibrosis. COPD genetic risk loci clustered into groups of quantitative imaging features and comorbidity associations. Our analyses provide further support to the genetic susceptibility and heterogeneity of COPD.

Rationale: As the third leading cause of death in the United States, the impact of chronic obstructive pulmonary disease (COPD) makes identification of its molecular mechanisms of great importance. Genome-wide association studies (GWASs) have identified multiple genomic regions associated with COPD. However, genetic variation only explains a small fraction of the susceptibility to COPD, and sub–genome-wide significant loci may play a role in pathogenesis. Objectives: Regulatory annotation with epigenetic evidence may give priority for further investigation, particularly for GWAS associations in noncoding regions. We performed integrative genomics analyses using DNA methylation profiling and genome-wide SNP genotyping from lung tissue samples from 90 subjects with COPD and 36 control subjects. Methods: We performed methylation quantitative trait loci (mQTL) analyses, testing for SNPs associated with percent DNA methylation and assessed the colocalization of these results with previous COPD GWAS findings using Bayesian methods in the R package coloc to highlight potential regulatory features of the loci. Measurements and Main Results: We identified 942,068 unique SNPs and 33,996 unique CpG sites among the significant (5% false discovery rate) cis-mQTL results. The genome-wide significant and subthreshold (P

SUMMARY Klinefelter syndrome (KS) (47 XXY) is a common sex-chromosome aneuploidy with an estimated prevalence of one in every 660 male births. Investigations into the associations between DNA methylation and the highly variable clinical manifestations of KS have largely focused on the supernumerary X chromosome; systematic investigations of the epigenome have been limited. We obtained genome-wide DNA methylation data from peripheral blood using the Illumina HumanMethylation450K platform in 5 KS (47 XXY) versus 102 male (46 XY) and 113 female (46 XX) control subjects participating in the COPDGene Study. Empirical Bayes-mediated models were used to test for differential methylation by KS status. CpG sites with a false-discovery rate

Tuberous sclerosis complex (TSC) is an autosomal disease caused by inactivating mutations in either of the tumor suppressor genes TSC1 or TSC2. TSC-associated tumor growth is present in multiple tissues and organs including brain, kidney, liver, heart, lungs, and skin. In the kidney, TSC angiomyolipomas have aberrant vascular structures with abnormal endothelial cells, suggesting a role for endothelial mTORC1 function. In the current report, a genetically engineered mouse model (GEMM) with a conditional knockout allele of Tsc1 with a Darpp32-Cre allele displayed accelerated formation of both kidney cystadenomas and paw hemangiosarcomas. All mutant mice developed hemangiosarcomas on multiple paws by 6 weeks of age. By 16 weeks of age, the average mutant hind paw was 4.0 mm in diameter, nearly double the size of control mice. Furthermore, the hemangiosarcomas and kidney cystadenomas were responsive to intraperitoneal rapamycin treatment. Immunoblotting and immunostaining for phospho-S6 (pS6) and phospho-CAD showed that the effect of rapamycin on tumor size was through inhibition of the mTOR signaling pathway. Finally, elevated VEGF mRNA levels were also observed in hemangiosarcoma specimens. Because paw hemangiosarcomas are easily detectable and scorable for size and growth, this novel mouse model enables accelerated in vivo drug testing for therapies of TSC-related tumors. Implications: These findings provide a strong rationale for simultaneous use of this conditional knockout mouse as an in vivo genetic model while seeking new cancer therapies for TSC-related tumors. Mol Cancer Res; 13(3); 548–55. ©2014 AACR.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disorder. COPD patients may have different clinical features, imaging characteristics and natural history. Multiple studies have investigated heterogeneity using statistical methods such as unsupervised clustering to define different subgroups of COPD based largely on clinical phenotypes. Some studies have performed clustering using genetic data or limited numbers of blood biomarkers. Our primary goal was to use proteomic data to find subtypes of COPD within clinically similar individuals. In the Treatment of Emphysema with a gamma-Selective Retinoid Agonist (TESRA) study, multiplex biomarker panels were run in serum samples collected prior to randomization. After implementing an algorithm to minimize missing values, the dataset included 396 COPD individuals and 87 biomarkers. Using hierarchical clustering, we identified 3 COPD subgroups, containing 267 (67.4%), 104 (26.3%), and 25 (6.3%) individuals, respectively. The third cluster had less emphysema on quantitative analysis of chest computed tomography scans (p=0.03) and worse disease-related quality of life based on the St. George's Respiratory Questionnaire (total score cluster 1: 45.6, cluster 2: 45.4, cluster 3: 56.6; p=0.01), despite similar levels of lung function impairment (forced expiratory volume in 1 second (49.2%, 49.2%, 48.2 % predicted, respectively). Enrichment analysis showed the biomarkers distinguishing cluster 3 mapped to platelet alpha granule and cell chemotaxis pathways. Thus, we identified a subgroup which has less emphysema but may have greater inflammation, which could be potentially targeted with anti-inflammatory therapies.

