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Martin Kulke,1 Felix Nagel,1 Lukas Schulig,2 Norman Geist,1 Marcel Gabor,1 Julia Mayerle,3 Markus M Lerch,4 Andreas Link,2 Mihaela Delcea1 1Institute of Biochemistry, University of Greifswald, Greifswald, Germany; 2Institute of Pharmacy, University of Greifswald, Greifswald, Germany; 3Department of Medicine II, Ludwig-Maximilian University of Munich, Munich, Germany; 4Department of Medicine a, University Medicine Greifswald, Greifswald, GermanyCorrespondence: Mihaela Delcea; Martin KulkeInstitute of Biochemistry, University of Greifswald, Felix-Hausdorff-Straße 4, Greifswald, 17487, GermanyTel +49 3834 420 4423Fax +49 3834 420 4377Email delceam@uni-greifswald.de; makulke@web.dePurpose: Although strongly related, the pathophysiological effect of the N34S mutation in the serine protease inhibitor Kazal type 1 (SPINK1) in chronic pancreatitis is still unknown. In this study, we investigate the conformational space of the human cationic trypsin-serine protease inhibitor complex.Methods: Simulations with molecular dynamics, replica exchange, and transition pathway methods are used.Results: Two main binding states of the inhibitor to the complex were found, which explicitly relate the influence of the mutation site to conformational changes in the active site of trypsin.Conclusion: Based on our result, a hypothesis is formulated that explains the development of chronic pancreatitis through accelerated digestion of the mutant by trypsin.Keywords: trypsin, molecular dynamics simulations, replica exchange, transition path sampling, umbrella sampling

The Krüppel-like transcription factor (KLF) BCL11B is characterized by a wide tissue distribution and crucial functions in key developmental and cellular processes, as well as in various pathologies including cancer and HIV infection. Although the basics of BCL11B activity and relevant interactions with other proteins have been uncovered, how this exclusively nuclear protein localizes to its compartment remained unclear. Here, we demonstrate that unlike other KLFs, BCL11B does not require the C-terminal DNA-binding domain to pass through the nuclear envelope but has an independent, previously unidentified, nuclear localization signal (NLS), which is located distantly from the zinc finger domains and fulfills the essential criteria of being an autonomous NLS. First, it can redirect a heterologous cytoplasmic protein to the nucleus. Second, its mutation causes aberrant localization of the protein of origin. Finally, we provide experimental and in silico evidences of the direct interaction with importin-α. The relative conservation of this motif allows formulating a consensus sequence (K/R)K-X13–14-KR+K++ (‘+’ indicates amino acids with similar chemical properties), which can be found in all BCL11B orthologs among vertebrates and in the closely related protein BCL11A.

Biocatalytic alkylations are important reactions to obtain chemo‐, regio‐ and stereoselectively alkylated compounds. This can be achieved using S‐adenosyl‐l‐methionine (SAM)‐dependent methyltransferases and SAM analogs. It was recently shown that a halide methyltransferase (HMT) from Chloracidobacterium thermophilum can synthesize SAM from SAH and methyl iodide. We developed an iodide‐based assay for the directed evolution of an HMT from Arabidopsis thaliana and used it to identify a V140T variant that can also accept ethyl‐, propyl‐, and allyl iodide to produce the corresponding SAM analogs (90, 50, and 70 % conversion of 15 mg SAH). The V140T AtHMT was used in one‐pot cascades with O‐methyltransferases (IeOMT or COMT) to achieve the regioselective ethylation of luteolin and allylation of 3,4‐dihydroxybenzaldehyde. While a cascade for the propylation of 3,4‐dihydroxybenzaldehyde gave low conversion, the propyl‐SAH intermediate could be confirmed by NMR spectroscopy., Biocatalytic alkylations are valuable for late‐stage functionalization but are limited by the availability of S‐adenosyl‐l‐methionine analogs. Directed evolution was used to create an engineered halide methyltransferase capable of converting cheap and readily available alkyl iodides into a number of SAM analogs. Used in cascades with methyltransferases, this enables chemo‐, regio‐ and stereoselective alkylations which are difficult to achieve by chemical means.

Clinical research in cardiology 110(8), 1150-1158 (2021). doi:10.1007/s00392-021-01867-2, Published by Springer, Berlin

Water consumption along value chains of goods and services has increased globally and led to increased attention on water footprinting. Most global water consumption is accounted for by evaporation (E), which is connected via bridges of atmospheric moisture transport to other regions on Earth. However, the resultant source-receptor relationships between different drainage basins have not yet been considered in water footprinting. Based on a previously developed data set on the fate of land evaporation, we aim to close this gap by using comprehensive information on evaporation recycling in water footprinting for the first time. By considering both basin internal evaporation recycling (BIER; >5% in 2% of the world's basins) and basin external evaporation recycling (BEER; >50% in 37% of the world's basins), we were able to use three types of water inventories (basin internal, basin external, and transboundary inventories), which imply different evaluation perspectives in water footprinting. Drawing on recently developed impact assessment methods, we produced characterization models for assessing the impacts of blue and green water evaporation on blue water availability for all evaluation perspectives. The results show that the negative effects of evaporation in the originating basins are counteracted (and partly overcompensated) by the positive effects of reprecipitation in receiving basins. By aggregating them, combined net impacts can be determined. While we argue that these offset results should not be used as a standalone evaluation, the water footprint community should consider atmospheric moisture recycling in future standards and guidelines.

