Acute fibrinolysis shutdown occurs early in septic shock and is associated with increased morbidity and mortality: results of an observational pilot study

Background: Septic coagulopathy represents a very dynamic disease entity, tilting from initial hypercoagulability towards a subsequent hypocoagulable disease state, entitled overt disseminated intravascular coagulation. Acute fibrinolysis shutdown has recently been described to be a crucial component of initial hypercoagulability in critically ill patients, although the underlying pathomechanisms, the specific temporal kinetics and its outcome relevance in patients with sepsis remain to be determined. Methods: In total, 90 patients (30 with septic shock, 30 surgical controls and 30 healthy volunteers) were enrolled. Blood samples were collected at sepsis onset or prior and immediately after the surgical procedure as well as 3 h, 6 h, 12 h, 24 h, 48 h and 7 d later, whereas blood samples from healthy volunteers were collected once. Besides viscoelastic and aggregometric point-of-care testing (POCT), enzyme-linked immunosorbent and thrombin generation assays and liquid chromatography–mass spectrometry-based measurements were performed. Results: As assessed by viscoelastic POCT, fibrinolysis shutdown occurred early in sepsis. Significant increases in tissue plasminogen activator had no effect on thromboelastometrical lysis indices (LIs). Contrariwise, plasminogen activator inhibitor-1 was already significantly increased at sepsis onset, which was paralleled by significantly increased LIs in patients suffering from septic shock in comparison with both control groups. This effect persisted throughout the 7-day observation period and was most pronounced in severely ill as well as non-surviving septic patients. Thromboelastometrical LI, therefore, proved to be suitable for early diagnosis [e.g. LI 45 min: area under the curve (AUC) up to 0.933] as well as prognosis (e.g. LI 60 min: AUC up to 1.000) of septic shock. Conclusions: Early inhibition of plasminogen activation leads to acute fibrinolysis shutdown with improved clot stability and is associated with increased morbidity and mortality in septic patients. Trial registration This study was approved by the local ethics committee (Ethics Committee of the Medical Faculty of Heidelberg; Trial-Code No. S247-2014/German Clinical Trials Register (DRKS)-ID: DRKS00008090; retrospectively registered: 07.05.2015). All study patients or their legal representatives signed written informed consent

Gut microbiome patterns correlate with higher postoperative complication rates after pancreatic surgery

Abstract Background Postoperative complications are of great relevance in daily clinical practice, and the gut microbiome might play an important role by preventing pathogens from crossing the intestinal barrier. The two aims of this prospective clinical pilot study were: (1) to examine changes in the gut microbiome following pancreatic surgery, and (2) to correlate these changes with the postoperative course of the patient. Results In total, 116 stool samples of 32 patients undergoing pancreatic surgery were analysed by 16S-rRNA gene next-generation sequencing. One sample per patient was collected preoperatively in order to determine the baseline gut microbiome without exposure to surgical stress and/or antibiotic use. At least two further samples were obtained within the first 10 days following the surgical procedure to observe longitudinal changes in the gut microbiome. Whenever complications occurred, further samples were examined. Based on the structure of the gut microbiome, the samples could be allocated into three different microbial communities (A, B and C). Community B showed an increase in Akkermansia, Enterobacteriaceae and Bacteroidales as well as a decrease in Lachnospiraceae, Prevotella and Bacteroides. Patients showing a microbial composition resembling community B at least once during the observation period were found to have a significantly higher risk for developing postoperative complications (B vs. A, odds ratio = 4.96, p < 0.01**; B vs. C, odds ratio = 2.89, p = 0.019*). Conclusions The structure of the gut microbiome is associated with the development of postoperative complications

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Identification and characterisation of enzymes that are associated with the COP9 signalosome

