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Statistical mechanics demands that the temperature of a system is positive provided that its internal energy has no upper bound. Yet if this condition is not met, it is possible to attain negative temperatures for which higher-order energy states are thermodynamically favored. Although negative temperatures have been reported in spin and Bose-Hubbard settings as well as in quantum fluids, the observation of thermodynamic processes in this regime has thus far remained elusive. Here, we demonstrate isentropic expansion-compression and Joule expansion for negative optical temperatures, enabled by purely nonlinear photon-photon interactions in a thermodynamic microcanonical photonic system. Our photonic approach provides a platform for exploring new all-optical thermal engines and could have ramifications in other bosonic systems beyond optics, such as cold atoms and optomechanics.

Background: Food loss and waste over-uses natural resources and is responsible for a considerable share of greenhouse gas (GHG) emissions. Moreover, increasing food prices and growing food insecurity internationally make instances of food wastage appear even more irresponsible and showcase the inefficiencies of the current food system. A new concept in the toolbox for fighting food loss and waste is upcycling – value enhancing use of ingredients otherwise wasted – that leads to upcycled food. However, not all products currently called ‘upcycled food’ live up to the resource-saving and value-adding promise entailed in the idea of ‘upcycling’, and products markedly differ in how ‘radical’ the upcycling is from a consumer perspective. Scope and approach: To shed light on this, we introduce a two-folded definition that hinges on current consumer edibility perception and has a view to changing consumer perception; We show that there are two types of upcycled food which each have a distinct role and contribution to preserving natural resources - one is avoiding resources are wasted that have gone into food production, thus constituting upcycled foods through alternative use, the other is diversifying and broadening the food resource base, thus constituting upcycled foods through novel use. We provide examples of these upcycled food types and highlight potential boundaries from a sustainable development goal perspective. Key findings and conclusions: Mainstreaming the idea of upcycling in food systems has huge potential for improving circularity in the food system. Untapping this potential needs collaboration across the whole value chain and taking a food system perspective, such as when being well-aware of the boundaries arising from the dynamic nature of the topic and the interdependencies.

Die Technologie Augmented Reality (AR) eröffnet durch Anreicherung der realen Welt mit virtuellen Informationen innovative pädagogisch-didaktische Gestaltungsmöglichkeiten in Lehr-Lern-Situationen. Insbesondere im Sachunterricht der Primarstufe besteht derzeit noch ein erhebliches Desiderat zur Erforschung des pädagogisch-didaktischen Potenzials des Einsatzes von AR sowie grundsätzlich zur Konzeption und Implementation entsprechender AR-Lehr-Lern-Tools. Ziel der in diesem Beitrag beschriebenen Studie ist die Erlangung grundlegender Erkenntnisse zur Einschätzung der «Pedagogical Usability» eines AR-Lehr-Lern-Tools für den naturwissenschaftlich-orientierten Sachunterricht durch Grundschullehrpersonen. Diese durch Befragung von Grundschullehrpersonen gewonnenen Einschätzungen wurden in einem qualitativen Design mittels eines leitfadengestützten Interviews erfasst und durch eine strukturierende qualitative Inhaltsanalyse ausgewertet. Insgesamt zeigt sich, dass das evaluierte AR-Lehr-Lern-Tool einige Teilaspekte der Pedagogical Usability aus Sicht der Lehrpersonen erfüllt (z. B. «Motivation» und «Student Control») und in anderen Teilen noch verbessert werden könnte (z. B. bzgl. «Applicability» und «Feedback»). Auffallend ist auch, dass die Lehrpersonen die pädagogisch-didaktischen Vorteile von AR nicht oder kaum erkennen und die Unterschiede zwischen verschiedenen AR-Technologien auch nur teilweise benennen oder erkennen können. Die Ergebnisse stellen eine erste Grundlage für weitere Untersuchungen zur Rolle der Lehrperson bei der Entwicklung, Evaluation und Implementierung hoch innovativer, technologiegestützter Lehr-Lern-Tools sowie für die Erforschung des pädagogisch-didaktischen Potenzials von AR zu weiteren Themen des Sachunterrichts dar.