In comparison to genome-wide association studies (GWAS), there has been poor replication of gene expression studies in chronic obstructive pulmonary disease (COPD). We performed microarray gene expression profiling on a large sample of resected lung tissues from subjects with severe COPD. Comparing 111 COPD cases and 40 control smokers, 204 genes were differentially expressed; none were at significant GWAS loci. The top differentially expressed gene was HMGB1, which interacts with AGER, a known COPD GWAS gene. Differentially expressed genes showed enrichment for putative interactors of the first three identified COPD GWAS genes IREB2, HHIP, and FAM13A, based on gene sets derived from protein and RNA binding studies, RNA-interference, a murine smoking model, and expression quantitative trait locus analyses. The gene module most highly associated for COPD in Weighted Gene Co-Expression Network Analysis (WGCNA) was enriched for B cell pathways, and shared seventeen genes with a mouse smoking model and twenty genes with previous emphysema studies. As in other common diseases, genes at COPD GWAS loci were not differentially expressed; however, using a combination of network methods, experimental studies and careful phenotype definition, we found differential expression of putative interactors of these genes, and we replicated previous human and mouse microarray results.

International audience; Chronic obstructive pulmonary disease (COPD) is a complex disease with strong environmental and genetic influences and sexually dimorphic features. Although genetic risk factors for COPD have been identified, much of the heritability remains unexplained. Sex-based genetic association studies may uncover additional COPD genetic risk factors. We studied current and former smokers from COPD case-control cohorts (COPDGene non-Hispanic whites and African Americans, Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-Points, and Genetics of Chronic Obstructive Lung Disease). COPD was defined as post-bronchodilator forced expiratory volume in 1 second/forced vital capacity less than 0.70 and forced expiratory volume in 1 second percent predicted less than 80. Testing was performed across all cohorts and combined in a meta-analysis adjusted for age, pack-years, and genetic ancestry. We first performed genome-wide single-nucleotide polymorphism (SNP)-by-sex interaction testing on the outcome of COPD affection status. We performed sex-stratified association testing for SNPs with interaction P less than 10-6. We examined over 8 million SNPs in four populations, including 6,260 subjects with COPD (40.6% female) and 5,269 smoking control subjects (47.3% female). The SNP rs9615358 in the cadherin gene CELSR1 approached genome-wide significance for an interaction with sex (P = 1.24 × 10-7). In the sex-stratified meta-analysis, this SNP was associated with COPD among females (odds ratio, 1.37 [95% confidence interval, 1.25-1.49]; P = 3.32 × 10-7) but not males (odds ratio, 0.90 [95% confidence interval, 0.79-1.01]; P = 0.06). CELSR1 is involved in fetal lung development. In a human fetal lung tissue dataset, we observed greater CELSR1 expression in female compared with male samples. This SNP-by-sex genome-wide association analysis identified the fetal lung development gene, CELSR1, as a potential sex-specific risk factor for COPD. Identifying sex-specific genetic risk factors may reveal new insights into sexually dimorphic features of COPD.

