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BackgroundSurvival after curative resection of early-stage lung adenocarcinoma (LUAD) varies and prognostic biomarkers are urgently needed.MethodsLarge-format tissue samples from a prospective cohort of 200 patients with resected LUAD were immunophenotyped for cancer hallmarks TP53, NF1, CD45, PD-1, PCNA, TUNEL and FVIII, and were followed for a median of 2.34 (95% CI 1.71–3.49) years.ResultsUnsupervised hierarchical clustering revealed two patient subgroups with similar clinicopathological features and genotype, but with markedly different survival: “proliferative” patients (60%) with elevated TP53, NF1, CD45 and PCNA expression had 50% 5-year overall survival, while “apoptotic” patients (40%) with high TUNEL had 70% 5-year survival (hazard ratio 2.23, 95% CI 1.33–3.80; p=0.0069). Cox regression and machine learning algorithms including random forests built clinically useful models: a score to predict overall survival and a formula and nomogram to predict tumour phenotype. The distinct LUAD phenotypes were validated in The Cancer Genome Atlas and KMplotter data, and showed prognostic power supplementary to International Association for the Study of Lung Cancer tumour–node–metastasis stage and World Health Organization histologic classification.ConclusionsTwo molecular subtypes of LUAD exist and their identification provides important prognostic information.

Animal studies have shown that the striatal cholinergic system plays a role in behavioral flexibility but, until recently, this system could not be studied in humans due to a lack of appropriate noninvasive techniques. Using proton magnetic resonance spectroscopy, we recently showed that the concentration of dorsal striatal choline (an acetylcholine precursor) changes during reversal learning (a measure of behavioral flexibility) in humans. The aim of the present study was to examine whether regional average striatal choline was associated with reversal learning. A total of 22 participants (mean age = 25.2 years, range = 18–32 years, 13 female) reached learning criterion in a probabilistic learning task with a reversal component. We measured choline at rest in both the dorsal and ventral striatum using magnetic resonance spectroscopy. Task performance was described using a simple reinforcement learning model that dissociates the contributions of positive and negative prediction errors to learning. Average levels of choline in the dorsal striatum were associated with performance during reversal, but not during initial learning. Specifically, lower levels of choline in the dorsal striatum were associated with a lower number of perseverative trials. Moreover, choline levels explained interindividual variance in perseveration over and above that explained by learning from negative prediction errors. These findings suggest that the dorsal striatal cholinergic system plays an important role in behavioral flexibility, in line with evidence from the animal literature and our previous work in humans. Additionally, this work provides further support for the idea of measuring choline with magnetic resonance spectroscopy as a noninvasive way of studying human cholinergic neurochemistry. SIGNIFICANCE STATEMENT Behavioral flexibility is a crucial component of adaptation and survival. Evidence from the animal literature shows that the striatal cholinergic system is fundamental to reversal learning, a key paradigm for studying behavioral flexibility, but this system remains understudied in humans. Using proton magnetic resonance spectroscopy, we showed that choline levels at rest in the dorsal striatum are associated with performance specifically during reversal learning. These novel findings help to bridge the gap between animal and human studies by demonstrating the importance of cholinergic function in the dorsal striatum in human behavioral flexibility. Importantly, the methods described here cannot only be applied to furthering our understanding of healthy human neurochemistry, but also to extending our understanding of cholinergic disorders.

International audience; A comprehensive characterization of lung adenocarcinoma (LADC) clinical features is currently missing. We prospectively evaluated Caucasian patients with early-stage LADC. Patients with LADC diagnosed between 2011 and 2015 were prospectively assessed for lung resection with curative intent. Fifty clinical, pathologic, radiologic, and molecular variables were recorded. Patients were followed till death/study conclusion. The main findings were compared to a separate cohort from France. Of 1943 patients evaluated, 366 were enrolled (18.8%; 181 female; 75 never-smokers; 28% of registered Bavarian cases over the study period). Smoking and obstruction were significantly more prevalent in GLAD compared with adult Bavarians (P < 0.0001). Ever-smoker tumors were preferentially localized to the upper lobes. We observed 120 relapses and 74 deaths over 704 cumulative follow-up years. Median overall and disease-free survival were >7.5 and 3.6 years, respectively. Patients aged 65 years, resected >60 days postdiagnosis, with abnormal FVC/DLCO VA , N2/N3 stage, or solid histology had significantly decreased survival estimates. These were fit into a weighted locoregional LADC death risk score that outperformed pTNM7 in predicting survival in the GLAD and in our second cohort. We define the clinical gestalt of locoregional LADC and provide a new clinical tool to predict survival, findings that may aid future management and research design

Rationale: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease characterized by lung epithelial cell injury, increased (myo) fibroblast activation, and extracellular matrix deposition. Extracellular vesicles (EVs) regulate intercellular communication by carrying a variety of signaling mediators, including WNT (wingless/integrated) proteins. The relevance of EVs in pulmonary fibrosis and their potential contribution to disease pathogenesis, however, remain unexplored.Objectives: To characterize EVs and study the role of EV-bound WNT signaling in IPF.Methods: We isolated EVs from BAL fluid (BALF) from experimental lung fibrosis as well as samples from IPF, non-IPF interstitial lung disease (ILD), non-ILD, and healthy volunteers from two independent cohorts. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Primary human lung fibroblasts (phLFs) were used for EV isolation and analyzed by metabolic activity assays, cell counting, quantitative PCR, and Western blotting upon WNT gain- and loss-of-function studies.Measurements and Main Results: We found increased EVs, particularly exosomes, in BALF from experimental lung fibrosis as well as from patients with IPF. WNT5A was secreted on EVs in lung fibrosis and induced by transforming growth factor-beta in primary human lung fibroblasts. The phLF-derived EVs induced phLF proliferation, which was attenuated by WNT5A silencing and antibody-mediated inhibition and required intact EV structure. Similarly, EVs from IPF BALF induced phLF proliferation, which was mediated by WNT5A.Conclusions: Increased EVs function as carriers for signaling mediators, such as WNT5A, in IPF and thus contribute to disease pathogenesis. Characterization of EV secretion and composition may lead to novel approaches to diagnose and develop treatments for pulmonary fibrosis.

Dorsolateral prefrontal cortex (dlPFC) is proposed to drive brain-wide focus by biasing processing in favour of task-relevant information. A longstanding debate concerns whether this is achieved through enhancing processing of relevant information and/or by inhibiting irrelevant information. To address this, we applied transcranial magnetic stimulation (TMS) during fMRI, and tested for causal changes in information coding. Participants attended to one feature, whilst ignoring another feature, of a visual object. If dlPFC is necessary for facilitation, disruptive TMS should decrease coding of attended features. Conversely, if dlPFC is crucial for inhibition, TMS should increase coding of ignored features. Here, we show that TMS decreases coding of relevant information across frontoparietal cortex, and the impact is significantly stronger than any effect on irrelevant information, which is not statistically detectable. This provides causal evidence for a specific role of dlPFC in enhancing task-relevant representations and demonstrates the cognitive-neural insights possible with concurrent TMS-fMRI-MVPA., Jade Jackson et al. use fMRI concurrent with transcranial magnetic stimulation (TMS) in an attention task to evaluate whether the right dorsolateral prefrontal cortex (dlPFC) is involved in facilitation of relevant information, or suppression of irrelevant information. Their results suggest that the dlPFC is causally involved in processing relevant information in an attention task and highlight the utility of a dual fMRI-TMS approach.

