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Background: Definite diagnosis and therapeutic management of cholangiocarcinoma (CCA) remains a challenge. The aim of the current study was to investigate feasibility and potential impact on clinical management of targeted sequencing of intraductal biopsies.
Methods: Intraductal biopsies with suspicious findings from 16 patients with CCA in later clinical course were analyzed with targeted sequencing including tumor and control benign tissue (n = 55 samples). A CCA-specific sequencing panel containing 41 genes was designed and a dual strand targeted enrichment was applied.
Results: Sequencing was successfully performed for all samples. In total, 79 mutations were identified and a mean of 1.7 mutations per tumor sample (range 0–4) as well as 2.3 per biopsy (0–6) were detected and potentially therapeutically relevant genes were identified in 6/16 cases. In 14/18 (78%) biopsies with dysplasia or inconclusive findings at least one mutation was detected. The majority of mutations were found in both surgical specimen and biopsy (68%), while 28% were only present in biopsies in contrast to 4% being only present in the surgical tumor specimen.
Conclusion: Targeted sequencing from intraductal biopsies is feasible and potentially improves the diagnostic yield. A profound genetic heterogeneity in biliary dysplasia needs to be considered in clinical management and warrants further investigation.
Translational impact: The current study is the first to demonstrate the feasibility of sequencing of intraductal biopsies which holds the potential to impact diagnostic and therapeutical management of patients with biliary dysplasia and neoplasia.
Acute myeloid leukemia (AML) is characterized by uncontrolled proliferation and accumulation of immature myeloblasts, which impair normal hematopoiesis. While this definition categorizes the disease into a distinctive group, the large number of different genetic and epigenetic alterations actually suggests that AML is not a single disease, but a plethora of malignancies. Still, most AML patients are not treated with targeted medication but rather by uniform approaches such as chemotherapy. The identification of novel treatment options likely requires the identification of cancer cell vulnerabilities that take into account the different genetic and epigenetic make-up of the individual tumors. Here we show that STK3 depletion by knock-down, knock-out or chemical inhibition results in apoptotic cells death in some but not all AML cell lines and primary cells tested. This effect is mediated by a premature activation of cyclin dependent kinase 1 (CDK1) in presence of elevated cyclin B1 levels. The anti-leukemic effects seen in both bulk and progenitor AML cells suggests that STK3 might be a promising target in a subset of AML patients.
Monoclonal antibodies (mAb) are promising therapeutics in multiple sclerosis and multiple new candidates have been developed, hence increasing the need for some agreement for preclinical mAb studies. We systematically analyzed publications of experimental autoimmune encephalomyelitis (EAE) studies showing effects of monoclonal antibodies. A PubMed search retrieved 570 records, out of which 122 studies with 253 experiments were eligible based on experimental design, number of animals and presentation of time courses of EAE scores. Analysis of EAE models, treatment schedules, single and total doses, routes of administration, and onset of treatment from pre-immunization up to 35 days after immunization revealed high heterogeneity. Total doses ranged from 0.1 to 360 mg/kg for observation times of up to 35 days after immunization. About half of experiments (142/253) used total doses of 10–70 mg/kg. Employing this range, we tested anti-Itga4 as a reference mAb at varying schedules and got no, mild or substantial EAE-score reductions, depending on the mouse strain and onset of the treatment. The result agrees with the range of outcomes achieved in 10 reported anti-Itga4 experiments. Studies comparing low and high doses of various mAbs or early vs. late onset of treatment did not reveal dose-effect or timing-effect associations, with a tendency towards better outcomes with preventive treatments starting within the first week after immunization. The systematic comparison allows for extraction of some “common” design characteristics, which may be helpful to further assess the efficacy of mAbs and role of specific targets in preclinical models of multiple sclerosis.
Objective: Most patients suffering with rheumatic diseases who undergo surgical treatment are receiving immune-modulating therapy. To determine whether these medications affect their outcomes a national registry was established in Germany by the German Society of Surgery (DGORh). Data from the first 1000 patients were used in a pilot study to identify relevant corisk factors and to determine whether such a registry is suitable for developing accurate and relevant recommendations.
Design and participants: Data were collected from patients undergoing surgical treatments with their written consent. A second consent form was used, if complications occurred. During this pilot study, in order to obtain a quicker overview, risk factors were considered only in patients with complications. Only descriptive statistical analysis was employed in this pilot study due to limited number of observed complications and inhomogeneous data regarding the surgery and the medications the patients received. Analytical statistics will be performed to confirm the results in a future outcome study.
Results: Complications occurred in 26 patients and were distributed equally among the different types of surgeries. Twenty one of these patients were receiving immune-modulating therapy at the time, while five were not. Infections were observed in 2.3% of patients receiving and in 5.1% not receiving immunosuppression.
Conclusions: Due to the low number of cases, inhomogeneity in the diseases and the treatments received by the patients in this pilot study, it is not possible to develop standardised best-practice recommendations to optimise their care. Based on this observation we conclude that in order to be suitable to develop accurate and relevant recommendations a national registry must include the most important and relevant variables that impact the care and outcomes of these patients.
Ich hatte im Wintersemester 1968 in Frankfurt mit dem Pharmaziestudium angefangen. Das pharmazeutische Institut lag ja damals in der Georg-Voigt-Straße, also nicht weit entfernt vom Zentrum der Studentenunruhen, dem Uni-Hauptgebäude mit der Aula. Daher haben wir einiges von den Protesten mitbekommen. ...