Background The growing interest in the secondary use of electronic health record (EHR) data has increased the number of new data integration and data sharing infrastructures. The present work has been developed in the context of the German Medical Informatics Initiative, where 29 university hospitals agreed to the usage of the Health Level Seven Fast Healthcare Interoperability Resources (FHIR) standard for their newly established data integration centers. This standard is optimized to describe and exchange medical data but less suitable for standard statistical analysis which mostly requires tabular data formats. Objectives The objective of this work is to establish a tool that makes FHIR data accessible for standard statistical analysis by providing means to retrieve and transform data from a FHIR server. The tool should be implemented in a programming environment known to most data analysts and offer functions with variable degrees of flexibility and automation catering to users with different levels of FHIR expertise. Methods We propose the fhircrackr framework, which allows downloading and flattening FHIR resources for data analysis. The framework supports different download and authentication protocols and gives the user full control over the data that is extracted from the FHIR resources and transformed into tables. We implemented it using the programming language R [1] and published it under the GPL-3 open source license. Results The framework was successfully applied to both publicly available test data and real-world data from several ongoing studies. While the processing of larger real-world data sets puts a considerable burden on computation time and memory consumption, those challenges can be attenuated with a number of suitable measures like parallelization and temporary storage mechanisms. Conclusion The fhircrackr R package provides an open source solution within an environment that is familiar to most data scientists and helps overcome the practical challenges that still hamper the usage of EHR data for research.

HighlightsVision-based approach for detecting outlet flow of tile drains in a laboratory environment.Method accurately detects and estimates flow within 12% or less of ground truth flow rate.A real-time application was developed that provides estimated flow rates from collected video.Abstract. This article presents a computer vision-based approach for monitoring water flow at outlet points of a tile drain system. The approach relies only on video capture of events at outlet points, thus a camera can be installed remotely and without contact with water. The algorithm detects, identifies, and tracks flows by motion, shape, and color features and measures flow rate based on a proposed model and two provided dimensions. The software was tested in a laboratory environment with three different target flow rate conditions: 0.312, 0.946, and 1.58 L/s (5, 15, and 25 gal/min). Flow rates reported by the computer vision approach are within 12% of the ground-truth flow rate baseline. The results of this work show that computer vision can be used as a reliable method for monitoring outlet flows from free-standing outlet structures under laboratory conditions. This work opens the possibility of applying computer vision techniques in tile drain monitoring from outlet points with mobile video recording devices in the field. Keywords: Keywords., Morphological transformations, Outflow detection, Outlet flow, Real-time application.

Background and Aims: Serum galectin-3 is regarded as an inflammatory marker in patients with chronic liver diseases. Hepatitis C virus (HCV) infection is associated with higher levels of inflammatory molecules which ameliorate by efficient treatment with direct-acting antivirals (DAAs). The aim of this study was to compare serum galectin-3 levels between HCV patients before treatment with DAAs and at the time of sustained virologic response at 12 weeks post-treatment (SVR12). Methods: Hepatitis B and human immunodeficiency virus-negative HCV infected patients not treated with HCV therapies before were recruited at the University Hospital of Regensburg. Galectin-3 was measured by enzyme-linked immunosorbent assay in the serum of patients with chronic HCV infection, before treatment initializing, at four and twelve weeks after the start of DAA therapy and at SVR12. Associations of serum galectin-3 with C-reactive protein (CRP), leukocyte count and measures of liver disease severity were analyzed. Liver fibrosis was assessed by acoustic radiation force impulse, the aspartate aminotransferase/platelet ratio index, and the fibrosis-4 score. Results: In the serum of 81 HCV patients, galectin-3 did not correlate with viral load, viral genotype, CRP, leukocyte count, or the model for end stage liver disease score. Therapy with DAAs effectively diminished viral load within four weeks in all patients. The median value of the serum galectin-3 was 3.0 (Q1:2.0, Q3:4.0) ng/ml before therapy and declined to 2.4 (Q1: 1.7, Q3: 3.4) ng/ml at SVR12 (p

The neural correlates of major depressive disorder (MDD) remain disputed. In the absence of reliable biological markers, the dysfunction and interaction of neural networks have been proposed as pathophysiological neural mechanisms in depression. Here, we examined the functional connectivity (FC) of brain networks. 51 healthy volunteers (mean age 33.57 ± 7.80) and 55 individuals diagnosed with MDD (mean age 33.89 ± 11.00) participated by performing a resting-state (rs) fMRI scan. Seed to voxel FC analyses were performed. Compared to healthy control (HC), MDD patients showed higher connectivity between the hippocampus and the anterior cingulate cortex (ACC) and lower connectivity between the insula and the ACC. The MDD group displayed lower connectivity between the inferior parietal lobule (IPL) and the superior frontal gyrus (SFG). The current data replicate previous findings regarding the cortico-limbic network (hippocampus - ACC connection) and the salience network (insula - ACC connection) and provide novel insight into altered rsFC in MDD, in particular involving the hippocampus - ACC and the insula - ACC connection. Furthermore, altered connectivity between the IPL and SFG indicates that the processing in higher cognitive processes such as attention and working memory is affected in MDD. These data further support dysfunctional neuronal networks as an interesting pathophysiological marker in depression.