The correspondence of cell state changes in diseased organs to peripheral protein signatures is currently unknown. Here, we generated and integrated single‐cell transcriptomic and proteomic data from multiple large pulmonary fibrosis patient cohorts. Integration of 233,638 single‐cell transcriptomes (n = 61) across three independent cohorts enabled us to derive shifts in cell type proportions and a robust core set of genes altered in lung fibrosis for 45 cell types. Mass spectrometry analysis of lung lavage fluid (n = 124) and plasma (n = 141) proteomes identified distinct protein signatures correlated with diagnosis, lung function, and injury status. A novel SSTR2+ pericyte state correlated with disease severity and was reflected in lavage fluid by increased levels of the complement regulatory factor CFHR1. We further discovered CRTAC1 as a biomarker of alveolar type‐2 epithelial cell health status in lavage fluid and plasma. Using cross‐modal analysis and machine learning, we identified the cellular source of biomarkers and demonstrated that information transfer between modalities correctly predicts disease status, suggesting feasibility of clinical cell state monitoring through longitudinal sampling of body fluid proteomes., Multi‐modal integration of single‐cell RNA‐seq data from lung tissue and proteomic data from body fluids across independent lung fibrosis patient cohorts revealed biomarker signatures that correspond with cell state changes during disease progression.

RATIONALE: The bronchial epithelium is constantly challenged by inhalative insults including cigarette smoke (CS), a key risk factor for lung disease. In vitro exposure of bronchial epithelial cells using CS extract (CSE) is a widespread alternative to whole CS (wCS) exposure. However, CSE exposure protocols vary considerably between studies, precluding direct comparison of applied doses. Moreover, they are rarely validated in terms of physiological response in vivo and the relevance of the findings is often unclear. METHODS: We tested six different exposure settings in primary human bronchial epithelial cells (phBECs), including five CSE protocols in comparison with wCS exposure. We quantified cell-delivered dose and directly compared all exposures using expression analysis of 10 well-established smoke-induced genes in bronchial epithelial cells. CSE exposure of phBECs was varied in terms of differentiation state, exposure route, duration of exposure, and dose. Gene expression was assessed by quantitative Real-Time PCR (qPCR) and Western Blot analysis. Cell type-specific expression of smoke-induced genes was analyzed by immunofluorescent analysis. RESULTS: Three surprisingly dissimilar exposure types, namely chronic CSE treatment of differentiating phBECs, acute CSE treatment of submerged basal phBECs, and wCS exposure of differentiated phBECs performed best, resulting in significant upregulation of seven (chronic CSE) and six (acute wCS, acute submerged CSE exposure) out of 10 genes. Acute apical or basolateral exposure of differentiated phBECs with CSE was much less effective despite similar doses used. CONCLUSIONS: Our findings provide guidance for the design of human in vitro CS exposure models in experimental and translational lung research.

Malignant pleural mesothelioma (MPM) arises from mesothelial cells lining the pleural cavity of asbestos-exposed individuals and rapidly leads to death. MPM harbors loss-of-function mutations in BAP1, NF2, CDKN2A, and TP53, but isolated deletion of these genes alone in mice does not cause MPM and mouse models of the disease are sparse. Here, we show that a proportion of human MPM harbor point mutations, copy number alterations, and overexpression of KRAS with or without TP53 changes. These are likely pathogenic, since ectopic expression of mutant KRAS(G12D) in the pleural mesothelium of conditional mice causes epithelioid MPM and cooperates with TP53 deletion to drive a more aggressive disease form with biphasic features and pleural effusions. Murine MPM cell lines derived from these tumors carry the initiating KRAS(G12D) lesions, secondary Bap1 alterations, and human MPM-like gene expression profiles. Moreover, they are transplantable and actionable by KRAS inhibition. Our results indicate that KRAS alterations alone or in accomplice with TP53 alterations likely play an important and underestimated role in a proportion of patients with MPM, which warrants further exploration., European Union (EU); Horizon 2020; European Research Council 2010 Starting Independent Investigator; ERC Proof of Concept 2015 (ERC-PoC-2015); German Research Society; ALTERNATIVE; German Ministry for Education and Research; German Center for Lung Research Translational Research Grant; Greek State Scholarship Foundation Program; European Union (EU); European Union Social Fund; Greek National Fund; Reinforcement of Postdoctoral Researchers-1 stand 2nd cycles; General Secretariat for Research and Innovation; Hellenic Foundation for Research and Innovation; REPSIRE European Respiratory Society Fellowship; Institut National de la Sante et de la Recherche Medicale (INSERM); Centre National de la Recherche Scientifique (CNRS); Institut de Recherche en Sante Respiratoire des Pays de la Loire; National Research Agency Programme d' Investissements d' Avenir; Pays de la Loire Region Research Program; Ligue Contrele Cancer; Chancellerie des Universites de Paris; Projekt DEAL

Individuals who score high in self-reported Intolerance of Uncertainty (IU) display difficulties updating threat associations to safe associations. Here we sought to determine whether individuals who score high in IU can learn and retain new safety associations if given more exposure. We recorded skin conductance response, pupil dilation and expectancy ratings during an associative threat learning task with acquisition, same-day extinction and next-day extinction phases. Participants (n = 144) were assigned to either a regular exposure (32 trials of same-day and next-day extinction) or extended exposure condition (48 trials of same-day and next-day extinction). We failed to replicate previous work showing that IU is associated with poorer safety-learning indexed via SCR, although the results were at trend and in the expected direction. We found preliminary evidence for promoted safety-retention in individuals with higher Inhibitory IU in the extended exposure condition, relative to individuals with higher Inhibitory IU in the regular exposure condition, indexed via SCR. These findings further our current understanding of the role of IU in safety-learning and -retention, informing models of IU and exposure-based treatments.

Fibroblasts play an important role in lung homeostasis and disease. In lung fibrosis, fibroblasts adopt a proliferative and migratory phenotype, with increased expression of alpha-smooth muscle actin (alpha SMA) and enhanced secretion of extracellular matrix components. Comprehensive profiling of fibroblast heterogeneity is limited because of a lack of specific cell-surface markers. We have previously profiled the surface proteome of primary human lung fibroblasts. Here, we sought to define and quantify a panel of cluster of differentiation (CD) markers in primary human lung fibroblasts and idiopathic pulmonary fibrosis (IPF) lung tissue, using immunofluorescence and FACS analysis. Fibroblast function was assessed by analysis of replicative senescence. We observed the presence of distinct fibroblast phenotypes in vivo, characterized by various combinations of Desmin, alpha SMA, CD36, or CD97 expression. Most markers demonstrated stable expression over passages in vitro, but significant changes were observed for CD36, CD54, CD82, CD106, and CD140a. Replicative senescence of fibroblasts was observed from passage 10 onward. CD36- and CD97-positive but alpha SMA-negative cells were present in remodeled areas of IPF lungs. Transforming growth factor (TGF)-beta treatment induced alpha SMA and collagen I expression but repressed CD36 and CD97 expression. We identified a panel of stable surface markers in human lung fibroblasts, applicable for positive-cell isolation directly from lung tissue. TGF-beta exposure represses CD36 and CD97 expression, despite increasing alpha SMA expression; we therefore identified complex surface protein changes during fibroblast-myofibroblast activation. Coexistence of quiescence and activated fibroblast subtypes in the IPF lung suggests dynamic remodeling of fibroblast activation upon subtle changes to growth factor exposure in local microenvironmental niches.