Reactive oxygen species (ROS) are known to trigger drug release from arylboronate-containing ROS-responsive prodrugs. In cancer cells, elevated levels of ROS can be exploited for the selective activation of prodrugs via Baeyer-Villiger type oxidation rearrangement sequences. Here, we report a proof of concept to demonstrate that these cascades can as well be initiated by cold physical plasma (CPP). An analog of a recently reported fluorouracil prodrug based on the less toxic drug 5-fluorocytosine (5-FC) was synthesized with a view to laboratory safety reasons and used as a model compound to prove our hypothesis that CPP is suitable as a trigger for the prodrug activation. Although the envisioned oxidation and rearrangement with successive loss of boronic acid species could be achieved by plasma treatment, the anticipated spontaneous liberation of 5-FC was inefficient in the model case. However, the obtained results suggest that custom-tailored CPP-responsive prodrugs might become an evolving research field.

We demonstrate an acoustic device to mechanically probe a population of red blood cells at the single cell level. The device operates by exciting a surface acoustic wave in a microfluidic channel creating a stationary acoustic wave field of nodes and antinodes. Erythrocytes are attracted to the nodes and are deformed. Using a stepwise increasing and periodically oscillating acoustic field we study the static and dynamic deformation of individual red blood cells one by one. We quantify the deformation by the Taylor deformation index D and relaxation times τ1 and τ2 that reveal both the viscous and elastic properties of the cells. The precision of the measurement allows us to distinguish between individual cells in the suspension and provides a quantitative viscoelastic fingerprint of the blood sample at single cell resolution. The method overcomes limitations of other techniques that provide averaged values and has the potential for high-throughput.

Because isoenzymes of the experimentally and therapeutically extremely relevant sirtuin family show high similarity, addressing the unique selectivity pocket of sirtuin 2 is a promising strategy towards selective inhibitors. An unrelated approach towards selective inhibition of isoenzymes with varied tissue distribution is targeted drug delivery or spatiotemporal activation by photochemical activation. Azologization of two nicotinamide‐mimicking lead structures was undertaken to combine both approaches and yielded a set of 33 azobenzenes and azopyridines that have been evaluated for their photochemical behaviour and bioactivity. For some compounds, inhibitory activity reached the sub‐micromolar range in their thermodynamically favoured E form and could be decreased by photoisomerization to the metastable Z form. Besides, derivatization with long‐chain fatty acids yielded potent sirtuin 2 inhibitors, featuring another intriguing aspect of azo‐based photoswitches. In these compounds, switching to the Z isomer increased aqueous solubility and thereby enhanced biological activity by up to a factor of 21. The biological activity of two compounds was confirmed by hyperacetylation of sirtuin specific histone proteins in a cell‐based activity assay., Switched into action: Besides fine‐tuning molecular geometry for enzyme inhibition, photoinduced isomerization of azopyridine‐based nicotinamide mimics results in improved aqueous solubility, enforcing the effect on target protein sirtuin 2. The resulting Sirt2 inhibitor could be used to study binding events in cellular systems with spatiotemporal control by two independent molecular and supramolecular effects.

Die Wurzeln der Arzneimittelforschung reichen zuruck bis in die Anfange der Menschheitsgeschichte. Von jeher war es der Traum, auf gezieltem Weg zu Therapeutika zu gelangen. Schon fur die ersten Hochkulturen ist der Einsatz pflanzlicher, mineralischer oder tierischer Drogen belegt. Im Mittelalter suchten die Alchimisten nach dem Lebenselixier, das alle Krankheiten zu heilen vermag – leider vergebens. Bis zum Anfang des 19. Jahrhunderts beschrankte sich die Arzneimitteltherapie indessen auf die Verabreichung nicht-standardisierter Naturstoffgemische und anorganischer Chemikalien. Viele der damals verfolgten Konzepte entspringen der traditionellen.

