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Seven different instruments and measurement methods were used to examine the immersion freezing of bacterial ice nuclei from Snomax® (hereafter Snomax), a product containing ice active protein complexes from non-viable Pseudomonas syringae bacteria. The experimental conditions were kept as similar as possible for the different measurements. Of the participating instruments, some examined droplets which had been made from suspensions directly, and the others examined droplets activated on previously generated Snomax particles, with particle diameters of mostly a few hundred nanometers and up to a few micrometers in some cases. Data were obtained in the temperature range from −2 to −38 °C, and it was found that all ice active protein complexes were already activated above −12 °C. Droplets with different Snomax mass concentrations covering 10 orders of magnitude were examined. Some instruments had very short ice nucleation times down to below 1 s, while others had comparably slow cooling rates around 1 K min−1. Displaying data from the different instruments in terms of numbers of ice active protein complexes per dry mass of Snomax, nm, showed that within their uncertainty the data agree well with each other as well as to previously reported literature results. Two parameterizations were taken from literature for a direct comparison to our results, and these were a time dependent approach based on a contact angle distribution Niedermeier et al. (2014) and a modification of the parameterization presented in Hartmann et~al.~(2013) representing a time independent approach. The agreement between these and the measured data were good, i.e. they agreed within a temperature range of 0.6 K or equivalently a range in nm of a factor of 2. From the results presented herein, we propose that Snomax, at least when carefully shared and prepared, is a suitable material to test and compare different instruments for their accuracy of measuring immersion freezing.
Seven different instruments and measurement methods were used to examine the immersion freezing of bacterial ice nuclei from Snomax® (hereafter Snomax), a product containing ice-active protein complexes from non-viable Pseudomonas syringae bacteria. The experimental conditions were kept as similar as possible for the different measurements. Of the participating instruments, some examined droplets which had been made from suspensions directly, and the others examined droplets activated on previously generated Snomax particles, with particle diameters of mostly a few hundred nanometers and up to a few micrometers in some cases. Data were obtained in the temperature range from −2 to −38 °C, and it was found that all ice-active protein complexes were already activated above −12 °C. Droplets with different Snomax mass concentrations covering 10 orders of magnitude were examined. Some instruments had very short ice nucleation times down to below 1 s, while others had comparably slow cooling rates around 1 K min−1. Displaying data from the different instruments in terms of numbers of ice-active protein complexes per dry mass of Snomax, nm, showed that within their uncertainty, the data agree well with each other as well as to previously reported literature results. Two parameterizations were taken from literature for a direct comparison to our results, and these were a time-dependent approach based on a contact angle distribution (Niedermeier et al., 2014) and a modification of the parameterization presented in Hartmann et al. (2013) representing a time-independent approach. The agreement between these and the measured data were good; i.e., they agreed within a temperature range of 0.6 K or equivalently a range in nm of a factor of 2. From the results presented herein, we propose that Snomax, at least when carefully shared and prepared, is a suitable material to test and compare different instruments for their accuracy of measuring immersion freezing.
Background: Juvenile dermatomyositis (JDM) is the most common inflammatory myopathy in childhood and a major cause of morbidity among children with pediatric rheumatic diseases. The management of JDM is very heterogeneous. The JDM working group of the Society for Pediatric Rheumatology (GKJR) aims to define consensus- and practice-based strategies in order to harmonize diagnosis, treatment and monitoring of JDM.
Methods: The JDM working group was established in 2015 consisting of 23 pediatric rheumatologists, pediatric neurologists and dermatologists with expertise in the management of JDM. Current practice patterns of management in JDM had previously been identified via an online survey among pediatric rheumatologists and neurologists. Using a consensus process consisting of online surveys and a face-to-face consensus conference statements were defined regarding the diagnosis, treatment and monitoring of JDM. During the conference consensus was achieved via nominal group technique. Voting took place using an electronic audience response system, and at least 80% consensus was required for individual statements.
Results: Overall 10 individual statements were developed, finally reaching a consensus of 92 to 100% regarding (1) establishing a diagnosis, (2) case definitions for the application of the strategies (moderate and severe JDM), (3) initial diagnostic testing, (4) monitoring and documentation, (5) treatment targets within the context of a treat-to-target strategy, (6) supportive therapies, (7) explicit definition of a treat-to-target strategy, (8) various glucocorticoid regimens, including intermittent intravenous methylprednisolone pulse and high-dose oral glucocorticoid therapies with tapering, (9) initial glucocorticoid-sparing therapy and (10) management of refractory disease.
Conclusion: Using a consensus process among JDM experts, statements regarding the management of JDM were defined. These statements and the strategies aid in the management of patients with moderate and severe JDM.