Background Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease. Repetitive injury and reprogramming of the lung epithelium are thought to be critical drivers of disease progression, contributing to fibroblast activation, extracellular matrix remodeling, and subsequently loss of lung architecture and function. To date, Pirfenidone and Nintedanib are the only approved drugs known to decelerate disease progression, however, if and how these drugs affect lung epithelial cell function, remains largely unexplored. Methods We treated murine and human 3D ex vivo lung tissue cultures (3D-LTCs; generated from precision cut lung slices (PCLS)) as well as primary murine alveolar epithelial type II (pmATII) cells with Pirfenidone or Nintedanib. Murine 3D-LTCs or pmATII cells were derived from the bleomycin model of fibrosis. Early fibrotic changes were induced in human 3D-LTCs by a mixture of profibrotic factors. Epithelial and mesenchymal cell function was determined by qPCR, Western blotting, Immunofluorescent staining, and ELISA. Results Low μM concentrations of Nintedanib (1 μM) and mM concentrations of Pirfenidone (2.5 mM) reduced fibrotic gene expression including Collagen 1a1 and Fibronectin in murine and human 3D-LTCs as well as pmATII cells. Notably, Nintedanib stabilized expression of distal lung epithelial cell markers, especially Surfactant Protein C in pmATII cells as well as in murine and human 3D-LTCs. Conclusions Pirfenidone and Nintedanib exhibit distinct effects on murine and human epithelial cells, which might contribute to their anti-fibrotic action. Human 3D-LTCs represent a valuable tool to assess anti-fibrotic mechanisms of potential drugs for the treatment of IPF patients. Electronic supplementary material The online version of this article (10.1186/s12931-018-0876-y) contains supplementary material, which is available to authorized users.

Blockade of lymphotoxin beta-receptor (LT beta R) signalling restores WNT signalling and epithelial repair in a model of chronic obstructive pulmonary disease. Lymphotoxin beta-receptor (LT beta R) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures(1,2), which are associated with severe chronic inflammatory diseases that span several organ systems(3-6). How LT beta R signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LT beta R blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LT beta R ligands in adaptive and innate immune cells, enhanced non-canonical NF-kappa B signalling, and enriched LT beta R target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LT beta R signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LT beta R signalling dampened epithelial non-canonical activation of NF-kappa B, reduced TGF beta signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/beta-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LT beta R signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures(1) and inhibition of apoptosis with tissue-regenerative strategies.

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue's stiffness, which is crucial for subsequent slicing. 500 µm thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Previous studies of change blindness have suggested a distinction between detection and localisation of changes in a visual scene. Using a simple paradigm with an array of coloured squares, the present study aimed to further investigate differences in event-related potentials (ERPs) between trials in which participants could detect the presence of a colour change but not identify the location of the change (sense trials), versus those where participants could both detect and localise the change (localise trials). Individual differences in performance were controlled for by adjusting the difficulty of the task in real time. Behaviourally, reaction times for sense, blind, and false alarm trials were distinguishable when comparing across levels of participant certainty. In the EEG data, we found no significant differences in the visual awareness negativity ERP, contrary to previous findings. In the N2pc range, both awareness conditions (localise and sense) were significantly different to trials with no change detection (blind trials), suggesting that this ERP is not dependent on explicit awareness. Within the late parietal positivity range, all conditions were significantly different. These results suggest that changes can be ‘sensed’ without knowledge of the location of the changing object, and that participant certainty scores can provide valuable information about the perception of changes in change blindness.

Transforming growth factor-β (TGF-β)-induced fibroblast-to-myofibroblast differentiation contributes to remodeling in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis, but whether this impacts the ability of fibroblasts to support lung epithelial repair remains little explored. We pretreated human lung fibroblasts [primary (phFB) or MRC5 cells] with recombinant human TGF-β to induce myofibroblast differentiation, then cocultured them with adult mouse lung epithelial cell adhesion molecule-positive cells (EpCAM+) to investigate their capacity to support epithelial organoid formation in vitro. While control phFB and MRC5 lung fibroblasts supported organoid formation of mouse EpCAM+cells, TGF-β pretreatment of both phFB and MRC5 impaired organoid-supporting ability. We performed RNA sequencing of TGF-β-treated phFB, which revealed altered expression of key Wnt signaling pathway components and Wnt/β-catenin target genes, and modulated expression of secreted factors involved in mesenchymal-epithelial signaling. TGF-β profoundly skewed the transcriptional program induced by the Wnt/β-catenin activator CHIR99021. Supplementing organoid culture media recombinant hepatocyte growth factor or fibroblast growth factor 7 promoted organoid formation when using TGF-β pretreated fibroblasts. In conclusion, TGF-β-induced myofibroblast differentiation results in Wnt/β-catenin pathway skewing and impairs fibroblast ability to support epithelial repair likely through multiple mechanisms, including modulation of secreted growth factors.

RATIONALE\nChronic obstructive pulmonary disease patients and in particular smokers are more susceptible to respiratory infections contributing to acute exacerbations of disease. The immunoproteasome is a specialized type of proteasome destined to improve major histocompatibility complex (MHC) class I-mediated antigen presentation for the resolution of intracellular infections.\nOBJECTIVES\nTo characterize immunoproteasome function in COPD and its regulation by cigarette smoke.\nMETHODS\nImmunoproteasome expression and activity were determined in bronchoalveolar lavage (BAL) and lungs of human donors, COPD, and IPF patients, as well as in cigarette smoke-exposed mice. Smoke-mediated alteration of immunoproteasome activity and MHC I surface expression were analysed in human blood-derived macrophages. Immunoproteasome-specific MHC I antigen presentation was evaluated in spleen and lung immune cells that had been smoke-exposed in vitro or in vivo.\nMEASUREMENTS AND MAIN RESULTS\nImmunoproteasome and MHC I mRNA expression was reduced in BAL cells of COPD patients and in isolated alveolar macrophages of COPD and IPF patients. Exposure of immune cells to cigarette smoke extract in vitro reduced immunoproteasome activity and impaired immunoproteasome-specific MHC I antigen presentation. In vivo, acute cigarette smoke exposure dynamically regulated immunoproteasome function and MHC I antigen presentation in mouse BAL cells. End-stage COPD lungs showed markedly impaired immunoproteasome activities.\nCONCLUSIONS\nWe here show for the first time that the activity of the immunoproteasome is impaired by cigarette smoke resulting in reduced MHC I antigen presentation. Regulation of immunoproteasome function by cigarette smoke may thus alter adaptive immune responses and add to prolonged infections and exacerbations in COPD and IPF.

// Arne Warth 1, # , Volker Endris 1 , Albrecht Stenzinger 1 , Roland Penzel 1 , Alexander Harms 1 , Thomas Duell 2 , Amir Abdollahi 3, + , Michael Lindner 4 , Peter Schirmacher 1 , Thomas Muley 5, # , Hendrik Dienemann 6, # , Ludger Fink 7, # , Alicia Morresi-Hauf 8 , Nicole Pfarr 1, 10, § , Wilko Weichert 1, 9, 10, +, § 1 Institute of Pathology, Heidelberg University, Heidelberg, Germany 2 Department of Pneumology and Thoracic Oncology, Asklepios Hospital, Munich-Gauting, Germany 3 Department of Radiation Oncology, Heidelberg University, Heidelberg, Germany 4 Department of Thoracic Surgery, Asklepios Hospital, Munich-Gauting, Germany 5 Translational Research Unit, Thoraxklinik at Heidelberg University, Heidelberg, Germany 6 Department of Thoracic Surgery, Thoraxklinik at Heidelberg University, Heidelberg, Germany 7 Institute of Pathology, Wetzlar, Germany 8 Institute of Pathology, Asklepios Hospital, Munich-Gauting, Germany 9 National Center for Tumor Diseases (NCT), Heidelberg, Germany 10 Institute of Pathology, Technical University (TUM), Munich, Germany # Translational Lung Research Centre Heidelberg, Member of the German Center for Lung Research (DZL-TLRC) + Member of the German Cancer Consortium (DKTK) § shared senior authorship Correspondence to: Arne Warth, email: arne.warth@med.uni-heidelberg.de Wilko Weichert, email: wilko.weichert@tum.de Keywords: lung cancer, molecular evolution, driver mutations, therapy Received: November 06, 2015 Accepted: March 28, 2016 Published: April 20, 2016 ABSTRACT Background: Large scale sequencing efforts defined common molecular alterations in non-small cell lung cancer (NSCLC) and revealed potentially druggable mutations. Yet, systematic data on the changes of the respective molecular profiles under standard therapy in NSCLC are limited. Results: 14 out of 68 observed coding mutations (21%) and 6 out of 33 (18%) copy number variations (CNV) were lost or gained during therapy. Mutational and CNV changes clustered in 6/37 (16%) and 3/37 (8%) patients. Changes in clinically relevant mutations were rare but present in single cases for genes such as BRAF and PIK3CA . The type of radiochemotherapy but not the duration of therapy impacted on the frequency of mutational changes. Methods: We established a lung cancer specific next-generation sequencing panel covering ~7500 hotspots of 41 genes frequently mutated in NSCLC and performed ultradeep multigene sequencing of 37 corresponding pre- and post-therapeutic formalin fixed paraffin-embedded specimens to discover mutational changes and copy number variations under neo-adjuvant radio- (RTX) and/or chemotherapy (CTX). Conclusion: We unraveled changes in common driver gene candidates in NSCLC under neo-adjuvant therapy. Our data shed first light on the genetic changes of NSCLC under conventional therapy and might be taken into account when the relevance of sequential biopsy approaches is discussed.