Über die Bedeutung des geistigen Erbes von 68 wird 50 Jahre später trefflich gestritten. Mögen die politischen Ideen des Aufbruchs und das daran anknüpfende Handeln heute für Kontroversen sorgen, so erfreut sich hingegen der Sound der Revolution, der damals zum Ärger orthodoxer Kommunisten ganz klar von der Rockmusik dominiert wurde, einer nahezu ungebrochenen Begeisterung: Das "»Weiße Album" der Beatles, "Electric Ladyland" von Jimi Hendrix oder "Beggars Banquet" von den Rolling Stones, alle 68 erschienen, werden heute mehr denn je bewundert. Im Zeichen einer grassierenden "Retromania" (Simon Reynolds) gewinnt der mit Mythen und Heldengeschichten aufgeladene Sound der Vergangenheit mit zunehmendem zeitlichen Abstand sogar noch an Bedeutung. In ihrer Frühphase ist die Popmusik noch ein Wettstreit verschiedener Stile, Techniken und auch Bands. Ihre steigende gesellschaftliche Akzeptanz lässt aber spätestens in den 70er Jahren Zweifel laut werden an ihrer weiterhin behaupteten und auch inszenierten Widerständigkeit.
Das Private ist politisch! – 68 war der Slogan eine auf Ganze gerichtete Perspektive der Kritik und ein Impuls für die Revolutionierung des Alltagslebens: Es ging bei den Diskussionen und Analysen um die "Weltherrschaft des Kapitals" und den Zusammenhang von Faschismus und Kapitalismus immer zugleich um die Manifestationen von Herrschaft und Unterdrückung in den Kapillaren des Alltäg lichen. In den Blick gerieten dabei nicht zuletzt die Autoritätsstrukturen der bürgerlichen Familie und damit die (im Sinne marxistischer Gesellschaftsanalyse) sogenannte "Nebenwidersprüche" des Privaten: das Verhältnis der Geschlechter, Fragen von Liebe und Sexualität, Kindererziehung, Eigentumsfragen und Besitzdenken. Der Slogan wurde darüber hinaus zum Leitspruch der Frauenbewegung, die vor Augen führt, wie politisch das Private gerade in Fragen von Nachwuchs und Alltagsorganisation ist. ...
In den zahlreichen Beiträgen zum "Jubeljahr der 1968er-Bewegung" kommen oft ehemalige Aktive, Historikerinnen und Experten zu Wort. Doch wie blicken eigentlich Aktivistinnen und Aktivisten des 21. Jahrhunderts auf diese Zeit zurück? Dieser Frage hat sich ein zweijähriges Forschungsprojekt am Institut für Politikwissenschaft der Goethe-Universität gewidmet.
Weg mit den Talaren! Hoch die Doktorhüte! : vom Wandel der Zeremonialität an deutschen Universitäten
(2018)
"Unter den Talaren – Muff von 1000 Jahren": Dieser Slogan steht wie kaum ein anderer für die Aufbruchstimmung der 68er-Generation. Damit zielten die Hamburger Studenten auf das Verschweigen der NS-Vergangenheit ab, anschließend ging es aber auch den Talaren selbst an den Kragen. Seit einigen Jahren wird im zeremoniellen Raum der Universitäten vielfach experimentiert: Die Fächer bilden eine jeweils eigene Feierkultur aus – auf der Suche nach einem Übergang von der Universität in das berufliche Leben.
Herr Wolff, in der Ausgabe von Forschung Frankfurt zum Jubiläumsjahr werden Sie mit dem Satz zitiert: "Die Offenheit der Debatten, die die 68er erkämpft haben, lässt sich nicht mehr zurücknehmen." Vor Kurzem wurde an der Goethe-Universität heftig über Meinungsfreiheit gestritten. Die Frage war: Darf man den Polizeigewerkschafter Rainer Wendt zur Diskussion an die Uni einladen. Hat Ihre Aussage nach wie vor Bestand? ...
Mit "Sit-in" und "Teach-in" zur Weltrevolution : Erinnerungen an den Sprachgebrauch der "68er"
(2018)
"Sit-in" und "Teach-in"? – Wer 1968 noch kein Zeitgenosse war, wird beides nur für zwei der im 20. Jahrhundert immer beliebter werdenden, aber oft unverstandenen Fremdwörter aus dem Englischen halten. Heute sind beide weitgehend vergessen, so brandaktuell sie auch einmal waren. Mit ihnen wurde nicht weniger gemeint als eine Sitzblockade vor Hörsälen und die Verhinderung einer regulären Lehrveranstaltung, indem man sie in ein Agitationsforum "umfunktionierte". Die traditionellen Vorlesungen wurden ohnehin als "säkularisierte Predigten" verhöhnt. Als ersten Frankfurter Hochschullehrer traf es ausgerechnet den Staatsrechtler Carlo Schmid, immerhin einen der Väter des Grundgesetzes, der seine Vorlesung abbrechen musste. ...
Kurz vor Mitternacht am 2. April 1968 bricht im Kaufhaus M. Schneider auf der Zeil ein Feuer aus. Kurz darauf ertönt der Feueralarm im Kaufhof an der Hauptwache. Menschen werden nicht verletzt, der Schaden beträgt nach heutigem Geldwert rund eine Million Euro. Unter den Tätern sind Andreas Baader und Gudrun Ensslin. Beide werden zu drei Jahren Zuchthaus verurteilt. Die Brandanschläge auf die Frankfurter Kaufhäuser sind die Geburtsstunde der Roten Armee Fraktion – das wissen wir heute. ...