Eisnukleierende Partikel (INP) sind ein wichtiger Bestandteil des atmosphärischen Aerosols. Trotz ihrer geringen Konzentrationen in der Atmosphäre haben sie Einfluss auf die Bildung von Eiskristallen und auf den Niederschlag. Durch Änderungen in Anzahlkonzentration oder anderer Eigenschaften der INP können sich Wolkenparameter wie Lebensdauer und Tröpfchendichte ändern, was weiter eine Ursache für Änderungen im globalen Strahlungshaushalt sein kann.
Der Anteil zum globalen Strahlungshaushalt durch „Wolken-Anpassungen aufgrund von Aerosolen“, stellt weiterhin die größte Unsicherheit des Strahlungsantriebes dar. Aus diesem Grund sind Messungen und Studien über atmosphärische Aerosole und INP notwendig. Im Rahmen dieser Arbeit wurde der Eiskeimzähler FINCH („fast ice nucleus chamber“) grundlegend überholt und für Messungen von INP optimiert. FINCH ist ein in-situ Eiskeimzähler der durch Mischung unterschiedlicher Luftströme eine Übersättigung der Probeluft mit Wasserdampf erzeugt, um auf diese Weise die zu untersuchenden Aerosolpartikel zu Eiskristallen wachsen zu lassen. Am Ende einer Wachstumskammer werden die Partikel durch eine Optik, dem FINCH-OPS, anhand von Streueigenschaften klassifiziert und ausgewertet. Um FINCH im erwarteten Umfang benutzen zu können, wurden am F-OPS der Laser und die zur Detektion des Streulichts benutzen Photomultiplier ersetzt. Weiter wurde die Software zur Detektion der Partikel neu entwickelt. Durch diese Änderungen ist es möglich Partikelanzahl, Partikelgröße sowie eine Information über die Form der Partikel abzuleiten. Über einen weiteren Photomultiplier im F-OPS ist es zudem möglich eine Information über Fluoreszenz des Partikels zu gewinnen, um so auf einen biologischen Ursprung des Partikels zu schließen. Vorangegangene Probleme durch elektromagnetische Einstrahlung und dadurch entstandene Inkonsistenzen während Messungen konnten im Rahmen dieser Arbeit identifiziert und ausgeschlossen werden. Ebenfalls konnten die zur Flusskühlung benutzen Wärmetaucher als Ursache für Verunreinigungen und Kontamination ausgemacht werden. Auch dieser, für Messungen ungeeignete Zustand, wurde im Rahmen dieser Arbeit behoben.
Ausführliche Charakterisierungsmessungen konnte die Funktionsfähigkeit des F-OPS, als einzelnes Messgerät ohne FINCH-Kammer, belegen. Durch Messungen mit einer steuerbaren Lichtquelle in der Optik konnte zudem die elektrische Verarbeitung sowie die Zählqualität der Optik verifiziert werden. Weiter kann durch diese Experimente gezeigt werden, dass mit dem F-OPS größenaufgelöst gemessen werden kann.
Auch in den ersten Streulichtexperimenten mit Testaerosol kann die Funktionsfähigkeit der Optik gezeigt werden. Für Partikelgröße von Dp > 400 nm wird eine Zähleffizienz von 25% eines TSI 3025 CPCs erreicht. Die über den F-OPS abgeleitete Partikelgröße kann durch Messungen mit monodispersen Aerosolpartikeln und einer parallelen Messung mit einem TSI 3330 OPS parametrisiert werden. Weiter kann die Fluoreszenz von ausgewählten Referenzstoffen gezeigt werden.
Im Betrieb von F-OPS hinter der FINCH-Kammer, also FINCH als Komplettsystem, wurden weitere Charakterisierungsmessungen durchgeführt. Es wurde festgestellt, dass die Anzahlkonzentration der Partikel nach Schließen eines Ventils exponentiell abfällt. Die Partikel folgen demnach nicht nur einem laminaren Fluss durch die Kammer. 50% der Partikel haben nach ca. 13 s die Kammer verlassen. Auch wurde ermittelt, dass ca. 40% der Partikel in der Kammer verloren gehen.
Erste Aktivierungsexperimente zeigen, dass Eispartikel in der Kammer auf eine Größe von Dp > 6 µm anwachsen. Durch Nullfiltermessungen in Aktivierungsphasen, sowie Ändern des zur Aktivierung notwendigen feuchten Flusses, wird zudem gezeigt, dass die Aktivierung der INP durch Mischung erfolgt und außerdem keine Fremdpartikel aktiviert werden. Die neue Steuerung der Kammer lässt es zu Temperaturen in etwa einer Stunde gezielt anzusteuern. Es wird gezeigt, dass Schwankungen in der Sättigung hauptsächlich durch Temperaturphänomene beeinflusst werden.