Human sample, from patients or healthy donors, are a valuable link between basic research and clinic. Especially in translational research, they play an essential role in understanding development and progression of diseases as well as in developing new diagnostic and therapeutic tools. Stored in biobanks, fast access to appropriate material becomes possible. However, biobanking in a clinical context faces several challenges. In practice, collecting samples during clinical routine does not allow to strictly adhere to protocols of sample collection in all aspects. This may influence sample quality to variable degrees. Time from sample draw to asservation is a variable factor, and influences of prolonged storage at ambient temperature of tissues are not well understood. We investigated whether delays between 5 minutes and 3 hours, and the use of RNAlater RNA-preserving reagent would lead to a relevant drop in sample quality, measured by quantitative mRNA expression analysis. Our findings suggest that even under ambient conditions, delays up to 3 hours do not have a major impact on sample quality as long as the tissue remains intact.

Simultaneous multi-slice (SMS) imaging is a popular technique for increasing acquisition speed in echo-planar imaging (EPI) fMRI. However, SMS data are prone to motion sensitivity and slice leakage artefacts, which spread signal between simultaneously acquired slices. Relevant to motion sensitivity, artefacts from moving anatomic structures propagate along the phase-encoding (PE) direction. This is particularly relevant for eye movement. As signal from the eye is acquired along with signal from simultaneously excited slices during SMS, there is potential for signal to spread in-plane and between spatially remote slices. After identifying an artefact temporally coinciding with signal fluctuations in the eye and spatially distributed in correspondence with multiband slice acceleration and parallel imaging factors, we conducted a series of small experiments to investigate eye movement artefacts in SMS data and the contribution of PE direction to the invasiveness of these artefacts. Five healthy adult volunteers were scanned during a blinking task using a standard SMS-EPI protocol with posterior-to-anterior (P ≫ A), anterior-to-posterior (A ≫ P) or right-to-left (R ≫ L) PE direction. The intensity of signal fluctuations (artefact severity) was measured at expected artefact positions and control positions. We demonstrated a direct relationship between eye movements and artefact severity across expected artefact regions. Within-brain artefacts were apparent in P ≫ A- and A ≫ P-acquired data but not in R ≫ L data due to the shift in artefact positions. Further research into eye motion artefacts in SMS data is warranted but researchers should exercise caution with SMS protocols. We recommend rigorous piloting of SMS protocols and switching to R ≫ L/L ≫ R PE where feasible.

Animal studies have shown that acetylcholine (ACh) levels in the dorsal striatum play a role in reversal learning. However, this has not been studied in humans due to a lack of appropriate non-invasive techniques. Proton magnetic resonance spectroscopy (1H-MRS) can be used to measure metabolite levels in humans in vivo. Although it cannot be used to study ACh directly, 1H-MRS can be used to study choline, an ACh precursor which is linked to activity-dependent ACh release. The aim of this study was to use functional-1H-MRS (fMRS) to measure changes in choline levels in the human dorsal striatum during performance of a probabilistic reversal learning task. We demonstrate a task-dependent decrease in choline, specifically during reversal, but not initial, learning. We interpret this to reflect a sustained increase in ACh levels, which is in line with findings from the animal literature. This task-dependent change was specific to choline and was not observed in control metabolites. These findings provide support for the use of fMRS in the in vivo study of the human cholinergic system.

Background In idiopathic pulmonary fibrosis (IPF), fibroblasts gain a more migratory phenotype and excessively secrete extracellular matrix (ECM), ultimately leading to alveolar scarring and progressive dyspnea. Here, we analyzed the effects of deficiency of FK506-binding protein 10 (FKBP10), a potential IPF drug target, on primary human lung fibroblast (phLF) adhesion and migration. Methods Using siRNA, FKBP10 expression was inhibited in phLF in absence or presence of 2ng/ml transforming growth factor-β1 (TGF-β1) and 0.1mM 2-phosphoascorbate. Effects on cell adhesion and migration were monitored by an immunofluorescence (IF)-based attachment assay, a conventional scratch assay, and single cell tracking by time-lapse microscopy. Effects on expression of key players in adhesion dynamics and migration were analyzed by qPCR and Western Blot. Colocalization was evaluated by IF microscopy and by proximity ligation assays. Results FKBP10 knockdown significantly attenuated adhesion and migration of phLF. Expression of collagen VI was decreased, while expression of key components of the focal adhesion complex was mostly upregulated. The effects on migration were 2-phosphoascorbate-dependent, suggesting collagen synthesis as the underlying mechanism. FKBP10 colocalized with collagen VI and coating culture dishes with collagen VI, and to a lesser extent with collagen I, abolished the effect of FKBP10 deficiency on migration. Conclusions These findings show, to our knowledge for the first time, that FKBP10 interacts with collagen VI and that deficiency of FKBP10 reduces phLF migration mainly by downregulation of collagen VI synthesis. The results strengthen FKBP10 as an important intracellular regulator of ECM remodeling and support the concept of FKBP10 as drug target in IPF.

Cues from the extracellular matrix (ECM) and their functional interplay with cells play pivotal roles for development, tissue repair, and disease. However, the precise nature of this interplay remains elusive. We used an innovative 3D cell culture ECM model by decellularizing 300-µm-thick ex vivo lung tissue scaffolds (d3D-LTCs) derived from diseased and healthy mouse lungs, which widely mimics the native (patho)physiological in vivo ECM microenvironment. We successfully repopulated all d3D-LTCs with primary human and murine fibroblasts, and moreover, we demonstrated that the cells also populated the innermost core regions of the d3D-LTCs in a real 3D fashion. The engrafted fibroblasts revealed a striking functional plasticity, depending on their localization in distinct ECM niches of the d3D-LTCs, affecting the cells’ tissue engraftment, cellular migration rates, cell morphologies, and protein expression and phosphorylation levels. Surprisingly, we also observed fibroblasts that were homing to the lung scaffold’s interstitium as well as fibroblasts that were invading fibrotic areas. To date, the functional nature and even the existence of 3D cell matrix adhesions in vivo as well as in 3D culture models is still unclear and controversial. Here, we show that attachment of fibroblasts to the d3D-LTCs evidently occurred via focal adhesions, thus advocating for a relevant functional role in vivo. Furthermore, we found that protein levels of talin, paxillin, and zyxin and phosphorylation levels of paxillin Y118, as well as the migration-relevant small GTPases RhoA, Rac, and CDC42, were significantly reduced compared with their attachment to 2D plastic dishes. In summary, our results strikingly indicate that inherent physical or compositional characteristics of the ECM act as instructive cues altering the functional behavior of engrafted cells. Thus, d3D-LTCs might aid to obtain more realistic data in vitro, with a high relevance for drug discovery and mechanistic studies alike.