On-line solid-phase supercritical fluid extraction (SFE) and chromatography (SFC) coupled to mass spectrometry (MS) has been evaluated for its usefulness with respect to metabolic profiling and pharmacological investigations of ketamine in humans. The aim of this study was to develop and validate a rapid, highly selective and sensitive SFE–SFC–MS method for the quantification of ketamine and its metabolites in miniature amounts in human urine excluding liquid-liquid extraction (LLE). Several conditions were optimized systematically following the requirements of the European Medicines Agency: selectivity, carry-over, calibration curve parameters (LLOQ, range and linearity), within- and between-run accuracy and precision, dilution integrity, matrix effect, and stability. The method, which required a relatively small volume of human urine (20 μL per sample), was validated for pharmacologically and toxicologically relevant concentrations ranging from 25.0 to 1000 ng/mL (r2 > 0.995). The lower limit of quantification (LLOQ) for all compounds was found to be as low as 0.5 ng. In addition, stability of analytes during removal of water from the urine samples using different conditions (filter paper or ISOLUTE® HM-N) was studied. In conclusion, the method developed in this study can be successfully applied to studies of ketamine metabolites in humans, and may pave the way for routine application of on-line SFE–SFC–MS in clinical investigations.

Flupirtine, an opener of neuronal voltage gated potassium channels (KV7.2/3), has been used as a therapeutic alternative for pain treatment in patients refractory to NSAIDs and opioids. Because flupirtine is associated with rare but fatal drug-induced liver injury that may result from the formation of toxic metabolites upon metabolic oxidation, we synthesized novel derivatives with the goal of identifying equally active and ultimately safer KV7.2/3 channel openers. Four thioether analogues were designed to lack a nitrogen atom that would be a prerequisite for the formation of toxic para-quinone diimines, and form sulfoxide and sulfone metabolites instead. KV7.2/3 channel opening activity and hepatotoxicity data of twelve novel flupirtine analogues, four thioethers and their respective sulfoxide and sulfone metabolites are reported.

Simple Summary Protein expression profiles of 10 HDAC/Sirtuin isoenzymes in two panels of human cancer cell lines were compared with each other and with the potencies of various anticancer drugs by Pearson and Spearman correlation analysis to identify patterns of enzyme expression and anticancer activity. Furthermore, the NCI COMPARE database was used to identify possible correlations between the mRNA expression in a 60 cancer cell panel and the potency of the same anticancer drugs. While several interesting correlations were found within both data sets, none of these correlations were identical in the two sets of data, suggesting that protein and mRNA expression profiles are not comparable. Combination treatments with several HDAC inhibitors with a number of the anticancer drugs revealed interesting synergistic effects that were in keeping with some of the correlations predicted by our protein expression analysis. Abstract Inhibiting the activity of histone deacetylase (HDAC) is an ongoing strategy in anticancer therapy. However, to our knowledge, the relationships between the expression of HDAC proteins and the antitumor drug sensitivity of cancer cells have not been studied until now. In the current work, we investigated the relative expression profiles of 10 HDAC isoenzymes comprising the classes I–III (HDAC1/2/4/6; Sirt1/2/3/5/6/7) in a panel of 17 cancer cell lines, including the breast, cervix, oesophageal, lung, oral squamous, pancreas, as well as urinary bladder carcinoma cells. Correlations between the data of mRNA expression for these enzymes obtained from the National Cancer Institute (NCI) 60 cancer cell line program were also examined. Next, we performed univariate analysis between the expression patterns of HDAC/Sirt isoenzymes with the sensitivity of a 16 cell panel of cancer cell lines towards several antitumor drugs. In a univariate correlation analysis, we found a strong relation between Sirt2 expression and cytotoxicity caused by busulfan, etoposide, and hydroxyurea. Moreover, it was identified that Sirt5 correlates with the effects exerted by oxaliplatin or topotecan, as well as between HDAC4 expression and these two drugs. Correlations between the data of mRNA expression for enzymes with the potencies of the same anticancer agents obtained from the NCI 60 cancer cell line program were also found, but none were the same as those we found with our protein expression data. Additionally, we report here the effects upon combination of the approved HDAC inhibitor vorinostat and one other known inhibitor trichostatin A as well as newer hetero-stilbene and diazeno based sirtuin inhibitors on the potency of cisplatin, lomustine, and topotecan. For these three anticancer drugs, we found a significantly enhanced cytotoxicity when co-incubated with HDAC inhibitors, demonstrating a potentially beneficial influence of HDAC inhibition on anticancer drug treatment.

BACKGROUND Receptor for advanced glycation end products (RAGE) and its cleavage fragment soluble RAGE (sRAGE) are opposite players in inflammation. Enhanced monocytic RAGE expression and decreased plasma sRAGE levels are associated with higher mortality in infarction-related cardiogenic shock. Active matrix metalloproteinase-9 (MMP-9) has been implied in RAGE ectodomain cleavage and subsequently sRAGE shedding in vitro. We investigated MMP-9 activity in myocardial infarction-induced cardiogenic shock with regard to RAGE/sRAGE regulation. METHODS AND RESULTS We determined MMP-9 serum activity by zymography and tissue inhibitor of matrix metalloproteinases (TIMP-1) expression by Western blot and correlated it to RAGE/sRAGE data in patients with cardiogenic shock after acute myocardial infarction (CS, n = 30), in patients with acute myocardial infarction without shock (AMI, n = 20) and in healthy volunteers (n = 20).MMP-9 activity is increased in AMI (P = 0.02 versus controls), but significantly decreased in CS with lowest levels in non-survivors (n = 13, P = 0.02 versus AMI). In all patients, MMP-9 activity correlated inversely with RAGE expression on circulating monocytes (r = -0.57; P = 0.0001; n = 50).TIMP-1 levels showed an inverse regulation in comparison to active MMP-9 with significantly decreased levels in AMI as compared with controls (P = 0.02 versus controls) and highest levels in non-survivors of CS (P