Purpose Phosphatidylinositide-3-kinase (PI3K) regulates proliferation and apoptosis; somatic PIK3CA-mutations may activate these processes. Aim of this study was to determine the prevalence of PIK3CA-mutations in a cohort of early stage breast cancer patients and the association to the course of disease. Patients and methods From an unselected cohort of 1270 breast cancer patients (PiA, Prognostic Assessment in routine application, NCT 01592825) 1123 tumours were tested for the three PIK3CA hotspot-mutations H1047R, E545K, and E542K by qPCR. Primary objectives were the prevalence of somatic PIK3CA-mutations and their association to tumour characteristics. Secondary objective was the association of PIK3CA-mutations to recurrence-free interval (RFI) and overall survival. Results PIK3CA-mutation rate was 26.7% (300 of 1123). PIK3CA-mutations were significantly more frequent in steroid hormone-receptor (SHR)-positive HER2-negative (31.4%), and G1 and G2 tumours (32.8%). Overall, we did not observe a significant association of PIK3CA-mutations to RFI. In SHR-positive BCs with PIK3CA-mutations, a strong trend for impaired RFI was observed (adjusted HR 1.64, 95% CI 0.958–2.807), whilst in SHR-negative BCs PIK3CA-mutations were insignificantly associated with improved RFI (adjusted HR 0.49; 95% CI 0.152–1.597). Of note, we observed a significantly detrimental prognostic impact of PIK3CA-mutations on RFI in SHR-positive, HER2-negative BCs if only aromatase inhibitors were administered as adjuvant therapy (adjusted HR 4.44, 95% CI 1.385–13.920), whilst no impact was observed in tamoxifen treated patients. Conclusion This cohort study speficies the overall mutation rate of PIK3CA in early breast cancer. The impact of PIK3CA-mutations on RFI and OS was heterogeneous. Our results suggest that estrogen deprivation failes to be active in case of PIK3CA-mutation.

Purpose To evaluate efficacy and safety of vaccination with StroVac compared to placebo in patients with recurrent urinary tract infections (rUTI). Material and methods We performed a prospective, double-blinded, placebo-controlled study in patients with uncomplicated rUTI. Patients received three single intramuscular injections with StroVac every two weeks. Primary endpoint was the number of bacterial urinary tract infections (UTI) over 13.5 months after randomization and adjusted by the respective “baseline” value when comparing verum and placebo group. Secondary endpoints were the number of patients with non-recurrence, time to first recurrence, frequency of recurrences, and patients' self-assessment of quality of life using a validated questionnaire. Results 376 patients were randomized to both groups between January 2012 and March 2015. Mean age was 44.4 years. Patients were mainly female (98.4%). In the StroVac group (n = 188), the number of UTIs was reduced from 5.5 to 1.2, in the placebo group (n = 188) from 5.4 to 1.3 (p = 0.63). In patients with ≥ 7 UTIs prior to study inclusion, StroVac was statistically significantly superior to placebo (p = 0.048). However, in all other secondary endpoints, no statistical differences between the two groups could be seen (all p > 0.3). Conclusion StroVac reduced the number of clinically relevant UTIs like in former studies but did not show statistically significant better results than the chosen placebo. Most likely, that was due to a, since confirmed, prophylactic effect of the chosen placebo itself. Therefore, placebo-controlled and double-blinded studies using a different ineffective placebo preparation are needed to determine the importance of StroVac in prophylaxis of rUTI.

Paper presented in CLEO Europe 2023 in Munich.