Chronic obstructive pulmonary disease (COPD) is characterised by a progressive loss of lung tissue. Inducing repair processes within the adult diseased lung is of major interest and Wnt/β-catenin signalling represents a promising target for lung repair. However, the translation of novel therapeutic targets from model systems into clinical use remains a major challenge.We generated murine and patient-derived three-dimensional (3D) ex vivo lung tissue cultures (LTCs), which closely mimic the 3D lung microenvironment in vivo. Using two well-known glycogen synthase kinase-3β inhibitors, lithium chloride (LiCl) and CHIR 99021 (CT), we determined Wnt/β-catenin-driven lung repair processes in high spatiotemporal resolution using quantitative PCR, Western blotting, ELISA, (immuno)histological assessment, and four-dimensional confocal live tissue imaging.Viable 3D-LTCs exhibited preserved lung structure and function for up to 5 days. We demonstrate successful Wnt/β-catenin signal activation in murine and patient-derived 3D-LTCs from COPD patients. Wnt/β-catenin signalling led to increased alveolar epithelial cell marker expression, decreased matrix metalloproteinase-12 expression, as well as altered macrophage activity and elastin remodelling. Importantly, induction of surfactant protein C significantly correlated with disease stage (per cent predicted forced expiratory volume in 1 s) in patient-derived 3D-LTCs.Patient-derived 3D-LTCs represent a valuable tool to analyse potential targets and drugs for lung repair. Enhanced Wnt/β-catenin signalling attenuated pathological features of patient-derived COPD 3D-LTCs.

Nanoparticles allow for controlled and targeted drug delivery to diseased tissues and therefore bypass systemic side effects. Spatiotemporal control of drug release can be achieved by nanocarriers that respond to elevated levels of disease-specific enzymes. For example, matrix metalloproteinase 9 (MMP9) is overexpressed in tumors, is known to enhance the metastatic potency of malignant cells, and has been associated with poor prognosis of lung cancer. Here, we report the synthesis of mesoporous silica nanoparticles (MSNs) tightly capped by avidin molecules via MMP9 sequence-specific linkers to allow for site-selective drug delivery in high-expressing MMP9 tumor areas. We provide proof-of-concept evidence for successful MMP9-triggered drug release from MSNs in human tumor cells and in mouse and human lung tumors using the novel technology of ex vivo 3D lung tissue cultures. This technique allows for translational testing of drug delivery strategies in diseased mouse and human tissue. Using this method we show MMP9-mediated release of cisplatin, which induced apoptotic cell death only in lung tumor regions of Kras mutant mice, without causing toxicity in tumor-free areas or in healthy mice. The MMP9-responsive nanoparticles also allowed for effective combinatorial drug delivery of cisplatin and proteasome inhibitor bortezomib, which had a synergistic effect on the (therapeutic) efficiency. Importantly, we demonstrate the feasibility of MMP9-controlled drug release in human lung tumors.

Leiomyoma of the pulmonary artery represents a curiosity in the literature. We describe a case of a 54-year-old female patient who presented with recurrent cough of a few weeks' duration. Computed tomography of the thorax located a smooth, limited tumor in the left thorax near the interlobar space. Thoracoscopic exploration showed a tumor mass, fused with the pulmonary artery. After anterolateral thoracotomy, a complete resection of the tumor was performed. The histopathologic examination showed the presence of a vascular leiomyoma of the tunica media of the pulmonary artery.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with poor prognosis and limited therapeutic options. The incidence of IPF increases with age, and ageing-related mechanisms such as cellular senescence have been proposed as pathogenic drivers. The lung alveolar epithelium represents a major site of tissue injury in IPF and senescence of this cell population is probably detrimental to lung repair. However, the potential pathomechanisms of alveolar epithelial cell senescence and the impact of senolytic drugs on senescent lung cells and fibrosis remain unknown. Here we demonstrate that lung epithelial cells exhibit increased P16 and P21 expression as well as senescence-associated β-galactosidase activity in experimental and human lung fibrosis tissue and primary cells. Primary fibrotic mouse alveolar epithelial type (AT)II cells secreted increased amounts of senescence-associated secretory phenotype (SASP) factors in vitro, as analysed using quantitative PCR, mass spectrometry and ELISA. Importantly, pharmacological clearance of senescent cells by induction of apoptosis in fibrotic ATII cells or ex vivo three-dimensional lung tissue cultures reduced SASP factors and extracellular matrix markers, while increasing alveolar epithelial markers. These data indicate that alveolar epithelial cell senescence contributes to lung fibrosis development and that senolytic drugs may be a viable therapeutic option for IPF., Alveolar epithelial cell senescence occurs in IPF and senolytic treatment attenuates experimental lung fibrosis http://ow.ly/nFlz30bsmNm

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic interstitial lung disease (ILD) characterized by lung tissue scarring and high morbidity. Lung epithelial injury, myofibroblast activation, and deranged repair are believed to be key processes involved in disease onset and progression, but the exact molecular mechanisms behind IPF remain unclear. Several drugs have been shown to slow disease progression, but treatments that halt or reverse IPF progression have not been identified. Ex vivo models of human lung have been proposed for drug discovery, one of which is precision-cut lung slices (PCLS). Although PCLS production from IPF explants is possible, IPF explants are rare and typically represent end-stage disease. Here we present a novel model of early fibrosis-like changes in human PCLS derived from patients without ILD/IPF using a combination of profibrotic growth factors and signaling molecules (transforming growth factor-β, tumor necrosis factor-α, platelet-derived growth factor-AB, and lysophosphatidic acid). Fibrotic-like changes of PCLS were qualitatively analyzed by histology and immunofluorescence and quantitatively by water-soluble tetrazolium-1, RT-qPCR, Western blot analysis, and ELISA. PCLS remained viable after 5 days of treatment, and fibrotic gene expression ( FN1, SERPINE1, COL1A1, CTGF, MMP7, and ACTA2) increased as early as 24 h of treatment, with increases in protein levels at 48 h and increased deposition of extracellular matrix. Alveolar epithelium reprogramming was evident by decreases in surfactant protein C and loss of HOPX. In summary, using human-derived PCLS, we established a novel ex vivo model that displays characteristics of early fibrosis and could be used to evaluate novel therapies and study early-stage IPF pathomechanisms.

Introduction: In lung cancer, interleukin-22 (IL-22) expression within primary tissue has been demonstrated, but the frequency and the functional consequence of IL-22 signaling have not been addressed. This study aims at analyzing the cellular effects of IL-22 on lung carcinoma cell lines and the prognostic impact of IL-22 tis- sue expression in lung cancer patients. Methods: Biological effects of IL-22 signaling were investigated in seven lung cancer cell lines by Western blot, flow cytometry, real- time polymerase chain reaction, and proliferation assays. Tumor tissue specimens of two cohorts with a total of 2300 lung cancer patients were tested for IL-22 expression by immunohistochemistry. IL-22 serum concentrations were analyzed in 103 additional patients by enzyme-linked immunosorbent assay. Results: We found the IL-22 receptor 1 (IL-22-R1) to be expressed in six of seven lung cancer cell lines. However IL-22 signaling was functional in only four cell lines, where IL-22 induced signal trans- ducer activator of transcription 3 phosphorylation and increased cell proliferation. Furthermore, IL-22 induced the expression of anti- apoptotic B-cell lymphoma 2, but did not rescue tumor cells from carboplatin-induced apoptosis. Cisplatin-resistant cell lines showed a significant up-regulation of IL-22-R1 along with a stronger prolifera- tive response to IL-22 stimulation. IL-22 was preferentially expressed in small- and large-cell lung carcinoma (58% and 46% of cases, respectively). However, no correlation between IL-22 expression by immunohistochemistry and prognosis was observed. Conclusion: IL-22 is frequently expressed in lung cancer tissue. Enhanced IL-22-R1 expression and signaling in chemotherapy- refractory cell lines are indicative of a protumorigenic function of IL-22 and may contribute to a more aggressive phenotype.