Die Ausstellung "Freiraum der Kunst", die bis 8. Juli 2018 im Museum Giersch der GoetheUniversität zu sehen ist, zeichnet die Geschichte der "studiogalerie" nach. Dieses vom Allgemeinen Studentenausschuss (AStA) betriebene Forum präsentierte im Studentenhaus auf dem Campus Bockenheim von 1964 bis 1968 Ausstellungen und Veranstaltungen der nationalen und internationalen Avantgarde. Malerei und Objekte der Licht-Kunst und Kinetischen Kunst, der Konkreten Kunst, des Neuen Realismus, der Op-Art, der Hard-Edge- und Farbmalerei, aber auch FluxusKonzerte und Happenings sollten als studentischer Beitrag zur Demokratisierung von Kunst und Gesellschaft verstanden werden. Legendär wurde die Ausstellung "Serielle Formationen" von 1967. Im Zuge der Radikalisierung der Frankfurter Studentenschaft kamen die Aktivitäten jedoch 1968 zum Erliegen.
Unter dem Schlagwort "68er-Bewegung" werden verschiedene linksgerichtete Protestbewegungen, Bürgerrechtsdemonstrationen und antiautoritäre Aktionen zusammengefasst, die ab Mitte der 1960er in Deutschland und zahlreichen anderen Ländern stattfanden und auf eine Umwälzung bestehender sozialer und politischer Strukturen zielten. ...
Jedes Mal, wenn ich in Frankfurt bin, eine Stadt, in der ich von 1960 bis 1980 gelebt habe und wo ich Ostern 1968, direkt nach meiner Entlassung aus der Bundeswehr, sofort an den großen Osterdemonstrationen teilgenommen habe – die Antwort auf das Attentat auf Rudi Dutschke –, zieht es mich in das alte Universitätsviertel an der Bockenheimer Warte. ...
Ende 1967 ist er noch Pressereferent beim AStA der Goethe-Universität, da ereilt ihn zum Jahreswechsel der Ruf des Uni-Rektors Walter Rüegg: Klaus Viedebantt, Student der Volkskunde, übernimmt im Januar 1968 den Posten des "Pressereferenten der Akademischen Presse- und Informationsstelle der Universität". ...
Auch im Fachbereich Rechtswissenschaft an der Goethe-Universität kam es 1968 zu heftigen Turbulenzen. Dabei zeigten die meisten jüngeren Professoren des Fachbereichs durchaus Verständnis für manche der studentischen Forderungen. Einige Reformansätze scheiterten, andere wurden erst durch feinere Nachjustierungen funktional.
Die weltweiten Proteste von 1968 waren in verschiedenen Ländern unterschiedlich ausgeprägt. Geeint wurden sie durch einen Gesellschaftsentwurf, der sich nicht nur vom Kapitalismus unterschied, sondern auch vom Kommunismus und den Leitideen der Sozialdemokratie. Zu den wichtigsten Exponenten in Europa zählen Daniel Cohn-Bendit, Tariq Ali und Rudi Dutschke, die die Massen mobilisierten.
Die Erinnerung an das Jahr 1968 kann nur eine Form annehmen: die der Collage. Ein halbes Jahrhundert nach jenen Ereignissen könnte das beispielsweise so aussehen: Manches, was inzwischen in Publikationen und Dokumentationen vorliegt, mit Selbsterlebtem verbinden, um auf diese Weise einen Eindruck von der Vielschichtigkeit und dem gelegentlich surrealistisch wirkenden Zusammen- und Gegeneinanderspiel von "Frankfurter Schule" und Studentenbewegung zu vermitteln.
Vor 50 Jahren probte die Studentenbewegung den Umsturz. Der Vietnamkrieg und die Forderung nach Emanzipation brachten weltweit junge Menschen auf die Straße, Bildungsmisere und Notstandsgesetze waren spezifisch deutsche Protestthemen. Für Frankfurt charakteristisch war die Auseinandersetzung mit der Frankfurter Schule und ihren Vertretern.
Purpose: Anastomotic leakage is a major surgical complication following esophagectomy and gastric pull-up. Specific risk factors such as celiac trunk (TC) stenosis and high calcification score of the aorta have been identified, but no data are available on their relative prognostic values. This retrospective study aimed to compare and evaluate calcification score versus stenosis quantification with regards to prognostic impact on anastomotic leakage.
Patients and methods: Preoperative contrast-enhanced computed tomography scans of 164 consecutive patients with primary esophageal cancer were evaluated by two radiologists to apply a calcification score (0–3 scale) assessing the aorta, the celiac axis and the right and left postceliac arteries. Concurrently, the presence and degree of stenosis of TC and superior mesenteric artery were recorded for stenosis quantification.
Results: Anastomotic leakage was noted in 14/164 patients and 12/14 showed stenosis of TC (n=11). The presence of TC stenosis was found to have a significant impact on anastomotic healing (p=0.004). The odds ratio for the prediction of anastomotic leakage by the degree of stenosis was 1.04 (95% CI, 1.02–1.07). Ten of 14 patients had aortic calcification scores of 1 or 2, but calcification scores of the aorta, the celiac axis and the right and left postceliac arteries did not correlate with the corresponding TC stenosis values and showed no influence on patient outcome as defined by the occurrence of anastomotic insufficiency (p=0.565, 0.855, 0.518 and 1.000, respectively). Inter-reader reliability of computed tomography analysis and absolute agreement on calcium scoring was mostly over 90%. No significant differences in preoperative comorbidities and patient characteristics were found between those with and without anastomotic leakage.