Abschließend wurden beispielhaft Laborexperimente mit Snomax© durchgeführt, welche sehr gut mit Werten aus der Literatur verglichen werden konnten. Auch eine erste Außenluftmessung am Jungfraujoch (Schweiz) wird in dieser Arbeit präsentiert. Die gemessenen INP-Konzentrationen im gezeigten Zeitraum liegen zwischen 1,4 ± 4,1 L-1 und 53 ± 30 L-1 und entsprechen somit wiederum bekannten Literaturwerten.
Background: Obesity and depression are both associated with changes in sleep/wake regulation, with potential implications for individualized treatment especially in comorbid individuals suffering from both. However, the associations between obesity, depression, and subjective, questionnaire-based and objective, EEG-based measurements of sleepiness used to assess disturbed sleep/wake regulation in clinical practice are not well known.
Objectives: The study investigates associations between sleep/wake regulation measures based on self-reported subjective questionnaires and EEG-derived measurements of sleep/wake regulation patterns with depression and obesity and how/whether depression and/or obesity affect associations between such self-reported subjective questionnaires and EEG-derived measurements.
Methods: Healthy controls (HC, NHC = 66), normal-weighted depressed (DEP, NDEP = 16), non-depressed obese (OB, NOB = 68), and obese depressed patients (OBDEP, NOBDEP = 43) were included from the OBDEP (Obesity and Depression, University Leipzig, Germany) study. All subjects completed standardized questionnaires related to daytime sleepiness (ESS), sleep quality and sleep duration once as well as questionnaires related to situational sleepiness (KSS, SSS, VAS) before and after a 20 min resting state EEG in eyes-closed condition. EEG-based measurements of objective sleepiness were extracted by the VIGALL algorithm. Associations of subjective sleepiness with objective sleepiness and moderating effects of obesity, depression, and additional confounders were investigated by correlation analyses and regression analyses.
Results: Depressed and non-depressed subgroups differed significantly in most subjective sleepiness measures, while obese and non-obese subgroups only differed significantly in few. Objective sleepiness measures did not differ significantly between the subgroups. Moderating effects of obesity and/or depression on the associations between subjective and objective measures of sleepiness were rarely significant, but associations between subjective and objective measures of sleepiness in the depressed subgroup were systematically weaker when patients comorbidly suffered from obesity than when they did not.
Conclusion: This study provides some evidence that both depression and obesity can affect the association between objective and subjective sleepiness. If confirmed, this insight may have implications for individualized diagnosis and treatment approaches in comorbid depression and obesity.
Treatment of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute leukemia (Ph+ ALL) has been revolutionized with the advent of tyrosine kinase inhibitors (TKIs). Most patients with CML achieve long-term survival similar to individuals without CML due to treatment with TKIs not only in frontline but also in further lines of therapy. The third-generation TKI ponatinib has demonstrated efficacy in patients with refractory CML and Ph+ ALL. Ponatinib is currently the most potent TKI in this setting demonstrating activity against T315I mutant clones. However, ponatinib’s safety data revealed a dose-dependent, increased risk of serious cardiovascular (CV) events. Guidance is needed to evaluate the benefit–risk profile of TKIs, such as ponatinib, and safety measures to prevent treatment-associated CV events. An expert panel of German hematologists and cardiologists summarize current evidence regarding ponatinib’s efficacy and CV safety profile. We propose CV management strategies for patients who are candidates for ponatinib.
The survivin suppressant YM155 is a drug candidate for neuroblastoma. Here, we tested YM155 in 101 neuroblastoma cell lines (19 parental cell lines, 82 drug-adapted sublines). 77 cell lines displayed YM155 IC50s in the range of clinical YM155 concentrations. ABCB1 was an important determinant of YM155 resistance. The activity of the ABCB1 inhibitor zosuquidar ranged from being similar to that of the structurally different ABCB1 inhibitor verapamil to being 65-fold higher. ABCB1 sequence variations may be responsible for this, suggesting that the design of variant-specific ABCB1 inhibitors may be possible. Further, we showed that ABCC1 confers YM155 resistance. Previously, p53 depletion had resulted in decreased YM155 sensitivity. However, TP53-mutant cells were not generally less sensitive to YM155 than TP53 wild-type cells in this study. Finally, YM155 cross-resistance profiles differed between cells adapted to drugs as similar as cisplatin and carboplatin. In conclusion, the large cell line panel was necessary to reveal an unanticipated complexity of the YM155 response in neuroblastoma cell lines with acquired drug resistance. Novel findings include that ABCC1 mediates YM155 resistance and that YM155 cross-resistance profiles differ between cell lines adapted to drugs as similar as cisplatin and carboplatin.