In many cases, native states of proteins may be predicted with sufficient accuracy by molecular dynamics simulations (MDSs) with modern force fields. Enhanced sampling methods based on MDS are applied for exploring the phase space of a protein sequence and to overcome barriers on rough conformational energy landscapes. The minimum free energy state is obtained with sampling algorithms providing sufficient convergence and accuracy. A reliable but computationally very expensive method is replica exchange molecular dynamics, with many modifications to this approach presented in the past. Recently, we demonstrated how our temperature intervals with global exchange of replicas hybrid (TIGER2h) solvent sampling algorithm made a good compromise between efficiency and accuracy. There, all states are sampled under full explicit solvent conditions with a freely chosen number of replicas, whereas an implicit solvent is used during the swap decisions. This hybrid method yielded a much better approximation to the agreement with calculations in an explicit solvent than fully implicit solvent simulations. Here, we present an extension of TIGER2h and add a few layers of explicit water molecules around the peptide for the energy calculations, whereas the dynamics in fully explicit water is maintained. We claim that these water layers better reproduce steric effects, the polarization of the solvent, and the resulting reaction field energy than typical implicit solvent models. By investigating the protein-solvent interactions across comprehensive thermodynamic state ensembles, we found a strong conformational dependence of this reaction field energy. All simulations were performed with nanoscale molecular dynamics on two peptides, the α-helical peptide (AAQAA)3 and the β-hairpin peptide HP7. A production-ready TIGER2hs implementation is supplied, approaching the accuracy of full explicit solvent sampling at a fraction of computational resources.

The use of light as an external trigger to change ligand shape and as a result its bioactivity, allows the probing of pharmacologically relevant systems with spatiotemporal resolution. In this context, aromatic diazeno compounds are well suited for the design of photoswitchable ligands due to the long thermal relaxation half‐lives of the photoinduced Z configuration, and tunability of the absorption wavelength λ max. In search for sirtuin inhibitors, a hetero-stilbene lead resulting from the screening of a compound that was originally designed as kinase inhibitor, was remodelled to its diazeno analogue. By this azologization, the shape of the molecule was left unaltered whereas the photoswitching ability was improved. As anticipated, the highly analogous compound showed similar activity in its thermodynamically stable stretched-out E form. Irradiation of this isomer triggers isomerisation to the Z configuration with a bent geometry causing a considerably shorter end‐to‐end distance. The resulting affinity shifts are intended to enable real‐time photomodulation of sirtuins in vitro.

High-pressure gas chromatography is not a novel concept, but it is an intriguing one: the eco-friendly chimera seeks to combine the advantages of GC and HPLC, which has led to the development of supercritical fluid chromatography (SFC). But while the chromatographic age of supercritical fluids has been advertised by proponents and manufacturers for more than 50 years, SFC has largely been deprecated as science fiction due to complex method development, a perceived lack of robustness, and a tendency to fall short of expectations. For the longest time, separation scientists were justifiably skeptical—if not blissfully unaware—of the reformation process taking place within SFC, which has only recently escaped scientific anonymity/infamy by gaining industrial significance as the method of choice for chiral and semi-preparative separations. Indeed, the truly exceptional performance and a multitude of adjustable parameters make SFC arguably the most versatile of industrially employed chromatographic techniques. Here, the principal reasons for the renewed interest in SFC (what is SFC good for?) are reviewed, before addressing the underlying physical principles and instrumental requirements (what makes SFC work?). A discussion of the fundamental limitations (what is the catch?) follows. The frontiers between different types of chromatography are fading and may soon give way to unified or convergent analysis that is expected to provide a more accurate analytical vision than any singular form of chromatography.