Conclusion: Measurement of TC stenosis in preoperative contrast-enhanced computed tomography scans proved to be more reliable than calcification scores in predicting anastomotic leakage and should, therefore, be used in the risk assessment of patients undergoing esophagectomy and gastric pull-up.
Purpose: All-ceramic restorations required extensive tooth preparation. The purpose of this in vitro study was to investigate a minimally invasive preparation and thickness of monolithic zirconia crowns, which would provide sufficient mechanical endurance and strength.
Materials and methods: Crowns with thickness of 0.2 mm (group 0.2, n=32) or of 0.5 mm (group 0.5, n=32) were milled from zirconia and fixed with resin-based adhesives (groups 0.2A, 0.5A) or zinc phosphate cements (groups 0.2C, 0.5C). Half of the samples in each subgroup (n=8) underwent thermal cycling and mechanical loading (TCML)(TC: 5℃ and 55℃, 2×3,000 cycles, 2 min/cycle; ML: 50 N, 1.2×106 cycles), while the other samples were stored in water (37℃/24 h). Survival rates were compared (Kaplan-Maier). The specimens surviving TCML were loaded to fracture and the maximal fracture force was determined (ANOVA; Bonferroni; α=.05). The fracture mode was analyzed.
Results: In both 0.5 groups, all crowns survived TCML, and the comparison of fracture strength among crowns with and without TCML showed no significant difference (P=.628). Four crowns in group 0.2A and all of the crowns in group 0.2C failed during TCML. The fracture strength after 24 hours of the cemented 0.2 mm-thick crowns was significantly lower than that of adhesive bonded crowns. All cemented crowns provided fracture in the crown, while about 80% of the adhesively bonded crowns fractured through crown and die.
Conclusion: 0.5 mm thick monolithic crowns possessed sufficient strength to endure physiologic performance, regardless of the type of cementation. Fracture strength of the 0.2 mm cemented crowns was too low for clinical application.
Mannan-induced Nos2 in macrophages enhances IL-17–driven psoriatic arthritis by innate lymphocytes
(2018)
Previous identification of the inducible nitric oxide synthase (NOS2) gene as a risk allele for psoriasis (Ps) and psoriatic arthritis (PsA) suggests a possible pathogenic role of nitric oxide (NO). Using a mouse model of mannan-induced Ps and PsA (MIP), where macrophages play a regulatory role by releasing reactive oxygen species (ROS), we found that NO was detectable before disease onset in mice, independent of a functional nicotinamide adenine dinucleotide phosphate oxidase 2 complex. MIP was suppressed by either deletion of Nos2 or inhibition of NO synthases with NG-nitro-L-arginine methyl ester, demonstrating that Nos2-derived NO is pathogenic. NOS2 expression was also up-regulated in lipopolysaccharide- and interferon-γ–stimulated monocyte subsets from patients with PsA compared to healthy controls. Nos2-dependent interleukin-1α (IL-1α) release from skin macrophages was essential for arthritis development by promoting IL-17 production of innate lymphoid cells. We conclude that Nos2-derived NO by tissue macrophages promotes MIP, in contrast to the protective effect by ROS.
Selective BRAF inhibitors such as vemurafenib have become a treatment option in patients with Langerhans cell Histiocytosis (LCH). To date, only 14 patients receiving vemurafenib for LCH have been reported. Although vemurafenib can stabilize the clinical condition of these patients, it does not seem to cure the patients, and it is unknown, when and how to stop vemurafenib treatment. We present a girl with severe multisystem LCH who responded only to vemurafenib. After 8 months of treatment, vemurafenib was tapered and replaced by prednisone and vinblastine, a strategy which has not been described to date. Despite chemotherapy, early relapse occurred, but remission was achieved by re-institution of vemurafenib. Further investigation needs to address the optimal duration of vemurafenib therapy in LCH and whether and which chemotherapeutic regimen may prevent disease relapse after cessation of vemurafenib.
Human papilloma virus (HPV) infection is linked to cervical cancer, which represents the world's fourth most common cancer in women. So far, no detailed map of the worldwide HPV research architecture has been constructed. Hence, this study focuses on the chronological development and geographical distribution of the global HPV-specific publications and evaluates citation-based parameters as well as socioeconomic features of the publishing countries.
In total, 29,330 HPV-related publications were identified. The US was the leading country with 12,270 publications. Only high-income-countries were found in the ranking of the fifteen most active countries with Germany, France, and Japan among the top five. Analysis of HPV research activity in relation to the economic strength demonstrated a lead position of Finland and Sweden with an average of 2248.78 and 1924.67 HPV-related publications per GDP in 1000 bn US-$, respectively. The most active upper-middle-income country was Mexico (416.78 HPV-related publications per GDP in 1000 bn US-$). India as lower-middle-income country reached a value of 279.78 HPV-related publications per GDP in 1000 bn US-$. Collaboration analysis pointed to the US as the center of the 4517 international HPV collaborations.
The worldwide HPV-research landscape is dominated by North American and Western European countries. By contrast, a high prevalence of HPV-related cervical cancer is documented for low-income countries. Hence, HPV-related public health interventions and prevention research specifically tailored to these countries needs to be fostered by monetary support and international collaborations.