Silica-based optical fibres are a workhorse of nonlinear optics, providing ready access to a range of nonlinear phenomena including solitons and self-phase modulation. However, they have one fundamental limitation: due to the amorphous nature of silica, they do not exhibit second-order nonlinearity, except for negligible contributions from surfaces. Here we demonstrate second-harmonic generation in functionalized optical fibres by using a monolayer of highly nonlinear MoS2 directly grown on the fibre’s core. The MoS2-functionalized fibre exhibits a second-order susceptibility (χ(2)) value of 44 pm V–1 and a second-harmonic generation conversion efficiency of 0.2 × 10–3 m−2 W−1. This approach is scalable and can be generalized to other transition metal dichalcogenides and a wide range of waveguide systems. Our results demonstrate a new approach towards efficient in-fibre second-harmonic generation sources and may establish a platform for χ(2)-based nonlinear fibre optics, optoelectronics, photonics platforms, integrated optical architectures and active fibre networks.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and imported in a posttranslational manner. Intricate protein import machineries have evolved that catalyze the different stages of translocation. In humans, PEX5L was found to be an essential component of the peroxisomal translocon. PEX5L is the main receptor for substrate proteins carrying a peroxisomal targeting signal (PTS). Substrates are bound by soluble PEX5L in the cytosol after which the cargo-receptor complex is recruited to peroxisomal membranes. Here, PEX5L interacts with the docking protein PEX14 and becomes part of an integral membrane protein complex that facilitates substrate translocation into the peroxisomal lumen in a still unknown process. In this study, we show that PEX5L containing complexes purified from human peroxisomal membranes constitute water-filled pores when reconstituted into planar-lipid membranes. Channel characteristics were highly dynamic in terms of conductance states, selectivity and voltage- and substrate-sensitivity. Our results show that a PEX5L associated pore exists in human peroxisomes, which can be activated by receptor-cargo complexes.

IntroductionKeratinocytes form a multilayer barrier that protects the skin from invaders or injuries. The barrier function of keratinocytes is in part mediated by the production of inflammatory modulators that promote immune responses and wound healing. Skin commensals and pathogens such as Staphylococcus aureus secrete high amounts of phenol-soluble modulin (PSM) peptides, agonists of formyl-peptide receptor 2 (FPR2). FPR2 is crucial for the recruitment of neutrophils to the sites of infection, and it can influence inflammation. FPR1 and FPR2 are also expressed by keratinocytes but the consequences of FPR activation in skin cells have remained unknown.MethodsSince an inflammatory environment influences S. aureus colonization, e. g. in patients with atopic dermatitis (AD), we hypothesized that interference with FPRs may alter keratinocyte-induced inflammation, proliferation, and bacterial colonization of the skin. To assess this hypothesis, we investigated the effects of FPR activation and inhibition in keratinocytes with respect to chemokine and cytokine release as well as proliferation and skin wound gap closure.ResultsWe observed that FPR activation induces the release of IL-8, IL-1α and promotes keratinocyte proliferation in a FPR-dependent manner. To elucidate the consequence of FPR modulation on skin colonization, we used an AD-simulating S. aureus skin colonization mouse model using wild-type (WT) or Fpr2-/- mice and demonstrate that inflammation enhances the eradication of S. aureus from the skin in a FPR2-dependent way. Consistently, inhibition of FPR2 in the mouse model or in human keratinocytes as well as human skin explants promoted S. aureus colonization.DiscussionOur data indicate that FPR2 ligands promote inflammation and keratinocyte proliferation in a FPR2-dependent manner, which is necessary for eliminating S. aureus during skin colonization.

Supplementary materials for a vision-based application to estimate outlet flow rate, duration, and occurrence from perched tile drain outlet structures. This submission includes a supplemental video of how the Windows application works and additional images of the flow rate detections.

Long-chain fatty acids with antimicrobial properties are abundant on the skin and mucosal surfaces, where they are essential to restrict the proliferation of opportunistic pathogens such asStaphylococcus aureus. These antimicrobial fatty acids (AFAs) elicit bacterial adaptation strategies, which have yet to be fully elucidated. Characterizing the pervasive mechanisms used byS. aureusto resist AFAs could open new avenues to prevent pathogen colonization. Here, we identify theS. aureuslipase Lip2 as a novel resistance factor against AFAs. Lip2 detoxifies AFAs via esterification with cholesterol. This is reminiscent of the activity of the fatty acid-modifying enzyme (FAME), whose identity has remained elusive for over three decades.In vitro, Lip2-dependent AFA-detoxification was apparent during planktonic growth and biofilm formation. Our genomic analysis revealed that prophage-mediated inactivation of Lip2 was more common in blood and nose isolates than in skin strains, suggesting a particularly important role of Lip2 for skin colonization. Accordingly, in a mouse model ofS. aureusskin colonization, bacteria were protected from sapienic acid - a human-specific AFA - in a cholesterol- and lipase-dependent manner. These results suggest Lip2 is the long-sought FAME that exquisitely manipulates environmental lipids to promote bacterial growth. Our data support a model in whichS. aureusexploits and/or exacerbates lipid disorders to colonize otherwise inhospitable niches.