Drug induced liver injury (DILI) and tissue discoloration led to the recent discontinuation of the therapeutic use of the closely related drugs flupirtine and retigabine, respectively. Experience gained with these drugs strongly suggests that heterotetramer, voltage-gated potassium channels 2 and 3 (KV7.2/3) are valid targets for effective treatment of pain and epilepsy. Because the adverse effects are not related to the mechanism of action, it appears promising to investigate chemical modifications of these clinically validated, drug-like leads. In the present retro-metabolic drug design study, a series of 43 compounds were synthesized and characterized with regard to KV7.2/3 opening activity and efficacy. The most active compound 22d displays excellent potency (EC50 = 4 nM) and efficacy (154%) as a KV7.2/3 opener. Limited aqueous solubility hampered toxicity testing at concentrations higher than 63 μM, but this concentration was nontoxic to two hepatocellular cell lines (HEP-G2 and TAMH) in culture. The slightly less active but more soluble compound 25b (EC50 = 11 nM, efficacy 111%) showed an improved toxicity/activity ratio compared to flupirtine by three orders of magnitude and represents an attractive lead structure for the development of safer analgesics and antiepileptics.

The highly structurally similar drugs flupirtine and retigabine have been regarded as safe and effective for many years but lately they turned out to exert intolerable side effects. While the twin molecules share the mode of action, both stabilize the open state of voltage-gated potassium channels, the form and severity of adverse effects is different. The analgesic flupirtine caused drug-induced liver injury in rare but fatal cases, whereas prolonged use of the antiepileptic retigabine led to blue tissue discoloration. Because the adverse effects seem unrelated to the mode of action, it is likely, that both drugs that occupied important therapeutic niches, could be replaced. Reasons for the clinically relevant toxicity will be clarified and future substitutes for these drugs presented in this review.

The potassium channel openers flupirtine and retigabine have proven to be valuable analgesics or antiepileptics. Their recent withdrawal due to occasional hepatotoxicity and tissue discoloration, respectively, leaves a therapeutic niche unfilled. Metabolic oxidation of both drugs gives rise to the formation of electrophilic quinones. These elusive, highly reactive metabolites may induce liver injury in the case of flupirtine and blue tissue discoloration after prolonged intake of retigabine. We examined which structural features can be altered to avoid the detrimental oxidation of the aromatic ring and shift oxidation toward the formation of more benign metabolites. Structure-activity relationship studies were performed to evaluate the K

Indoles are privileged structures in medicinal and bioorganic chemistry that are particularly well suited to serve as platforms for diversity. Among many other therapeutic areas, the indole scaffold has been used to design aromatic compounds useful to interfere with enzymes engaged in the regulation of substrate acylation status, such as sirtuins. However, the planarity of the indole ring is not necessarily optimal for all target enzymes, especially when functionalization with aromatic side chains is required. Replacement of flat scaffolds by nonplanar molecular cores dominated by sp3 hybridization is a common strategy to avoid the disadvantages associated with poor solubility and high promiscuity, while covering less-well-explored areas of chemical space. Thus, we synthesized fragment-like tetrahydroindoles suitable for fragment-based drug discovery as well as a well-characterized small library intended as multipurpose screening compounds. For proof of principle, these compounds were screened against sirtuins 1-3, enzymes known to be addressable by indoles. We found that 2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxamides are potent and selective SIRT2 inhibitors. Compound 16 t displayed an IC50 value of 0.98 μm and could serve as exquisite starting point for hit-to-lead profiling.

We present an acoustofluidic device for fluorescently triggered merging of surfactant-stabilized picoliter droplet pairs at high throughput. Droplets that exceed a preset fluorescence threshold level are selectively merged by a traveling surface acoustic wave (T-SAW) pulse. We characterize the operation of our device by analyzing the merging efficiency as a function of acoustic pulse position, duration, and acoustic pressure amplitude. We probe droplet merging at different droplet rates and find that efficient merging occurs above a critical acoustic power level. Our results indicate that the efficiency of acoustically induced merging of surfactant stabilized droplets is correlated with acoustic streaming velocity. Finally, we discuss how both time-averaged and instantaneous acoustic pressure fields can affect the integrity of surfactant layers. Our technique, by allowing the merging of up to 105 droplets per hour, shows great potential for integration into microfluidic systems for high-throughput and high-content screening applications.

Die oben gezeigten Arbeitsmaschinen und Industrieanwendungen mit mehr als 76 kW, werden heute vorwiegend durch Dieselmotoren bestritten. Ein kleiner Teil verwendet Gasmotoren mit LPG oder CNG. Weniger als 0,5 % bei mobilen Arbeitsmaschinen verwenden elektrische Antriebe.

Transcription factors play a crucial role in regulating biological processes such as cell growth, differentiation, organ development and cellular signaling. Within this group, proteins equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown to be critically important for efficient regulation of the BCL11B target genes and could therefore represent a promising target for novel drug therapies. Here, we report the structural basis for BCL11B–BCL11B interaction mediated by the N-terminal ZF domain. By combining structure prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of the single ZF domain and directly involved in forming the dimer interface. These findings not only provide deep insight into how BCL11B acquires its active structure but also represent an important step towards rational design or selection of potential inhibitors.