Background: Sphingolipids are versatile signaling molecules derived from membrane lipids of eukaryotic cells. Ceramides regulate cellular processes such as proliferation, differentiation and apoptosis and are involved in cellular stress responses. Experimental evidence suggests a pivotal role of sphingolipids in the pathogenesis of cardiovascular diseases, including ischemic stroke. A neuroprotective effect has been shown for beta-adrenergic antagonists in rodent stroke models and supported by observational clinical data. However, the exact underlying pathophysiological mechanisms are still under investigation. We aimed to examine the influence of propranolol on the ceramide metabolism in the stroke-affected brain.
Methods: Mice were subjected to 60 or 180 min transient middle cerebral artery occlusion (tMCAO) and infarct size, functional neurological deficits, glucose tolerance, and brain ceramide levels were assessed after 12, 24, and 72 h to evaluate whether the latter two processes occur in a similar time frame. Next, we assessed the effects of propranolol (10 mg/kg bw) at 0, 4 and 8 h after tMCAO and FTY720 (fingolimod; 1 mg/kg) on infarct size, functional outcome, immune cell counts and brain ceramide levels at 24 h after 60 min tMCAO.
Results: We found a temporal coincidence between stroke-associated impaired glucose tolerance and brain ceramide accumulation. Whereas propranolol reduced ischemic lesion size, improved functional outcome and reduced brain ceramide accumulation without an effect on circulating immune cells, FTY720 showed the known neuroprotective effect and strong reduction of circulating immune cells without affecting brain ceramide accumulation.
Conclusions: Propranolol ameliorates both stroke-associated impairment of glucose tolerance and brain ceramide accumulation which are temporally linked, strengthening the evidence for a role of the sympathetic nervous system in regulating post-stroke glucose metabolism and its metabolic consequences in the brain.
5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) is an established pharmacological activator of AMP-activated protein kinase (AMPK). Both, AICAR and AMPK were reported to attenuate inflammation. However, AICAR is known for many AMPK-independent effects, although the mechanisms remain incompletely understood. Here we report a potent suppression of lipopolysaccharide (LPS)-induced inflammatory gene expression by AICAR in primary human macrophages, which occurred independently of its conversion to AMPK-activating 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl monophosphate. Although AICAR did not interfere with activation of cytosolic signalling cascades and nuclear translocation of nuclear factor - κB (NFκB) by LPS, it prevented the recruitment of NFκB and RNA polymerase II to target gene promoters. AICAR also inhibited signal transducer and activator of transcription 3 (STAT3)-dependent induction of interleukin (IL) IL-6 and IL-10 targets, while leaving STAT6 and HIF1α-dependent gene expression in IL-4 and dimethyloxalylgylcine-treated macrophages intact. This points to a transcription factor-specific mode of action. Attenuated gene expression correlated with impaired NFκB and STAT3, but not HIF-binding in electrophoretic mobility shift assays in vitro. Conclusively, AICAR interferes with DNA binding of NFκB and STAT3 to modulate inflammatory responses.
Inducible gene expression is an important tool in molecular biology research to study protein function. Most frequently, the antibiotic doxycycline is used for regulation of so-called tetracycline (Tet)-inducible systems. In contrast to stable gene overexpression, these systems allow investigation of acute and reversible effects of cellular protein induction. Recent reports have already called for caution when using Tet-inducible systems as the employed antibiotics can disturb mitochondrial function and alter cellular metabolism by interfering with mitochondrial translation. Reprogramming of energy metabolism has lately been recognized as an important emerging hallmark of cancer and is a central focus of cancer research. Therefore, the scope of this study was to systematically analyze dose-dependent metabolic effects of doxycycline on a panel of glioma cell lines with concomitant monitoring of gene expression from Tet-inducible systems. We report that doxycycline doses commonly used with inducible expression systems (0.01–1 µg/mL) substantially alter cellular metabolism: Mitochondrial protein synthesis was inhibited accompanied by reduced oxygen and increased glucose consumption. Furthermore, doxycycline protected human glioma cells from hypoxia-induced cell death. An impairment of cell growth was only detectable with higher doxycycline doses (10 µg/mL). Our findings describe settings where doxycycline exerts effects on eukaryotic cellular metabolism, limiting the employment of Tet-inducible systems.
Sphingosine kinase (SK) catalyses the formation of sphingosine 1-phosphate (S1P), which acts as a key regulator of inflammatory and fibrotic reactions, mainly via S1P receptor activation. Here, we show that in the human renal proximal tubular epithelial cell line HK2, the profibrotic mediator transforming growth factor β (TGFβ) induces SK-1 mRNA and protein expression, and in parallel, it also upregulates the expression of the fibrotic markers connective tissue growth factor (CTGF) and fibronectin. Stable downregulation of SK-1 by RNAi resulted in the increased expression of CTGF, suggesting a suppressive effect of SK-1-derived intracellular S1P in the fibrotic process, which is lost when SK-1 is downregulated. In a further approach, the S1P transporter Spns2, which is known to export S1P and thereby reduces intracellular S1P levels, was stably downregulated in HK2 cells by RNAi. This treatment decreased TGFβ-induced CTGF and fibronectin expression, and it abolished the strong induction of the monocyte chemotactic protein 1 (MCP-1) by the pro-inflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1β. Moreover, it enhanced the expression of aquaporin 1, which is an important water channel that is expressed in the proximal tubules, and reverted aquaporin 1 downregulation induced by IL-1β/TNFα. On the other hand, overexpression of a Spns2-GFP construct increased S1P secretion and it resulted in enhanced TGFβ-induced CTGF expression. In summary, our data demonstrate that in human renal proximal tubular epithelial cells, SK-1 downregulation accelerates an inflammatory and fibrotic reaction, whereas Spns2 downregulation has an opposite effect. We conclude that Spns2 represents a promising new target for the treatment of tubulointerstitial inflammation and fibrosis.