ZusammenfassungJedes Jahr ereignen sich in Deutschland mehrere Hundert tödliche Arbeitsunfälle, wobei sich die Anzahl in den vergangenen Jahren rückläufig zeigte. Den beiden hier vorgestellten Arbeitsunfällen lag ein sehr ungewöhnlicher Unfallmechanismus zugrunde, der insbesondere im Fall 1 eine den Gegebenheiten angepasste Sektionstechnik erforderte.Fall 1Beim Reinigen eines Hackschnitzelsilos wurde ein 57-jähriger Mann von einer sich sehr langsam drehenden Förderschnecke erfasst und getötet. Durch vorsichtiges Abwickeln und Herauslösen von auf der Schnecke befindlichen Gewebsanteilen konnte gezeigt werden, dass der Erstkontakt am rechten Bein stattgefunden hat, und dass der Oberkörper wohl aufgrund einer Einwickelung der getragenen Jacke in die Schnecke geraten ist. Einblutungen am rechten Handrücken und einzelne Bluteinatmungsherde in den Lungen belegten, dass der Verstorbene zumindest zu Beginn des Geschehens noch geatmet bzw. einen intakten Kreislauf gehabt hat.Fall 2Ein 17-jähriger Mann war beim Befüllen eines Schachtes mit Hackschnitzeln mit seinem linken Bein in eine Förderschnecke geraten. Schwer verletzt wurde er in ein Krankenhaus eingeliefert, wo er trotz Notoperation verstarb.

Bacteria and their viruses (phages) use quorum sensing (QS) systems to coordinate group behavior. InStaphylococcus aureus, QS plays a critical role in the transition from colonization to infection and involves the accumulation of auto-inducing peptides (AIPs). Humans and animals are also colonized by non-aureus staphylococci (NAS) that produce AIPs, many of which inhibitS. aureusQS. We found that QS induction is necessary forS. aureussusceptibility to the lytic phage, Stab20 and that in mixed communities with NAS producing inhibitory AIPs,S. aureusis protected from phage infection. The primary phage receptors inS. aureusare wall teichoic acids (WTA) substituted with α- and/or β-linked N- acetylglucosamine (GlcNAc). We show that QS induction reduces α-GlcNAc substitutions and enables Stab20 infection through binding to β-glycosylated WTA. However, in the presence of inhibitory AIPs or during co-culture with NAS, QS induction and Stab20 infection are impeded. Our results highlight how cross-species communication can significantly impact bacterial susceptibility to phages and may explain occasional failures observed when phages are used as antimicrobials in for example phage therapy.

Supplementary Data from Skp2 Directs Myc-Mediated Suppression of p27Kip1 yet Has Modest Effects on Myc-Driven Lymphomagenesis

Ten patients with confirmed COVID-19 disease were studied. Nine patients required intensive care treatment, among them four needed extracorporeal membrane oxygenation (ECMO). Contrast enhanced ultrasonography (CEUS) was performed by one experienced investigator as a bolus injection of up to 2.4 ml of sulphur hexafluoride microbubbles via a central venous catheter. B-Mode evaluation and strain elastography showed mural edema of the small bowel with a thickness of up to 10 mm in all patients. We applied color coded Doppler sonography (CCDS) and power mode with flow-adapted parameters and early, dynamic capillary arterial contrast enhancement of bowel wall structures