0\. Titelblatt und Inhaltsverzeichnis 1\. Einleitung 1 1.1. Das COP9 Signalosom 3 1.2. Die Rolle des COP9 Signalosoms im Ubiquitin/26S-Proteasom-System 4 1.3. Die Proteinkinasen PKD und CK2 14 2\. Zielsetzung 23 3\. Ergebnisse 24 3.1. Das COP9 Signalosom besitzt eine Kinaseaktivität 24 3.2. Identifizierung der Kinasen während der Komplexisolierung 26 3.3. CK2 und PKD binden an das COP9 Signalosom 30 3.4. CK2 und PKD phosphorylieren CSN-Untereinheiten, p53 und c-Jun 33 3.5. Inhibitoren der Kinasen 37 3.6. Humanes Mdm2 wird durch die CSN-assoziierten Kinasen phosphoryliert 38 3.7. Mdm2 bindet an das COP9 Signalosom 39 3.8. Curcumin induziert die Auto-Ubiquitylierung von Mdm2 42 4\. Diskussion 45 4.1. Die Kinaseaktivität des COP9 Signalosoms 45 4.2. Die CSN-assoziierten Proteinkinasen CK2 und PKD regulieren den Proteinabbau über das Ubiquitin/26S-Proteasom-System 53 4.3. Das COP9 Signalosom beeinflusst die Aktivität der Ubiquitin-Ligase Mdm2 55 4.4. Das COP9 Signalosom als Plattform für die Bildung von Ubiquitinkonjugaten 59 5\. Material und Methoden 63 5.1. Molekularbiologische Methoden 63 5.2. Proteinbiochemische Methoden 67 5.3. Zellkultur 77 6\. Zusammenfassung 79 7\. Referenzen 82 8\. Abkürzungen 94 9\. Appendix 97 9.1. Danksagung 97 9.2. Lebenslauf 98 9.3. Publikationen 99Das COP9 Signalosom (CSN) ist von Hefe bis zum Menschen konserviert und besteht aus acht Untereinheiten (CSN1-8). Der aus humanen Erythrocyten gereinigte CSN-Komplex besitzt Kinaseaktivität und phosphoryliert Proteine wie c-Jun und p53 mit Konsequenzen für ihren Ubiquitin-abhängigen Abbau. In dieser Studie konnte gezeigt werden, dass die Proteinkinase CK2 und die Proteinkinase D (PKD) mit dem CSN-Komplex co-purifizieren. Die Bindung der beiden Kinasen an den Komplex konnte mit Immunopräzipitationen und Far-Westernblots sowohl in vitro als auch in Zellkultur nachgewiesen werden. Elektronenmikroskopische Untersuchungen mit einem gold-markierten ATP-Analogon weisen darauf hin, dass mindestens 10% der Komplexe mit Kinasen assoziiert sind. CK2 bindet an ΔCSN3(111-403) und CSN7, während PKD nur CSN3 bindet. Die PKD ist in der Lage CSN7 zu phosphorylieren, während die CK2 zusätzlich CSN2 phosphorylieren kann. In Kinase-Assays konnte gezeigt werden, dass c-Jun und p53 Substrate der beiden Kinasen sind. Durch die CK2-abhängige Phosphorylierung von p53 an Threonin 155 wird der Tumorsuppressor verstärkt über das Ubiquitin(Ub)/26S- Proteasom-System abgebaut. Curcumin, Emodin, DRB und Resveratrol blockieren die Komplex-assoziierte Kinaseaktivität und hemmen sowohl die CK2 als auch die PKD. Das Mdm2, eine Ubiquitin-Ligase, ist für den Abbau des Tumorsuppressors p53 verantwortlich. Immunopräzipitationen, Far-Westernblots und Immunofluoreszenzanalysen zeigen, dass das Onkoprotein an den CSN-Komplex bindet. Darüber hinaus wird das Mdm2 am Komplex phosphoryliert, höchstwahrscheinlich durch die CK2. Die Phosphorylierung wird dabei durch Curcumin inhibiert. Ferner konnte sowohl in Ubiquitylierungs-Assays als auch in MCF-7-Zellen gezeigt werden, dass Curcumin die Bildung von hochmolekularen Mdm2-Ubiquitin-Konjugaten induziert. Basierend auf diesen Daten kann ein Modell aufgestellt werden, in dem die Ubiquitin-Ligase Mdm2 und der p53-Abbau durch die CSN-assoziierten Kinasen reguliert werden. Durch Hemmung der gebundenen Kinasen wird das Mdm2 ubiquityliert, wahrscheinlich durch eine Stimulierung der Auto-Ubiquitylierungsaktivität, und dies führt in der Folge zu einem proteasomalen Abbau von Mdm2. Mit anderen Worten: Die Phosphorylierung von Mdm2 hemmt die Auto-Ubiquitylierung. Das COP9 Signalosom dient somit als Plattform für die Regulation von Ubiquitin-Ligasen und des Proteinabbaus durch das Ubiquitin/26S-Proteasom-System.The COP9 signalosome (CSN) is conserved from yeast to man and consists of eight subunits (CSN1-8). The purified CSN complex from human erythrocytes possesses kinase activity that phosphorylates proteins such as c-Jun and p53 with consequences for their ubiquitin (Ub) dependent degradation. In this study it is shown that the protein kinase CK2 and the protein kinase D (PKD) co-purify with the CSN. The association of these kinases with the complex was demonstrated by far-western blots and by immunoprecipitation in vitro and in cell culture experiments. As indicated by electron microscopy with a gold- labelled ATP-analogue, at least 10% of CSN particles are associated with kinases. CK2 binds to ΔCSN3(111-403) and CSN7, whereas PKD interacts only with full-length CSN3. The PKD modifies CSN7 while the CK2 also phosphorylates the CSN2 subunit. In kinase assays both kinases phosphorylate recombinant c-Jun and p53. The phosphorylation of p53 at Thr155 by the CK2 targets the tumour suppressor to degradation by the Ub/26S proteasome system. Curcumin, emodin, DRB and resveratrol block CSN-associated kinases and also inhibit the CK2 and PKD. The oncoprotein Mdm2, a RING finger ubiquitin ligase, is responsible for the ubiquitylation of the tumour suppressor p53. By far-western blots, immunoprecipitation experiments and immunofluorescence analysis it is shown that Mdm2 binds to the CSN complex in vitro and in cell culture experiments. Mdm2 is phosphorylated by the CSN-associated kinases, most likely by the CK2. The phosphorylation is inhibited by curcumin. In addition, curcumin induces the formation of high molecular weight Mdm2 ubiquitin conjugates in vitro and in MCF-7 cells. Based on these data we propose a model in which the Ub ligase Mdm2 and the degradation of p53 are regulated by the CSN-associated kinases. Inhibition of the kinases causes Mdm2 ubiquitin conjugate formation, most likely by stimulation of the auto-ubiquitylation activity, followed by the degradation of Mdm2. In other words, the phosphorylation by the CSN-associated kinases prevents the auto-ubiquitylation of Mdm2. Thus the COP9 signalosome serves as a scaffolding platform that regulates ubiquitin ligases and protein degradation via the Ub/26S proteasome system