Due to its remote and isolated location, Antarctica is home to a unique diversity of species. The harsh conditions have shaped a primarily highly adapted endemic fauna. This includes the notothenioid family Channichthyidae. Their exceptional physiological adaptations have made this family of icefish the focus of many studies. However, studies on their ecology, especially on their parasite fauna, are comparatively rare. Parasites, directly linked to the food chain, can function as biological indicators and provide valuable information on host ecology (e.g., trophic interactions) even in remote habitats with limited accessibility, such as the Southern Ocean. In the present study, channichthyid fish (Champsocephalus gunnari: n = 25, Chaenodraco wilsoni: n = 33, Neopagetopsis ionah: n = 3, Pagetopsis macropterus: n = 4, Pseudochaenichthys georgianus: n = 15) were collected off South Shetland Island, Elephant Island, and the tip of the Antarctic Peninsula (CCAML statistical subarea 48.1). The parasite fauna consisted of 14 genera and 15 species, belonging to the six taxonomic groups including Digenea (four species), Nematoda (four), Cestoda (two), Acanthocephala (one), Hirudinea (three), and Copepoda (one). The stomach contents were less diverse with only Crustacea (Euphausiacea, Amphipoda) recovered from all examined fishes. Overall, 15 new parasite-host records could be established, and possibly a undescribed genotype or even species might exist among the nematodes.
Biological invasions have been associated with niche changes; however, their occurrence is still debated. We assess whether climatic niches between native and non-native ranges have changed during the invasion process using two globally spread mosquitoes as model species, Aedes albopictus and Aedes aegypti. Considering the different time spans since their invasions (>300 vs. 30–40 years), niche changes were expected to be more likely for Ae. aegypti than for Ae. albopictus. We used temperature and precipitation variables as descriptors for the realized climatic niches and different niche metrics to detect niche dynamics in the native and non-native ranges. High niche stability, therefore, no niche expansion but niche conservatism was revealed for both species. High niche unfilling for Ae. albopictus indicates a great potential for further expansion. Highest niche occupancies in non-native ranges occurred either under more temperate (North America, Europe) or tropical conditions (South America, Africa). Aedes aegypti has been able to fill its native climatic niche in the non-native ranges, with very low unfilling. Our results challenge the assumption of rapid evolutionary change of climatic niches as a requirement for global invasions but support the use of native range-based niche models to project future invasion risk on a large scale.
The lysine-specific demethylase 1 (LSD1) is overexpressed in several cancers including rhabdomyosarcoma (RMS). However, little is yet known about whether or not LSD1 may serve as therapeutic target in RMS. We therefore investigated the potential of LSD1 inhibitors alone or in combination with other epigenetic modifiers such as histone deacetylase (HDAC) inhibitors. Here, we identify a synergistic interaction of LSD1 inhibitors (i.e., GSK690, Ex917) and HDAC inhibitors (i.e., JNJ-26481585, SAHA) to induce cell death in RMS cells. By comparison, LSD1 inhibitors as single agents exhibit little cytotoxicity against RMS cells. Mechanistically, GSK690 acts in concert with JNJ-26481585 to upregulate mRNA levels of the proapoptotic BH3-only proteins BMF, PUMA, BIM and NOXA. This increase in mRNA levels is accompanied by a corresponding upregulation of BMF, PUMA, BIM and NOXA protein levels. Importantly, individual knockdown of either BMF, BIM or NOXA significantly reduces GSK690/JNJ-26481585-mediated cell death. Similarly, genetic silencing of BAK significantly rescues cell death upon GSK690/JNJ-26481585 cotreatment. Also, overexpression of antiapoptotic BCL-2 or MCL-1 significantly protects RMS cells from GSK690/JNJ-26481585-induced cell death. Furthermore, GSK690 acts in concert with JNJ-26481585 to increase activation of caspase-9 and -3. Consistently, addition of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) significantly reduces GSK690/JNJ-26481585-mediated cell death. In conclusion, concomitant LSD1 and HDAC inhibition synergistically induces cell death in RMS cells by shifting the ratio of pro- and antiapoptotic BCL-2 proteins in favor of apoptosis, thereby engaging the intrinsic apoptotic pathway. This indicates that combined treatment with LSD1 and HDAC inhibitors is a promising new therapeutic approach in RMS.