Anamnese und klinischer Befund: Eine 23-jahrige Patientin wird mit dem Verdacht auf ein akutes Leberversagen in ein Krankenhaus der tertiaren Versorgung verlegt. Vorausgegangen war die Einnahme von Ciprofloxacin (2 × 500 mg / Tag) uber zwei Tage aufgrund von unspezifischen Allgemeinsymptome (Ubelkeit und Erbrechen). Die Beschwerden bestanden bereits seit einer Woche. Anfangs ist die Patientin neurologisch unauffallig, dann vermindert sich die Vigilanz im Verlauf bis hin zum Sopor. Bis auf eine Appendektomie im Jugendalter sind keine Vorerkrankungen bekannt. Untersuchungen: Die initiale korperliche und klinisch-neurologische Untersuchung ergibt keine auffalligen Befunde, auser einem Sklerenikterus und diskreten rechtsseitigen Oberbauchschmerzen. Laborchemisch zeigen sich deutlich angestiegene Transaminasen sowie eine Hyperammonamie und -bilirubinamie. Das toxikologische Screening ist unauffallig. Therapie und Verlauf: Es wird die Diagnose eines akuten, moglicherweise medikamentos induzierten, Leberversagens gestellt. Sofort wird eine Ammoniakdetoxifikation mit Laktulose eingeleitet. Die transjugulare Leberbiopsie ergibt eine ausgepragte, mit erheblichen Zytolysen einhergehende lobulare Hepatitis. Bei progredienter Vigilanzminderung wird eine Hirnodemtherapie mittels Glycerin und Mannitol eingeleitet. Hierunter steigt die Serumosmolaritat, der Ammoniakspiegel sinkt und die Patientin bessert sich neurologisch. Im Verlauf kommt es innerhalb von 48 Stunden zur vollstandigen klinischen Rekonstitution und die Lebersyntheseleistung bessert sich. Folgerung: Nachdem haufige Ursachen ausgeschlossen wurden, sollte bei einem akuten Leberversagen auch an eine Fluorchinolon-induzierte Leberschadigung gedacht und eine exakte Medikamentenanamnese erhoben werden.

During submission the author name Andreas Link was unfortunately omitted. The correct author list reads as follows.

Flupirtine, an opener of neuronal voltage gated potassium channels (KV7.2/3), has been used as a therapeutic alternative for pain treatment in patients refractory to NSAIDs and opioids. Because flupirtine is associated with rare but fatal drug-induced liver injury that may result from the formation of toxic metabolites upon metabolic oxidation, we synthesized novel derivatives with the goal of identifying equally active and ultimately safer KV7.2/3 channel openers. Four thioether analogues were designed to lack a nitrogen atom that would be a prerequisite for the formation of toxic para-quinone diimines, and form sulfoxide and sulfone metabolites instead. KV7.2/3 channel opening activity and hepatotoxicity data of twelve novel flupirtine analogues, four thioethers and their respective sulfoxide and sulfone metabolites are reported.

Itaconic acid is produced by mammalian leukocytes upon pro-inflammatory activation. It appears to inhibit bacterial growth and to rewire the metabolism of the host cell by inhibiting succinate dehydrogenase. Yet, it is unknown whether itaconic acid acts only intracellularly, locally in a paracrine fashion, or whether it is even secreted from the inflammatory cells at meaningful levels in peripheral blood of patients with severe inflammation or sepsis. The aim of this study was to determine the release rate of itaconic acid from pro-inflammatory activated macrophages in vitro and to test for the abundance of itaconic acid in bodyfluids of patients suffering from acute inflammation. We demonstrate that excretion of itaconic acid happens at a low rate and that it cannot be detected in significant amounts in plasma or urine of septic patients or in liquid from bronchial lavage of patients with pulmonary inflammation. We conclude that itaconic acid may serve as a pro-inflammatory marker in immune cells but that it does not qualify as a biomarker in the tested body fluids.

Picking values from long ordered lists, such as when setting a date or time, is a common task on smartphones. However, the system pickers and tables used for this require significant screen space for spinning and dragging, covering other information or pushing it off-screen. The Force Picker reduces this footprint by letting users increase and decrease values over a wide range using force touch for rate-based control. However, changing input direction this way is difficult. We propose three techniques to address this. With our best candidate, Thumb-Roll, the Force Picker lets untrained users achieve similar accuracy as a standard picker, albeit less quickly. Shrinking it to a single table row, 20% of the iOS picker height, slightly affects completion time, but not accuracy. Intriguingly, after 70 minutes of training, users were significantly faster with this minimized Thumb-Roll Picker compared to the standard picker, at the same accuracy and only 6% of the gesture footprint. We close with application examples.