Introduction. Tumor cells are influenced by their microenvironment, including immune infiltration. These tumor infiltrating lymphocytes (TILs) are considered to have prognostic and predictive value for patients with early breast cancer. The aim of this study was to evaluate the distribution of TILs and the association with survival in unselected sample of breast cancers. Patients and Methods. From a prospective, multicenter cohort of 1,270 breast cancer patients (PiA, Prognostic Assessment in routine application, NCT 01592825), 1,136 samples were evaluated for TIL infiltration inside the borders of the invasive tumor, following the recommendations of the international TILs working group. TILs were assigned to one of these three TILs groups: low TILs (60%) and additionally scored as a continuous parameter per 10% increment. Primary objective was the distribution of TILs in different groups dependent on hormone receptor and HER2 status of the tumor (IHC group). Second objective was the association of TILs with recurrence free interval (RFI) and overall survival (OS) in univariate and multivariate analyses. The median observation time was 62 months (1-123). Results. Dependent on the IHC types a specific distribution of TILs was found: more than 60% TILs were detected in 1.5% (12 of 828) of hormone receptor (HR) positive and HER2 negative tumors, 9.7% (17 of 175) of HER2 positive tumors with any HR status and 18.8% (25 of 133) of triple negative breast cancer (TNBC). Patients with HR positive and HER2 negative tumors showed no impact of TILs with regard to RFI and OS. In the HER2 positive group with more than 60% TILs, no RFI event was detected, and with less than 60% TILs, 15% of the patients had an RFI event. The probability of OS was 94% (TILs >60%) and 81% (TILS ≤60%). For TNBC and the same TIL cut off, 88% had no RFI and OS event, compared to 70% with no event for RFI and 74% for OS. Applying 10% increments of TILs to the entire cohort, the multivariate analysis revealed a hazard ratio of 0.895 (95% CI 0.796-1.007) for RFI and a significant hazard ratio of 0.890 (95% CI 0.794-0.997) for OS. Considering the IHC groups, a 10% increment of TILs in HER2 positive tumors led to an increase of RFI (0.792, 95% CI 0.601-1.043) and OS (0.713, 95% CI 0.533-0.955, p Citation Format: Kathleen Schüler, Daniel Bethmann, Tilmann Lantzsch, Christoph Uleer, Volker Hanf, Susanne Peschel, Jutta John, Marleen Pöhler, Joerg Buchmann, Karl-Friedrich Buerrig, Edith Weigert, Eva Johanna Kantelhardt, Christoph Thomssen, Martina Vetter. Tumor infiltrating lymphocytes as a prognostic factor [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-15.

Empathy and anthropomorphism, well-established components of the human-pet relationship, are considered to be especially related to pet animal welfare. We have developed a systematic and standardised approach to explore the effect of the human-pet relationship on animal welfare, focusing on pet birds. Based on a data set measuring the owner-bird relationship as well as bird welfare, cluster analysis and multivariate regressions were used to identify empirical types of bird owners and analyse their effect on bird welfare. Five empirical types of bird owners were identified based on the multi-dimensional relationship between owner and bird which consisted of: (i) the closeness-appreciating anthropomorphising owner; (ii) the closeness-appreciating socially supported owner; (iii) the anthropomorphising socially supported owner; (iv) the inattentive owner; and (v) the distance-appreciating owner. These differed in terms of the owner's tendency to anthropomorphism, the social support the bird provides to the owner, the empathy, attentiveness and respect of the owner towards the bird, and the bird's relationship with the owner. In particular, the inattentive type, but also both anthropomorphising types, raised serious questions as to the well-being of the pet bird. We found significant correlations to bird behaviour, such as imprinting aspects, aggressiveness towards humans, conspecifics and other pet animals, as well as behavioural disorders, such as locomotor stereotypies, courtship behaviour towards humans and feather-plucking.

Frontiers in physics 10, 880793 (2022). doi:10.3389/fphy.2022.880793 special issue: "Electronic and Nuclear Quantum Dynamics of Molecules in Intense Laser Fields", We report on the use of extreme ultraviolet (XUV, 30.3 nm) radiation from the Free-electron LASer in Hamburg (FLASH) and visible (Vis, 405 nm) photons from an optical laser to investigate the relaxation and fragmentation dynamics of fluorene ions. The ultrashort laser pulses allow to resolve the molecular processes occurring on the femtosecond timescales. Fluorene is a prototypical small polycyclic aromatic hydrocarbon (PAH). Through their infrared emission signature, PAHs have been shown to be ubiquitous in the universe, and they are assumed to play an important role in the chemistry of the interstellar medium. Our experiments track the ionization and dissociative ionization products of fluorene through time-of-flight mass spectrometry and velocity-map imaging. Multiple processes involved in the formation of each of the fragment ions are disentangled through analysis of the ion images. The relaxation lifetimes of the excited fluorene monocation and dication obtained through the fragment formation channels are reported to be in the range of a few tens of femtoseconds to a few picoseconds., Published by Frontiers Media, Lausanne