Social comparison, that is, the process of comparing oneself to other people, is a ubiquitous social cognitive mechanism; however, so far its neural correlates have remained unknown. The present study tested the hypothesis that social comparisons are supported by partly dissociated networks, depending on whether the dimension under comparison concerns a physical or a psychological attribute. We measured brain activity with functional MRI, whereas participants were comparing their own height or intelligence to that of individuals they personally know. Height comparisons were associated with higher activity in a frontoparietal network involved in spatial and numerical cognition. Conversely, intelligence comparisons recruited a network of midline areas that have been previously implicated in the attribution of mental states to oneself and others (Theory of mind). These findings suggest that social comparisons rely on diverse domain-specific mechanisms rather than on one unitary process.

TGF-β is important in lung injury and remodeling processes. TGF-β and Wingless/integrase-1 (WNT) signaling are interconnected; however, the WNT ligand-receptor complexes involved are unknown. Thus, we aimed to identify Frizzled (FZD) receptors that mediate TGF-β-induced profibrotic signaling. MRC-5 and primary human lung fibroblasts were stimulated with TGF-β1, WNT-5A, or WNT-5B in the presence and absence of specific pathway inhibitors. Specific small interfering RNA was used to knock down FZD8. In vivo studies using bleomycin-induced lung fibrosis were performed in wild-type and FZD8-deficient mice. TGF-β1 induced FZD8 specifically via Smad3-dependent signaling in MRC-5 and primary human lung fibroblasts. It is noteworthy that FZD8 knockdown reduced TGF-β1-induced collagen Iα1, fibronectin, versican, α-smooth muscle (sm)-actin, and connective tissue growth factor. Moreover, bleomycin-induced lung fibrosis was attenuated in FZD8-deficient mice in vivo. Although inhibition of canonical WNT signaling did not affect TGF-β1-induced gene expression in vitro, noncanonical WNT-5B mimicked TGF-β1-induced fibroblast activation. FZD8 knockdown reduced both WNT-5B-induced gene expression of fibronectin and α-sm-actin, as well as WNT-5B-induced changes in cellular impedance. Collectively, our findings demonstrate a role for FZD8 in TGF-β-induced profibrotic signaling and imply that WNT-5B may be the ligand for FZD8 in these responses.-Spanjer, A. I. R., Baarsma, H. A., Oostenbrink, L. M., Jansen, S. R., Kuipers, C. C., Lindner, M., Postma, D. S., Meurs, H., Heijink, I. H., Gosens, R., Königshoff, M. TGF-β-induced profibrotic signaling is regulated in part by the WNT receptor Frizzled-8.

Item does not contain fulltext This is the first study to combine psychometric and functional neuroimaging methods to study altered patterns of autobiographical memory in bipolar disorder (BD). All participants were interviewed with an expanded version of the Bielefelder Autobiographical Memory Inventory (Bielefelder Autobiographisches Gedachtnis Inventar 2004;Lisse: Swets and Zeitlinger). We then acquired functional magnetic resonance imaging data during a task of individually designed autobiographical recall. Compared with healthy controls, BD patients reported a stronger emotionality of autobiographical memories and more frequent recollections of autobiographical events during their everyday life. Furthermore, they failed to deactivate areas in the cuneus and lingual gyrus and showed decreased activation in the inferior frontal and precentral areas compared with the control group. More frequent intrusions from a person's past, which had a neural correlate in the lack of deactivation in some default mode network areas in BD patients, may contribute to manic or depressive symptoms.

The analysis of cortical and subcortical networks requires the identification of their nodes, and of the topology and dynamics of their interactions. Exploratory tools for the identification of nodes are available, e.g. magnetoencephalography (MEG) in combination with beamformer source analysis. Competing network topologies and interaction models can be investigated using dynamic causal modelling. However, we lack a method for the exploratory investigation of network topologies to choose from the very large number of possible network graphs. Ideally, this method should not require a pre-specified model of the interaction. Transfer entropy--an information theoretic implementation of Wiener-type causality--is a method for the investigation of causal interactions (or information flow) that is independent of a pre-specified interaction model. We analysed MEG data from an auditory short-term memory experiment to assess whether the reconfiguration of networks implied in this task can be detected using transfer entropy. Transfer entropy analysis of MEG source-level signals detected changes in the network between the different task types. These changes prominently involved the left temporal pole and cerebellum--structures that have previously been implied in auditory short-term or working memory. Thus, the analysis of information flow with transfer entropy at the source-level may be used to derive hypotheses for further model-based testing.

Understanding causal relationships, or effective connectivity, between parts of the brain is of utmost importance because a large part of the brain's activity is thought to be internally generated and, hence, quantifying stimulus response relationships alone does not fully describe brain dynamics. Past efforts to determine effective connectivity mostly relied on model based approaches such as Granger causality or dynamic causal modeling. Transfer entropy (TE) is an alternative measure of effective connectivity based on information theory. TE does not require a model of the interaction and is inherently non-linear. We investigated the applicability of TE as a metric in a test for effective connectivity to electrophysiological data based on simulations and magnetoencephalography (MEG) recordings in a simple motor task. In particular, we demonstrate that TE improved the detectability of effective connectivity for non-linear interactions, and for sensor level MEG signals where linear methods are hampered by signal-cross-talk due to volume conduction.

Extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN, CD147) is a multifunctional protein that has been implicated in cancer invasion and metastasis by the induction of MMPs. To address its role in primary tumors of human non-small-cell lung cancer we assessed whether EMMPRIN expression is associated with the expression of MMP-2 and MMP-9 and with patient survival. Primary tumors of 150 patients (65 adenocarcinomas, 58 squamous cell carcinomas, and 27 of other subtypes) with completely resected lung cancers were stained by immunohistochemistry. We assessed intensity, extent, and cellular localization of EMMPRIN staining and determined MMP-2 and MMP-9 expression. 145 tumors expressed EMMPRIN (strong expression in 61 tumors), which was predominantly localized at the tumor cell membranes in 102 (68%) patients. We could not determine any correlation between EMMPRIN expression and MMP-2 or MMP-9 expression. The prognostic relevance of EMMPRIN was evaluated by Kaplan-Meier and multivariate Cox regression analysis in patients with adenocarcinoma (n=57) and squamous cell carcinoma of the lung (n=56). The median follow-up period was 36.0 months (range 4-156 months). Staining scores for EMMPRIN and MMP-2 and MMP-9 derived from staining intensities and percentages of positive cells did not predict outcome of patients. In contrast, univariate survival analysis demonstrated that membranous localization of EMMPRIN was associated with shortened survival in patients with adenocarcinoma (P=0.03; log-rank test), but not in squamous cell carcinoma. For the former patients, membranous EMMPRIN expression was also an independent predictor of patient survival (P=0.04; Cox regression analysis). The findings point to a role of EMMPRIN for the progression of adenocarcinoma of the lung that is unrelated to its function as inducer of MMPs.

During the last decades, the study of cell behavior was largely accomplished in uncoated or extracellular matrix (ECM)-coated plastic dishes. To date, considerable cell biological efforts have tried to model in vitro the natural microenvironment found in vivo. For the lung, explants cultured ex vivo as lung tissue cultures (LTCs) provide a three-dimensional (3D) tissue model containing all cells in their natural microenvironment. Techniques for assessing the dynamic live interaction between ECM and cellular tissue components, however, are still missing. Here, we describe specific multidimensional immunolabeling of living 3D-LTCs, derived from healthy and fibrotic mouse lungs, as well as patient-derived 3D-LTCs, and concomitant real-time four-dimensional multichannel imaging thereof. This approach allowed the evaluation of dynamic interactions between mesenchymal cells and macrophages with their ECM. Furthermore, fibroblasts transiently expressing focal adhesions markers incorporated into the 3D-LTCs, paving new ways for studying the dynamic interaction between cellular adhesions and their natural-derived ECM. A novel protein transfer technology (FuseIt/Ibidi) shuttled fluorescently labeled α-smooth muscle actin antibodies into the native cells of living 3D-LTCs, enabling live monitoring of α-smooth muscle actin-positive stress fibers in native tissue myofibroblasts residing in fibrotic lesions of 3D-LTCs. Finally, this technique can be applied to healthy and diseased human lung tissue, as well as to adherent cells in conventional two-dimensional cell culture. This novel method will provide valuable new insights into the dynamics of ECM (patho)biology, studying in detail the interaction between ECM and cellular tissue components in their natural microenvironment.