Background Though the Expanded Disability Status Scale (EDSS) is commonly used to assess disability level in relapsing-remitting multiple sclerosis (RRMS), the criteria defining disability progression are used for patients with a wide range of baseline levels of disability in relatively short-term trials. As a result, not all EDSS changes carry the same weight in terms of future disability, and treatment benefits such as decreased risk of reaching particular disability milestones may not be reliably captured. The objectives of this analysis are to assess the probability of confirmed disability worsening to specific EDSS milestones (i.e., EDSS scores ≥3.0, ≥4.0, or ≥6.0) at 288 weeks in the Tysabri Observational Program (TOP) and to examine the impact of relapses occurring during natalizumab therapy in TOP patients who had received natalizumab for ≥24 months. Methods TOP is an ongoing, open-label, observational, prospective study of patients with RRMS in clinical practice. Enrolled patients were naive to natalizumab at treatment initiation or had received ≤3 doses at the time of enrollment. Intravenous natalizumab (300 mg) infusions were given every 4 weeks, and the EDSS was assessed at baseline and every 24 weeks during treatment. Results Of the 4161 patients enrolled in TOP with follow-up of at least 24 months, 3253 patients with available baseline EDSS scores had continued natalizumab treatment and 908 had discontinued (5.4% due to a reported lack of efficacy and 16.4% for other reasons) at the 24-month time point. Those who discontinued due to lack of efficacy had higher baseline EDSS scores (median 4.5 vs. 3.5), higher on-treatment relapse rates (0.82 vs. 0.23), and higher cumulative probabilities of EDSS worsening (16% vs. 9%) at 24 months than those completing therapy. Among 24-month completers, after approximately 5.5 years of natalizumab treatment, the cumulative probabilities of confirmed EDSS worsening by 1.0 and 2.0 points were 18.5% and 7.9%, respectively (24-week confirmation), and 13.5% and 5.3%, respectively (48-week confirmation). The risks of 24- and 48-week confirmed EDSS worsening were significantly higher in patients with on-treatment relapses than in those without relapses. An analysis of time to specific EDSS milestones showed that the probabilities of 48-week confirmed transition from EDSS scores of 0.0–2.0 to ≥3.0, 2.0–3.0 to ≥4.0, and 4.0–5.0 to ≥6.0 at week 288 in TOP were 11.1%, 11.8%, and 9.5%, respectively, with lower probabilities observed among patients without on-treatment relapses (8.1%, 8.4%, and 5.7%, respectively). Conclusions In TOP patients with a median (range) baseline EDSS score of 3.5 (0.0–9.5) who completed 24 months of natalizumab treatment, the rate of 48-week confirmed disability worsening events was below 15%; after approximately 5.5 years of natalizumab treatment, 86.5% and 94.7% of patients did not have EDSS score increases of ≥1.0 or ≥2.0 points, respectively. The presence of relapses was associated with higher rates of overall disability worsening. These results were confirmed by assessing transition to EDSS milestones. Lower rates of overall 48-week confirmed EDSS worsening and of transitioning from EDSS score 4.0–5.0 to ≥6.0 in the absence of relapses suggest that relapses remain a significant driver of disability worsening and that on-treatment relapses in natalizumab-treated patients are of prognostic importance.

The JumonjiC-domain-containing histone demethylase 2A (JMJD2A, KDM4A) is a key player in the epigenetic regulation of gene expression. Previous publications have shown that both elevated and lowered enzyme levels are associated with certain types of cancer, and therefore the definite role of KDM4A in oncogenesis remains elusive. To identify a novel molecular starting point with favorable physicochemical properties for the investigation of the physiological role of KDM4A, we screened a number of molecules bearing an iron-chelating moiety by using two independent assays. In this way, we were able to identify 2-(1H-tetrazol-5-yl)acetohydrazide as a novel fragment-like lead structure with low relative molecular mass (Mr =142 Da), low complexity, and an IC50 value of 46.6 μm in a formaldehyde dehydrogenase (FDH)-coupled assay and 2.4 μm in an antibody-based assay. Despite its small size, relative selectivity against two other demethylases could be demonstrated for this compound. This is the first example of a tetrazole group as a warhead in JMJD demethylases.

Aims Angiopoietin-2 (Ang-2) is a mediator of capillary leakage, and increased Ang-2 levels were associated with poor in-hospital outcome in a pilot study in cardiogenic shock (CS). In this larger study, we followed this hypothesis and aimed at assessing the predictive role of Ang-2 on short- and long-term mortality, investigating the effect of intra-aortic balloon pump (IABP) treatment on Ang-2 levels, and identifying clinical and procedural predictors of increased Ang-2. Methods and results In the IABP-SHOCK II-trial, 600 patients with CS complicating acute myocardial infarction were assigned to therapy with or without IABP. This substudy included 189 randomized patients with serial blood sampling performed at days 1, 2, and 3. No significant differences in Ang-2 levels were found between patients with or without IABP. The Ang-2 levels above the median at day 1 were associated with 30-day [hazard ratio (HR) 1.98, 95% confidence interval (CI) 1.26–3.10, P = 0.002) and 1-year mortality (HR 2.21, 95% CI 1.49–3.27, P < 0.001). Stratification of patients according to Ang-2 levels at day 3 increased these associations (30 days HR 5.15, 95% CI 2.80–9.45, P < 0.001; 1 year HR 5.24, 95% CI 3.19–8.58, P < 0.001). The Ang-2 concentrations were independent predictors for mortality in multivariate analysis (30 days HR 4.82, 95% CI 1.52–15.23, P = 0.007; 1 year HR 2.01, 95%CI 1.24–3.24, P = 0.005). Predictors of increased Ang-2 levels at day 3 were baseline Ang-2, development of acute kidney injury, bleeding events or transfusion, and impaired reperfusion. Conclusion In CS, high levels of Ang-2 are independently associated with poor short- and long-term outcome and associated with the reperfusion success as well as complications. Clinical Trial Registration URL: www.clinicaltrials.gov; unique identifier: NCT00491036