Purpose: Dasatinib and nilotinib are active in imatinib-resistant chronic myelocytic leukemia (CML) and many patients undergo sequential treatment. We aimed at modeling sequential tyrosine kinase inhibitor (TKI) resistance in vitro to compare the sequences imatinib-nilotinib-dasatinib and imatinib-dasatinib-nilotinib.Experimental Design: We designed an in vitro model for sequential TKI resistance in CML. Replicates of imatinib-resistant cell lines were treated with dasatinib or nilotinib. Second-line resistant replicates were exposed to third-line treatment.Results: Growth of all replicates in all three lines of treatment was associated with T315I. However, T315I occurred with low abundance and did not increase during sequential treatment. Nilotinib second-line more often gave rise to sequential resistance compared with dasatinib due to pre-existing P-loop mutations, especially at suboptimal drug concentration. In contrast, mutations predisposing to dasatinib resistance such as F317C/V and V299L did not occur before dasatinib exposure. Nilotinib third-line did not overcome imatinib-dasatinib resistance due to pre-existing T315I or P-loop/V299L or P-loop/F317 exchanges. Dasatinib third-line suppressed imatinib-nilotinib–resistant replicates with residual sensitivity.Conclusions: Sequential acquisition of BCR-ABL drug resistance mutations in CML might be underestimated. Resistance to sequential TKI monotherapy in vitro more often was associated with stepwise acquisition of drug-specific compound mutations compared with T315I. Pre-existing mutations strongly limited the activity of both third-line treatments, and the activity of nilotinib second-line in vitro critically depended on drug concentration. Clin Cancer Res; 19(11); 2962–72. ©2013 AACR.

Purpose: Histone deacetylase inhibitors (HDACi) are being studied in clinical trials with the aim to induce cellular differentiation, growth arrest, and apoptosis of tumor cells. Recent reports suggest that the multidrug resistance-1 (MDR1) gene is regulated by epigenetic mechanisms. To investigate whether additional drug transporters are regulated by HDACi and how this affects cytotoxicity, acute myeloid leukemia (AML) cells were examined.Experimental Design: AML cells were cultured in the presence of phenylbutyrate, valproate, suberoylanilide hydroxamic acid, or trichostatin A and analyzed for drug transporter expression and function as well as sensitivity to anticancer drugs.Results:MDR1, breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRP) 7 and 8 were induced in a dose- and time-dependent manner as shown by semiquantitative PCR. The pattern of gene induction was cell line specific. Phenylbutyrate induced P-glycoprotein and BCRP expression and the efflux of drugs as determined with labeled substrates. KG-1a cells treated with phenylbutyrate developed resistance to daunorubicin, mitoxantrone, etoposide, vinblastine, paclitaxel, topotecan, gemcitabine, and 5-fluorouracil; as a result drug-induced apoptosis was impaired. Chromatin immunoprecipitation revealed the hyperacetylation of histone proteins in the promoter regions of MDR1, BCRP, and MRP8 on valproate treatment. Furthermore, an alternative MRP8 promoter was induced by HDACi treatment.Conclusions: Exposure of AML cells to HDACi induces a drug resistance phenotype broader than the “classic multidrug resistance,” which might negatively affect treatment effectiveness.

Supplementary Data - PDF file 158K, Supplementary Table S1. Target mutations and ligation probes for mutation specific and wild type CKIT or PDGFRA hybridisation. Patient numbers corresponding to Figure 2, their specific tumor/target mutations and the sequences of the hybridisation probes. Supplementary Table S2. Overview of test results in relation to clinical setting for patients with active disease. Allocation of test results corresponding to Table 2 and to patient numbers corresponding to Figure 2. Supplementary Figure S1. LINE1 levels of GIST patients in complete remission versus progression and relapse. Shown are the median LINE1 fragment numbers in 2 microl plasma DNA

Purpose: In gastrointestinal stromal tumor (GIST), there is no biomarker available that indicates success or failure of therapy. We hypothesized that tumor-specific v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (CKIT)- or platelet-derived growth factor receptor-α (PDGFRA)–mutant DNA fragments can be detected and quantified in plasma samples of patients with GIST.Experimental Design: We prospectively collected 291 plasma samples from 38 subjects with GIST harboring activating mutations of CKIT or PDGFRA detected in tumor tissue, irrespective of current disease status or treatment. We used allele-specific ligation PCR to detect mutant free circulating DNA (fcDNA).Results: We were able to detect fcDNA harboring the tumor mutation in 15 of 38 patients. Patients with active disease displayed significantly higher amounts of mutant fcDNA compared with patients in complete remission (CR). The amount of mutant fcDNA correlated with disease course. We observed repeated positive test results or an increase of mutant fcDNA in five patients with progressive disease or relapse. A decline of tumor fcDNA or conversion from positive to negative was seen in five patients responding to treatment. A negative to positive conversion was seen in two patients with relapse and one patient with progression. In two cases, we aimed to identify additional mutations and found four additional exchanges, including mutations not known from sequentially conducted tumor biopsies.Conclusions: Our results indicate that fcDNA harboring tumor-specific mutations in the plasma of patients with GIST can be used as tumor-specific biomarker. The detection of resistance mutations in plasma samples might allow earlier treatment changes and obviates the need for repeated tumor biopsies. Clin Cancer Res; 19(17); 4854–67. ©2013 AACR.