Chronic obstructive pulmonary disease (COPD) occurs typically in current or former smokers, but only a minority of people with smoking history develops the disease. Besides environmental factors, genetics is an important risk factor for COPD. However, the relationship between genetics, environment and phenotypes is not well understood. Sample sizes for genome-wide expression studies based on lung tissue have been small due to the invasive nature of sample collection. Increasing evidence for the systemic nature of the disease makes blood a good alternative source to study the disease, but there have also been few large-scale blood genomic studies in COPD. Due to the complexity and heterogeneity of COPD, examining groups of interacting genes may have more relevance than identifying individual genes. Therefore, we used Weighted Gene Co-expression Network Analysis to find groups of genes (modules) that are highly connected. However, module definitions may vary between individual data sets. To alleviate this problem, we used a consensus module definition based on two cohorts, COPDGene and ECLIPSE. We studied the relationship between the consensus modules and COPD phenotypes airflow obstruction and emphysema. We also used these consensus module definitions on an independent cohort (TESRA) and performed a meta analysis involving all data sets. We found several modules that are associated with COPD phenotypes, are enriched in functional categories and are overrepresented for cell-type specific genes. Of the 14 consensus modules, three were strongly associated with airflow obstruction (meta p ≤ 0.0002), and two had some association with emphysema (meta p ≤ 0.06); some associations were stronger in the case-control cohorts, and others in the cases-only subcohorts. Gene Ontology terms that were overrepresented included "immune response" and "defense response." The cell types whose type-specific genes were overrepresented in modules (p < 0.05) included natural killer cells, dendritic cells, and neutrophils. Together, this is the largest investigation of gene blood expression in COPD with 469 cases in COPDGene, ECLIPSE and TESRA combined, with 6267 genes common to all data sets. Additional, we have 42 and 83 controls in COPDGene and ECLIPSE, respectively.

Tuberous sclerosis complex (TSC) is a disorder arising from mutation in the TSC1 or TSC2 gene, characterized by the development of hamartomas in various organs and neurological manifestations including epilepsy, intellectual disability and autism. TSC1/2 protein complex negatively regulates the mammalian target of rapamycin complex 1 (mTORC1) a master regulator of protein synthesis, cell growth and autophagy. Autophagy is a cellular quality-control process that sequesters cytosolic material in double membrane vesicles called autophagosomes and degrades it in autolysosomes. Previous studies in dividing cells have shown that mTORC1 blocks autophagy through inhibition of Unc-51-like-kinase1/2 (ULK1/2). Despite the fact that autophagy plays critical roles in neuronal homeostasis, little is known on the regulation of autophagy in neurons. Here we show that unlike in non-neuronal cells, Tsc2-deficient neurons have increased autolysosome accumulation and autophagic flux despite mTORC1-dependent inhibition of ULK1. Our data demonstrate that loss of Tsc2 results in autophagic activity via AMPK-dependent activation of ULK1. Thus, in Tsc2-knockdown neurons AMPK activation is the dominant regulator of autophagy. Notably, increased AMPK activity and autophagy activation are also found in the brains of Tsc1-conditional mouse models and in cortical tubers resected from TSC patients. Together, our findings indicate that neuronal Tsc1/2 complex activity is required for the coordinated regulation of autophagy by AMPK. By uncovering the autophagy dysfunction associated with Tsc2 loss in neurons, our work sheds light on a previously uncharacterized cellular mechanism that contributes to altered neuronal homeostasis in TSC disease.

Disorders of the dermis and the nails on the feet are common. Despite the simplicity of the skin and nail disorders of the foot, they can be debilitating and impact the patient's ability to ambulate and perform activities of daily living. Diagnosis in most cases is confirmed on physical examination alone. Diligent care of skin and nail disorders can prevent further pathology involving the deeper structures of the foot and allow the patient to fully participate in their usual activities.