Apparatus and Method for Enhancing an Audio Signal, Sound Enhancing System

An apparatus for enhancing an audio signal comprises a signal processor for processing the audio signal in order to reduce or eliminate transient and tonal portions of the processed signal and a decorrelator for generating a first decorrelated signal and a second decorrelated signal from the processed signal. The apparatus further comprises a combiner for weightedly combining the first and the second decorrelated signal and the audio signal or a signal derived from the audio signal by coherence enhancement using time variant weighting factors and to obtain a two-channel audio signal. The apparatus further comprises a controller for controlling the time variant weighting factors by analyzing the audio signal so that different portions of the audio signal are multiplied by different weighting factors and the two-channel audio signal has a time variant degree of decorrelation

Gankyrin Is an Ankyrin-repeat Oncoprotein That Interacts with CDK4 Kinase and the S6 ATPase of the 26 S Proteasome*

A yeast two-hybrid screen with the human S6 (TBP7, RPT3) ATPase of the 26 S proteasome has identified gankyrin, a liver oncoprotein, as an interacting protein. Gankyrin interacts with both free and regulatory complex-associated S6 ATPase and is not stably associated with the 26 S particle. Deletional mutagenesis shows that the C-terminal 78 amino acids of the S6 ATPase are necessary and sufficient to mediate the interaction with gankyrin. Deletion of an orthologous gene in Saccharomyces cerevisiae suggests that it is dispensable for cell growth and viability. Overexpression and precipitation of tagged gankyrin from cultured cells detects a complex containing co-transfected tagged S6 ATPase (or endogenous S6) and endogenous cyclin D-dependent kinase CDK4. The proteasomal ATPases are part of the AAA (ATPases associated with diverse cellular activities) family, members of which are molecular chaperones; gankyrin complexes may therefore influence CDK4 function during oncogenesis

Data on microcirculatory parameters of GTS- 21 treated rats assessed by intravital microscopy

This article contains animal experimental data associated with the research article entitled âGTS-21 reduces microvascular permeability during experimental endotoxemiaâ (Schmidt et al., 2017) [1] (supplementary datasets of baseline intravital microscopic measurements, baseline TNF-α levels and vital parameters of the evaluated experimental groups are provided). Beneficial anti-inflammatory effects of cholinergic mediators on microvascular inflammation have been demonstrated by intravital microscopic investigations (Schmidt et al., 2015) [2], therefore we evaluated the effect of the cholinergic mediator GTS-21 on microcirculatory alterations during endotoxemia [1]. The data regarding microcirculatory effects of GTS-21 treatment ((3-(2,4-Dimethoxybenzylidene)-anabaseine dihydrochloride; 1 mg/kg; i.v.) in non-endotoxemic animals are presented in this article

Concurrent Change in Serum Cholinesterase Activity and Midregional-Proadrennomedullin Level Could Predict Patient Outcome following Liver Transplantation

Background: After liver transplantation (LTX), patients are susceptible to opportunistic infections resulting in reduced outcomes within the early post-transplantation period. The postoperative monitoring of LTX patients has gained much importance in recent years. However, reliable plasmatic markers predicting 90-day outcomes are still lacking. Methods: In the post hoc analysis of a prospective, observational study, butyrylcholinesterase (BChE), mid-regional proadrenomedullin (MR-proADM), as well as conventional inflammatory markers (procalcitonin, C-reactive protein) were evaluated in 93 patients at seven consecutive timepoints within the first 28 days following LTX. Results: Persistently reduced activity of BChE and elevated MR-proADM levels indicated reduced 90-day survival following LTX. Furthermore, reduced BChE and increased MR-proADM activity could indicate early post-transplantation bacterial infections, whereas conventional inflammatory biomarkers showed no diagnostic efficacy within the observation period. Conclusion: Concurrent assessment of BChE and MR-proADM activity might serve as a bedside diagnostic tool for early bacterial infections following liver transplantation. Thus, a combined utilization of the two biomarkers may be a useful tool in the risk evaluation of patients following liver transplantation