Increased abundance and stiffness of the extracellular matrix, in particular collagens, is a hallmark of idiopathic pulmonary fibrosis (IPF). FK506-binding protein 10 (FKBP10) is a collagen chaperone, mutations of which have been indicated in the reduction of extracellular matrix stiffness (e.g., in osteogenesis imperfecta).To assess the expression and function of FKBP10 in IPF.We assessed FKBP10 expression in bleomycin-induced lung fibrosis (using quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunofluorescence), analyzed microarray data from 99 patients with IPF and 43 control subjects from a U.S. cohort, and performed Western blot analysis from 6 patients with IPF and 5 control subjects from a German cohort. Subcellular localization of FKBP10 was assessed by immunofluorescent stainings. The expression and function of FKBP10, as well as its regulation by endoplasmic reticulum stress or transforming growth factor-β1, was analyzed by small interfering RNA-mediated loss-of-function experiments, quantitative reverse transcriptase-polymerase chain reaction, Western blot, and quantification of secreted collagens in the lung and in primary human lung fibroblasts (phLF). Effects on collagen secretion were compared with those of the drugs nintedanib and pirfenidone, recently approved for IPF.FKBP10 expression was up-regulated in bleomycin-induced lung fibrosis and IPF. Immunofluorescent stainings demonstrated localization to interstitial (myo)fibroblasts and CD68(+) macrophages. Transforming growth factor-β1, but not endoplasmic reticulum stress, induced FKBP10 expression in phLF. The small interfering RNA-mediated knockdown of FKBP10 attenuated expression of profibrotic mediators and effectors, including collagens I and V and α-smooth muscle actin, on the transcript and protein level. Importantly, loss of FKBP10 expression significantly suppressed collagen secretion by phLF.FKBP10 might be a novel drug target for IPF.

Humans often evaluate their abilities by comparing their personal performance with that of others. For this process, it is critical whether the comparison turns out in one’s favor or against it. Here, we investigate how social comparisons of performance are encoded and integrated on the neural level. We collected functional magnetic resonance images while subjects answered questions in a knowledge quiz that was related to their profession. After each question, subjects received a feedback about their personal performance, followed by a feedback about the performance of a reference group who had been quizzed beforehand. Based on the subjects’ personal performance, we divided trials in downward and upward comparisons. We found that upward comparisons correlated with activity in the dorsolateral prefrontal cortex and the anterior insula. Downward comparisons were associated with increased activation in the ventral striatum (VS), the medial orbitofrontal cortex and the ventral anterior cingulate cortex (ACC). The extent to which subjects outperformed the reference group modulated the activity in the VS and in the dorsal ACC. We suggest that the co-activation of the VS and the dorsal ACC contributes to the integration of downward comparisons into the evaluation of personal performance.

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Deltaex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

In complex networks such as gene networks, traffic systems or brain circuits it is important to understand how long it takes for the different parts of the network to effectively influence one another. In the brain, for example, axonal delays between brain areas can amount to several tens of milliseconds, adding an intrinsic component to any timing-based processing of information. Inferring neural interaction delays is thus needed to interpret the information transfer revealed by any analysis of directed interactions across brain structures. However, a robust estimation of interaction delays from neural activity faces several challenges if modeling assumptions on interaction mechanisms are wrong or cannot be made. Here, we propose a robust estimator for neuronal interaction delays rooted in an information-theoretic framework, which allows a model-free exploration of interactions. In particular, we extend transfer entropy to account for delayed source-target interactions, while crucially retaining the conditioning on the embedded target state at the immediately previous time step. We prove that this particular extension is indeed guaranteed to identify interaction delays between two coupled systems and is the only relevant option in keeping with Wiener’s principle of causality. We demonstrate the performance of our approach in detecting interaction delays on finite data by numerical simulations of stochastic and deterministic processes, as well as on local field potential recordings. We also show the ability of the extended transfer entropy to detect the presence of multiple delays, as well as feedback loops. While evaluated on neuroscience data, we expect the estimator to be useful in other fields dealing with network dynamics.

Non-Small-Cell-Lung-Cancer (NSCLC) represents approximately 85% of all lung cancers and remains poorly understood. While signaling pathways operative during organ development, including Sonic Hedgehog (Shh) and associated Gli transcription factors (Gli1-3), have recently been found to be reactivated in NSCLC, their functional role remains unclear. Here, we hypothesized that Shh/Gli1-3 could mediate NSCLC autonomous proliferation and epithelial/stromal signaling in the tumoral tissue. In this context, we have investigated the activity of Shh/Gli1-3 signaling in NSCLC in both, cancer and stromal cells. We report here that inhibition of Shh signaling induces a significant decrease in the proliferation of NSCLC cells. This effect is mediated by Gli1 and Gli2, but not Gli3, through regulation of cyclin D1 and cyclin D2 expression. While exogenous Shh was unable to induce signaling in either A549 lung adenocarcinoma or H520 lung squamous carcinoma cells, both cells were found to secrete Shh ligand, which induced fibroblast proliferation, survival, migration, invasion, and collagen synthesis. Furthermore, Shh secreted by NSCLC mediates the production of proangiogenic and metastatic factors in lung fibroblasts. Our results thus provide evidence that Shh plays an important role in mediating epithelial/mesenchymal crosstalk in NSCLC. While autonomous Gli activity controls NSCLC proliferation, increased Shh expression by NSCLC is associated with fibroblast activation in tumor-associated stroma. Our study highlights the relevance of studying stromal-associated cells in the context of NSCLC regarding new prognosis and therapeutic options.

To-date, most invasion or migration assays use a modified Boyden chamber-like design to assess migration as single-cell or scratch assays on coated or uncoated planar plastic surfaces. Here, we describe a 96-well microplate-based, high-content, three-dimensional cell culture assay capable of assessing invasion dynamics and molecular signatures thereof. On applying our invasion assay, we were able to demonstrate significant effects on the invasion capacity of fibroblast cell lines, as well as primary lung fibroblasts. Administration of epidermal growth factor resulted in a substantial increase of cellular invasion, thus making this technique suitable for high-throughput pharmacological screening of novel compounds regulating invasive and migratory pathways of primary cells. Our assay also correlates cellular invasiveness to molecular events. Thus, we argue of having developed a powerful and versatile toolbox for an extensive profiling of invasive cells in a 96-well format. This will have a major impact on research in disease areas like fibrosis, metastatic cancers, or chronic inflammatory states.

Individuals with anorexia nervosa (AN) and bulimia nervosa (BN) tend to have disordered thinking and eating behaviours in regards to fat containing foods. This is the first study to investigate neuronal pathways that may contribute to altered fat consumption in eating disordered patients.We used functional magnetic resonance imaging (fMRI) to compare responses to a high-fat cream stimulus, water, and a non-caloric viscous stimulus (CMC) to control for response to viscosity in individuals recovered from AN (N = 15), BN (N = 14) and a healthy control sample (CW, N = 18).An interaction analysis (ANOVAR) comparing the three groups (AN, BN, CW) and the three conditions (cream, CMC, water) revealed significant differences in the left anterior ventral striatum (AVS). A post hoc analysis displayed a higher magnitude of response for the contrast cream/water in BN compared to AN or CW and for the contrast CMC/water in BN compared to AN.BN showed an exaggerated AVS response for the cream/water contrast in comparison to AN or CW. Moreover, BN showed an exaggerated AVS response for the CMC/water contrast in comparison to AN. These findings support the possibility that BN have an altered hedonic and/or motivational drive to consume fats.