Purpose: Epidermal growth factor receptor (EGFR) mutations in non–small cell lung cancer (NSCLC) might be predictive for clinical response to EGFR inhibitor treatment. However, retrospective analyses of EGFR mutations in clinical trials have shown inconclusive results and the effect of EGFR sequencing in NSCLC is still controversial. Because the vast majority of EGFR mutations described have not been functionally characterized, simple correlation of mutational status and treatment response may not provide reliable information about the predictive value of EGFR mutations. Thus, we aimed to characterize a comprehensive panel of clinically observed EGFR mutations.Experimental Design and Results: A panel of 30 EGFR mutations was cloned and characterized for kinase activity and the ability to confer growth factor independence. Interestingly, 4 of 30 EGFR mutations showed no kinase activity even after ligand stimulation and were not able to confer growth factor independence. Ba/F3 cells expressing activating EGFR mutants were then used to test the efficacy of EGFR inhibitors in a cell proliferation assay. IC50 values were calculated for gefitinib, erlotinib, and AEE788. We show that the sensitivity of EGFR mutations toward different inhibitors varies significantly, thus establishing a comprehensive sensitivity profile for each inhibitor.Conclusions: EGFR mutations identified in NSCLC patients display distinct biological features. The variability in kinase activity, transforming potential, and sensitivity to EGFR inhibitors has to be considered in clinical studies aiming to correlate mutational status and drug response. The identification of comprehensive drug resistance profiles opens the opportunity to test alternative EGFR inhibitors in vitro.

Supplementary Data from Histone Deacetylase Inhibitors Induce a Very Broad, Pleiotropic Anticancer Drug Resistance Phenotype in Acute Myeloid Leukemia Cells by Modulation of Multiple ABC Transporter Genes

Supplementary Data from Functional Analysis of Epidermal Growth Factor Receptor (EGFR) Mutations and Potential Implications for EGFR Targeted Therapy

PDF File - 120K, BCR-ABL kinase domain mutations recovered in the sequential screen with IC50-values for imatinib, dasatinib and nilotinib.

PDF file - 101K

PDF file - 96K, FLT uptake is a sensitive measure for response to HSP90 inhibition in NPM-ALK overexpressing lymphoma cells in vitro

Supplementary Material and Figure Legends

Supplementary Figures 1-4 from Stromal Niche Cells Protect Early Leukemic FLT3-ITD+ Progenitor Cells against First-Generation FLT3 Tyrosine Kinase Inhibitors

Supplementary Figure 1, Tables 1-7 from ADAM17 Regulates Epidermal Growth Factor Receptor Expression through the Activation of Notch1 in Non–Small Cell Lung Cancer

Sensitive in vivo imaging technologies applicable to the clinical setting are still lacking for adoptive T-cell–based immunotherapies, an important gap to fill if mechanisms of tumor rejection or escape are to be understood. Here, we propose a highly sensitive imaging technology to track human TCR-transgenic T cells in vivo by directly targeting the murinized constant TCR beta domain (TCRmu) with a zirconium-89 (89Zr)-labeled anti–TCRmu-F(ab')2 fragment. Binding of the labeled or unlabeled F(ab')2 fragment did not impair functionality of transgenic T cells in vitro and in vivo. Using a murine xenograft model of human myeloid sarcoma, we monitored by Immuno-PET imaging human central memory T cells (TCM), which were transgenic for a myeloid peroxidase (MPO)–specific TCR. Diverse T-cell distribution patterns were detected by PET/CT imaging, depending on the tumor size and rejection phase. Results were confirmed by IHC and semiquantitative evaluation of T-cell infiltration within the tumor corresponding to the PET/CT images. Overall, these findings offer a preclinical proof of concept for an imaging approach that is readily tractable for clinical translation. Cancer Res; 76(14); 4113–23. ©2016 AACR.