Borrelia burgdorferi sensu lato (Bbsl), the causative agent of Lyme disease, establishes an initial infection in the host’s skin following a tick bite, and then disseminates to distant organs, leading to multisystem manifestations. Tick-to-vertebrate host transmission requires that Bbsl survives during blood feeding. Complement is an important innate host defense in blood and interstitial fluid. Bbsl produces a polymorphic surface protein, CspA, that binds to a complement regulator, Factor H (FH) to block complement activation in vitro. However, the role that CspA plays in the Bbsl enzootic cycle remains unclear. In this study, we demonstrated that different CspA variants promote spirochete binding to FH to inactivate complement and promote serum resistance in a host-specific manner. Utilizing a tick-to-mouse transmission model, we observed that a cspA-knockout B. burgdorferi is eliminated from nymphal ticks in the first 24 hours of feeding and is unable to be transmitted to naïve mice. Conversely, ectopically producing CspA derived from B. burgdorferi or B. afzelii, but not B. garinii in a cspA-knockout strain restored spirochete survival in fed nymphs and tick-to-mouse transmission. Furthermore, a CspA point mutant, CspA-L246D that was defective in FH-binding, failed to survive in fed nymphs and at the inoculation site or bloodstream in mice. We also allowed those spirochete-infected nymphs to feed on C3-/- mice that lacked functional complement. The cspA-knockout B. burgdorferi or this mutant strain complemented with cspA variants or cspA-L246D was found at similar levels as wild type B. burgdorferi in the fed nymphs and mouse tissues. These novel findings suggest that the FH-binding activity of CspA protects spirochetes from complement-mediated killing in fed nymphal ticks, which ultimately allows Bbsl transmission to mammalian hosts.
Background: Cytogenetic aberrations such as deletion of chromosome 5q (del(5q)) represent key elements in routine clinical diagnostics of haematological malignancies. Currently established methods such as metaphase cytogenetics, FISH or array-based approaches have limitations due to their dependency on viable cells, high costs or semi-quantitative nature. Importantly, they cannot be used on low abundance DNA. We therefore aimed to establish a robust and quantitative technique that overcomes these shortcomings.
Methods: For precise determination of del(5q) cell fractions, we developed an inexpensive multiplex-PCR assay requiring only nanograms of DNA that simultaneously measures allelic imbalances of 12 independent short tandem repeat markers.
Results: Application of this method to n=1142 samples from n=260 individuals revealed strong intermarker concordance (R²=0.77–0.97) and reproducibility (mean SD: 1.7%). Notably, the assay showed accurate quantification via standard curve assessment (R²>0.99) and high concordance with paired FISH measurements (R²=0.92) even with subnanogram amounts of DNA. Moreover, cytogenetic response was reliably confirmed in del(5q) patients with myelodysplastic syndromes treated with lenalidomide. While the assay demonstrated good diagnostic accuracy in receiver operating characteristic analysis (area under the curve: 0.97), we further observed robust correlation between bone marrow and peripheral blood samples (R²=0.79), suggesting its potential suitability for less-invasive clonal monitoring.
Conclusions: In conclusion, we present an adaptable tool for quantification of chromosomal aberrations, particularly in problematic samples, which should be easily applicable to further tumour entities.
The human sense of smell is often analyzed as being composed of three main components comprising olfactory threshold, odor discrimination and the ability to identify odors. A relevant distinction of the three components and their differential changes in distinct disorders remains a research focus. The present data-driven analysis aimed at establishing a cluster structure in the pattern of olfactory subtest results. Therefore, unsupervised machine-learning was applied onto olfactory subtest results acquired in 10,714 subjects with nine different olfactory pathologies. Using the U-matrix, Emergent Self-organizing feature maps (ESOM) identified three different clusters characterized by (i) low threshold and good discrimination and identification, (ii) very high threshold associated with absent to poor discrimination and identification ability, or (iii) medium threshold, i.e., in the mid-range of possible thresholds, associated with reduced discrimination and identification ability. Specific etiologies of olfactory (dys)function were unequally represented in the clusters (p < 2.2 · 10−16). Patients with congenital anosmia were overrepresented in the second cluster while subjects with postinfectious olfactory dysfunction belonged frequently to the third cluster. However, the clusters provided no clear separation between etiologies. Hence, the present verification of a distinct cluster structure encourages continued scientific efforts at olfactory test pattern recognition.
Introduction: The 2017 update to the Global Initiative for Obstructive Lung Disease (GOLD) strategy document includes recommendations for treatment intensification or step-down in chronic obstructive pulmonary disease (COPD), although recognises that limited supporting information is available.
DACCORD is an ongoing observational, non-interventional study, recruiting patients following COPD maintenance treatment change or initiation, a subset of whom were receiving a long-acting β2-agonist (LABA) plus a long-acting muscarinic antagonist (LAMA) fixed-dose combination (FDC) on entry. Since there were no requirements in terms of prior medication (and no washout before commencing LABA/LAMA FDC), this provides an opportunity to generate "real world" data to test the GOLD 2017 recommendations.
Methods: To reduce heterogeneity, the current analyses include patients receiving indacaterol/glycopyrronium at baseline, and who, prior to the study, were receiving no COPD maintenance medication ("none"), LABA or LAMA monotherapy ("mono"), LABA plus inhaled corticosteroid (ICS; "LABA/ICS"), or triple therapy ("triple"). At the baseline visit, data collected included: demographic and disease characteristics; COPD Assessment Test (CAT); and exacerbations in the 6 months prior to entry. At 3, 6, 9 and 12 months data on exacerbations were collected, with CAT recorded at 3 and 12 months.
Results: A total of 2724 patients were included in the baseline analyses: 795, 954, 598 and 377 in the "none", "mono", "LABA/ICS" and "triple" subgroups, respectively. There were no clinically relevant differences in baseline demographics between the four groups. In terms of disease characteristics, the "triple" group had the highest proportion of patients with a disease duration of more than 1 year since diagnosis and with severe/very severe airflow limitation, but a similar percentage of non-exacerbators compared to the "none" group.