Background Transfer entropy (TE) is a measure for the detection of directed interactions. Transfer entropy is an information theoretic implementation of Wiener's principle of observational causality. It offers an approach to the detection of neuronal interactions that is free of an explicit model of the interactions. Hence, it offers the power to analyze linear and nonlinear interactions alike. This allows for example the comprehensive analysis of directed interactions in neural networks at various levels of description. Here we present the open-source MATLAB toolbox TRENTOOL that allows the user to handle the considerable complexity of this measure and to validate the obtained results using non-parametrical statistical testing. We demonstrate the use of the toolbox and the performance of the algorithm on simulated data with nonlinear (quadratic) coupling and on local field potentials (LFP) recorded from the retina and the optic tectum of the turtle (Pseudemys scripta elegans) where a neuronal one-way connection is likely present. Results In simulated data TE detected information flow in the simulated direction reliably with false positives not exceeding the rates expected under the null hypothesis. In the LFP data we found directed interactions from the retina to the tectum, despite the complicated signal transformations between these stages. No false positive interactions in the reverse directions were detected. Conclusions TRENTOOL is an implementation of transfer entropy and mutual information analysis that aims to support the user in the application of this information theoretic measure. TRENTOOL is implemented as a MATLAB toolbox and available under an open source license (GPL v3). For the use with neural data TRENTOOL seamlessly integrates with the popular FieldTrip toolbox.

To prevent postpneumonectomy bronchopleural fistula, coverage of the bronchial stump is recommended, especially for patients treated with neoadjuvant and adjuvant chemotherapy or radiochemotherapy. We compared outcomes after proximal pericardial fat pad coverage and coverage with pleura and surrounding tissues, by retrospective analysis of the records of 243 patients. Postpneumonectomy bronchopleural fistula occurred in 7/143 (4.9%) patients who had pericardial fat pad coverage, and in 6/100 (6.0%) treated by pleural covering. Bronchopleural fistula occurred in 11 patients within 21 days, in one after 2 months, and one after 6 months. Univariate analysis of comorbidities and risk factors did not show any significant differences between the groups. Advanced T stage and carcinomatous lymphangiosis at the resection margin were associated with a higher risk of bronchopleural fistula development, independent of the technique. Reinforcement of the bronchial stump by proximal pericardial fat pad coverage appears to be safe and feasible. It is comparable to coverage with pleura and surrounding tissues.

The purpose of this study was to examine our results of combined resection of the atrium and non-small-cell lung cancer using a concurrent and continuously updated database.A total of 35 patients underwent extended pulmonary resection with partial resection of the atrium. The main focus of the study was to define subgroups of patients who can potentially benefit from surgery.Pneumonectomy was performed in 31 cases, and the other 4 patients underwent a lesser resection. Postoperative morbidity was 20%, and the mortality rate was 9%. The median intensive care unit stay was 2 days and the hospital stay 13 days. The survival rates were 80% at 1 year, 21% at 3 years, and 16% at 5 years. The median survival of patients with low-grade tumors (G1/2) was 27 months, contrasted by only 15 months' survival for patients with high-grade tumors (P = 0.026). Multivariate analysis indicated that completeness of resection had a significant impact on survival (P = 0.042).Combined resection of lung and atrium is a complex surgical procedure, but it can be performed with fair morbidity and mortality rates, even in patients with an increased number of preoperative risk factors. Patients suffering from low-grade tumors benefit significantly from radical surgery. Future studies must define whether a multimodal therapeutic approach that includes induction therapy can prolong patient survival.

Laterality is a characteristic principle of the organization of the brain systems for language, and reduced hemispheric asymmetry has been considered a risk factor for schizophrenia. Here we sought support for the risk factor hypothesis by investigating whether reduced asymmetry of temporal lobe structure and function is also present in unaffected relatives. Sixteen schizophrenia patients, 16 age-matched first-degree relatives, and 15 healthy controls underwent high-resolution three-dimensional anatomical imaging and functional magnetic resonance imaging during auditory stimulation. Both the overall auditory cortex and planum temporale volumes and the lateralization to the left hemisphere were markedly reduced in patients. The decrease of lateralization correlated with increased severity of symptoms. In addition, both the overall functional activation in response to auditory stimulation and its asymmetry were reduced in the patients. Relatives had intermediate values between patients and controls on both structural and functional measures. This study provides added support for the idea that reduced hemispheric asymmetry is a biological risk factor for schizophrenia.

Person comparison is pervasive in social judgment and human decision making and yet its neural substrate is poorly explored. We measured brain activity when participants compared psychological (intelligence) and physical (height) characteristics of famous people and found activation of medial frontal, orbitofrontal and limbic areas and the temporoparietal junction. This network was largely driven by the psychological comparison, with activity being higher for intelligence than height comparison in several areas in medial prefrontal cortex, suggesting that their activation scales with the demand on person comparison. The person comparison network overlaps strikingly with that commonly described for classic theory of mind tasks. We interpret this overlap as indexing the use of perspective taking common to person comparison and theory of mind.

We conducted a series of psychophysical experiments to investigate the nature and specificity of behavioral interference between mental and manual rotation. Participants were asked to mentally rotate five different types of visual stimuli--hands, faces, tools, cubes, and natural objects--either clockwise or counterclockwise while they simultaneously manually rotated a wheel in the concordant or discordant direction. Our study clearly revealed object-specific interference between manual and mental rotation. In comparison with a neutral baseline condition without any manual rotation, both manual rotation directions generally impaired the mental rotation of cubes. In contrast, the mental rotation of hands was impaired only by discordant manual rotation. This object- and direction-specific interference between manual and mental rotation proved to be independent of task difficulty. We furthermore found an angular distance effect across different stimulus types.

Recent theoretical and empirical research on schizophrenia converges on the notion that core aspects of the pathophysiology of the disorder may arise from a dysfunction in the coordination of distributed neural activity. Synchronization of neural responses in the β-band (15–30 Hz) and γ-band range (30–80 Hz) has been implicated as a possible neural substrate for dysfunctional coordination in schizophrenia. To test this hypothesis, we examined the electroencephalography (EEG) activity in 19 patients with aDiagnostic and Statistical Manual of Mental Disorder, edition IV criteria, diagnosis of schizophrenia and 19 healthy control subjects during a Gestalt perception task. EEG data were analyzed for phase synchrony and induced spectral power as an index of neural synchronization. Schizophrenia patients were impaired significantly in the detection of images that required the grouping of stimulus elements into coherent object representations. This deficit was accompanied by longer reaction times in schizophrenia patients. Deficits in Gestalt perception in schizophrenia patients were associated with reduced phase synchrony in the β-band (20–30 Hz), whereas induced spectral power in the γ-band (40–70 Hz) was mainly intact. Our findings suggest that schizophrenia patients are impaired in the long-range synchronization of neural responses, which may reflect a core deficit in the coordination of neural activity and underlie the specific cognitive dysfunctions associated with the disorder.

Cancer/germline (CG) antigens represent promising targets for widely applicable mono- and multiantigen cancer vaccines for nonsmall cell lung cancer (NSCLC). Since little is known about their composite expression in this tumor type, we analyzed 7 CG genes (MAGE-A3, NY-ESO-1, LAGE-1, BRDT, HOM-TES-85, TPX-1 and LDHC) in 102 human NSCLC specimens. About 81% of NSCLC express at least 1 and half of the specimen at least 2 CG genes. Activation of most of these genes occurs more frequently in squamous cell cancer than in adenocarcinomas. Even though we found all genes but one to be regulated by genomic methylation, not all of them are co-expressed. In particular, combining CG genes not localized on the X-chromosome may provide effective treatment for an extended number of patients.