The survivin suppressant YM155 is a drug candidate for neuroblastoma. Here, we tested YM155 in 101 neuroblastoma cell lines (19 parental cell lines, 82 drug-adapted sublines). Seventy seven (77) cell lines displayed YM155 IC50s in the range of clinical YM155 concentrations. ABCB1 was an important determinant of YM155 resistance. The activity of the ABCB1 inhibitor zosuquidar ranged from being similar to that of the structurally different ABCB1 inhibitor verapamil to being 65-fold higher. ABCB1 sequence variations may be responsible for this, suggesting that the design of variant-specific ABCB1 inhibitors may be possible. Further, we showed that ABCC1 confers YM155 resistance. Previously, p53 depletion had resulted in decreased YM155 sensitivity. However, TP53-mutant cells were not generally less sensitive to YM155 than TP53 wild-type cells in this study. Finally, YM155 cross-resistance profiles differed between cells adapted to drugs as similar as cisplatin and carboplatin. In conclusion, the large cell line panel was necessary to reveal an unanticipated complexity of the YM155 response in neuroblastoma cell lines with acquired drug resistance. Novel findings include that ABCC1 mediates YM155 resistance and that YM155 cross-resistance profiles differ between cell lines adapted to drugs as similar as cisplatin and carboplatin.
Systematic protein localization and protein-protein interaction studies to characterize specific protein functions are most effectively performed using tag-based assays. Ideally, protein tags are introduced into a gene of interest by homologous recombination to ensure expression from endogenous control elements. However, inefficient homologous recombination makes this approach difficult in mammalian cells. Although gene targeting efficiency by homologous recombination increased dramatically with the development of designer endonuclease systems such as CRISPR/Cas9 capable of inducing DNA double-strand breaks with unprecedented accuracy, the strategies still require synthesis or cloning of homology templates for every single gene. Recent developments have shown that endogenous protein tagging can be achieved efficiently in a homology independent manner. Hence, combinations between CRISPR/Cas9 and generic tag-donor plasmids have been used successfully for targeted gene modifications in mammalian cells. Here, we developed a tool kit comprising a CRISPR/Cas9 expression vector with several EGFP encoding plasmids that should enable tagging of almost every protein expressed in mammalian cells. By performing protein-protein interaction and subcellular localization studies of mTORC1 signal transduction pathway-related proteins expressed in HEK293T cells, we show that tagged proteins faithfully reflect the behavior of their native counterparts under physiological conditions.
Simple Summary: In patients with myeloproliferative neoplasms (MPN) and in patients with kidney dysfunction, a higher rate of thrombosis has been reported compared with the general population. Furthermore, MPN patients are more prone to develop kidney dysfunction. In our study, we assessed the importance of specific risk factors for kidney dysfunction and thrombosis in MPN patients. We found that the rate of thrombosis is correlated with the degree of kidney dysfunction, especially in myelofibrosis. Significant associations for kidney dysfunction included arterial hypertension, MPN treatment, and increased inflammation, and those for thrombosis comprised arterial hypertension, non-excessive platelet counts, and antithrombotic therapy. The identified risk factor associations varied between MPN subtypes. Our data suggest that kidney dysfunction in MPN patients is associated with an increased risk of thrombosis, mandating closer monitoring, and, possibly, early thromboprophylaxis.
Abstract: Inflammation-induced thrombosis represents a severe complication in patients with myeloproliferative neoplasms (MPN) and in those with kidney dysfunction. Overlapping disease-specific attributes suggest common mechanisms involved in MPN pathogenesis, kidney dysfunction, and thrombosis. Data from 1420 patients with essential thrombocythemia (ET, 33.7%), polycythemia vera (PV, 38.5%), and myelofibrosis (MF, 27.9%) were extracted from the bioregistry of the German Study Group for MPN. The total cohort was subdivided according to the calculated estimated glomerular filtration rate (eGFR, (mL/min/1.73 m2)) into eGFR1 (≥90, 21%), eGFR2 (60–89, 56%), and eGFR3 (<60, 22%). A total of 29% of the patients had a history of thrombosis. A higher rate of thrombosis and longer MPN duration was observed in eGFR3 than in eGFR2 and eGFR1. Kidney dysfunction occurred earlier in ET than in PV or MF. Multiple logistic regression analysis identified arterial hypertension, MPN treatment, increased uric acid, and lactate dehydrogenase levels as risk factors for kidney dysfunction in MPN patients. Risk factors for thrombosis included arterial hypertension, non-excessive platelet counts, and antithrombotic therapy. The risk factors for kidney dysfunction and thrombosis varied between MPN subtypes. Physicians should be aware of the increased risk for kidney disease in MPN patients, which warrants closer monitoring and, possibly, early thromboprophylaxis.