Over the 1-year follow-up, the majority of patients in all four subgroups did not exacerbate (exacerbation rates 0.16, 0.19, 0.21, and 0.26 in the "none", "mono", "LABA/ICS" and "triple" groups, respectively). At 12 months, 61.4%, 65.0%, 71.0% and 52.4% of patients had a clinically relevant improvement in CAT score.
Conclusions: Overall, the results support the GOLD recommendations in suggesting that a switch from a mono-bronchodilator or LABA plus ICS to LABA/LAMA FDC is a valid treatment option for patients with COPD. The results also validate the use of a LABA/LAMA FDC as initial maintenance treatment for COPD, and provide first "real world" evidence to support the newly added "step down" recommendation (from triple to LABA/LAMA FDC).
Plant regeneration is essential for maintaining forest biodiversity and ecosystem functioning, which are globally threatened by human disturbance. Here we present the first integrative meta-analysis on how forest disturbance affects multiple ecological processes of plant regeneration including pollination, seed dispersal, seed predation, recruitment and herbivory. We analysed 408 pairwise comparisons of these processes between near-natural and disturbed forests. Human impacts overall reduced plant regeneration. Importantly, only processes early in the regeneration cycle that often depend on plant-animal interactions, i.e. pollination and seed dispersal, were negatively affected. Later processes, i.e. seed predation, recruitment and herbivory, showed overall no significant response to human disturbance. Conserving pollination and seed dispersal, including the animals that provide these services to plants, should become a priority in forest conservation efforts globally.
H2S is an important signalling molecule involved in diverse biological processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect the activity of target proteins. Like thiols, persulfides show reactivity towards electrophiles and behave similarly to other cysteine modifications in a biotin switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based method allowing the isolation of persulfide containing peptides in the mammalian proteome. With this method, we demonstrated that H2S donors differ in their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis revealed that persulfide formation affects all subcellular compartments and various cellular processes. Negatively charged amino acids appeared more frequently adjacent to cysteines forming persulfides. We confirmed our proteomic data using pyruvate kinase M2 as a model protein and showed that several cysteine residues are prone to persulfide formation finally leading to its inactivation. Taken together, the site-specific identification of persulfides on a proteome scale can help to identify target proteins involved in H2S signalling and enlightens the biology of H2S and its releasing agents.
Epithelial-to-mesenchymal transition (EMT) is supposed to be responsible for increased invasion and metastases in epithelial cancer cells. The activation of EMT genes has further been proposed to be important in the process of malignant transformation of primary CNS tumors. Since the cellular source and clinical impact of EMT factors in primary CNS tumors still remain unclear, we aimed at deciphering their distribution in vivo and clinico-pathological relevance in human gliomas.
We investigated 350 glioma patients for the expression of the key EMT factors SLUG and TWIST by immunohistochemistry and immunofluorescence related to morpho-genetic alterations such as EGFR-amplification, IDH-1 (R132H) mutation and 1p/19q LOH. Furthermore, transcriptional cluster and survival analyses were performed.
Our data illustrate that SLUG and TWIST are overexpressed in gliomas showing vascular proliferation such as pilocytic astrocytomas and glioblastomas. EMT factors are exclusively expressed by non-neoplastic pericytes/vessel-associated mural cells (VAMCs). They are not associated with patient survival but correlate with pericytic/VAMC genes in glioblastoma cluster analysis.
In summary, the upregulation of EMT genes in pilocytic astrocytomas and glioblastomas reflects the level of activation of pericytes/VAMCs in newly formed blood vessels. Our results underscore that the negative prognostic potential of the EMT signature in the group of diffuse gliomas of WHO grade II-IV does most likely not derive from glioma cells but rather reflects the degree of proliferating mural cells thereby constituting a potential target for future alternative treatment approaches.
The spindle assembly checkpoint (SAC) acts as a molecular safeguard in ensuring faithful chromosome transmission during mitosis, which is regulated by a complex interplay between phosphatases and kinases including PLK1. Adenomatous polyposis coli (APC) germline mutations cause aneuploidy and are responsible for familial adenomatous polyposis (FAP). Here we study the role of PLK1 in colon cancer cells with chromosomal instability promoted by APC truncation (APC-ΔC). The expression of APC-ΔC in colon cells reduces the accumulation of mitotic cells upon PLK1 inhibition, accelerates mitotic exit and increases the survival of cells with enhanced chromosomal abnormalities. The inhibition of PLK1 in mitotic, APC-∆C-expressing cells reduces the kinetochore levels of Aurora B and hampers the recruitment of SAC component suggesting a compromised mitotic checkpoint. Furthermore, Plk1 inhibition (RNAi, pharmacological compounds) promotes the development of adenomatous polyps in two independent ApcMin/+ mouse models. High PLK1 expression increases the survival of colon cancer patients expressing a truncated APC significantly.
Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo‐tomography
(2017)
We employed electron cryo‐tomography to visualize cytosolic ribosomes on the surface of mitochondria. Translation‐arrested ribosomes reveal the clustered organization of the TOM complex, corroborating earlier reports of localized translation. Ribosomes are shown to interact specifically with the TOM complex, and nascent chain binding is crucial for ribosome recruitment and stabilization. Ribosomes are bound to the membrane in discrete clusters, often in the vicinity of the crista junctions. This interaction highlights how protein synthesis may be coupled with transport. Our work provides unique insights into the spatial organization of cytosolic ribosomes on mitochondria.