Mobile communication is already an integral part of our lives, and its role will only continue to grow. While early mobile phone functions were limited mainly to simple address books and text messaging, today's predominant smartphones and tablets support almost endless functionality via third-party application software, bringing an insatiable hunger for higher download rates. Telecommunication associations all around the world [1] are, therefore, continuously working on new cellular network technologies. This comes along with the introduction of advanced digital modulation schemes with higher spectral efficiency; multiple-input, multiple-output (MIMO)-based radio interfaces; and carrier aggregation (CA) [2].

In patients with refractory cardiogenic shock (CS) mechanical assistance by venous-arterial extracorporeal membrane oxygenation (VA-ECMO) therapy may be considered to reach haemodynamic stabilization. In this first-in-man study, we analysed the applicability of the new i-cor VA-ECMO assist device equipped with a diagonal pump system.In total, 15 patients with refractory CS were treated with the i-cor assist device in three tertiary care centres. In 71%, CS was due to acute myocardial infarction (AMI). At baseline, patients were hypotensive (systolic/diastolic blood pressure 97 ± 4/62 ± 4 mm Hg) despite high doses of catecholamines. Under ECMO therapy, a significant reduction in vasopressor therapy and serum lactate levels was observed (norepinephrine: 0.69 ± 0.1 µg/kg/min at baseline vs 0.21 ± 0.08 µg/kg/min on the last day of treatment, p0.0001; serum lactate: 6.7 ± 1.4 mmol/l at baseline versus 1.3 ± 0.1 mmol/l on the last day, p0.001). Inspiratory oxygen concentration was significantly reduced during the course of VA-ECMO support (80.4 ± 7.0% at baseline vs 42.7 ± 2.4% on final day; p0.001). At baseline, three patients (20%) were on continuous haemodialysis treatment. Of the 12 patients without haemodialysis at baseline, only one patient required haemodialysis during the course of ECMO treatment. Glomerular filtration rate (GFR) significantly increased with treatment (41.2 ± 7.4 at baseline vs 69.0 ± 10.8 on last day; p=0.006). Bleeding at the insertion site was recorded in two patients (13.3%). Overall, 11 patients (73.3%) needed blood transfusion. Three patients (20%) developed signs of limb ischaemia that were fully reversible. Haemolysis was recorded in five patients (33%). None of the complications required the interruption of ECMO therapy. Overall mortality was 33.3% (five patients); two patients died during, and three patients after, ECMO therapy.This first-in-man analysis suggests that the i-cor ECMO device is successfully applicable in humans. The data set the stage for further evaluation of this novel system and provide the necessary basis to design randomised evaluations.

Patients with cardiogenic shock have a very high mortality. Here we report the first use of a percutaneous pulsatile cardiac assist device, based on a diagonal pump synchronized with the heart cycle by means of an electrocardiographic signal in adult pigs. Eight domestic pigs underwent mandatory ventilation. During sinus rhythm, there were no differences between pulsatile and nonpulsatile perfusion with regard to pulmonary artery pressure, pulmonary wedge pressure, central venous pressure, mean arterial pressure (MAP), mean pulse pressure, and mean coronary artery flow (CAF). After 2 min of complete cardiac arrest (ventricular fibrillation), circulatory support with the i-cor in venoarterial nonpulsatile extracorporeal membrane oxygenation (ECMO) mode (3 L/min) restored systemic circulation, with an increase of MAP to 78.3 mm Hg and CAF to 5.27 mL/min. After changing from ECMO settings to pulsatile mode (3 L/min, 75 bpm, pulse amplitude range 3500 rpm), MAP did not change significantly (75.6 mm Hg); however, CAF increased to 8.45 mL/min. After changing back to nonpulsatile mode, MAP remained stable (83.6 mm Hg), but CAF decreased to 4.85 mL/min. Thereafter, pulsatile cardiac assist was established with a reduced blood flow of 2.5 L/min, and the pulse amplitude range was extended to 4500 rpm. Under these conditions, MAP remained stable (71.0 mm Hg), but CAF significantly increased to 15.2 mL/min (P