Production of tailored hydroxylated prodiginine showing combinatorial activity with rhamnolipids against plant-parasitic nematodes.

Bacterial secondary metabolites exhibit diverse remarkable bioactivities and are thus the subject of study for different applications. Recently, the individual effectiveness of tripyrrolic prodiginines and rhamnolipids against the plant-parasitic nematode Heterodera schachtii, which causes tremendous losses in crop plants, was described. Notably, rhamnolipid production in engineered Pseudomonas putida strains has already reached industrial implementation. However, the non-natural hydroxyl-decorated prodiginines, which are of particular interest in this study due to a previously described particularly good plant compatibility and low toxicity, are not as readily accessible. In the present study, a new effective hybrid synthetic route was established. This included the engineering of a novel P. putida strain to provide enhanced levels of a bipyrrole precursor and an optimization of mutasynthesis, i.e., the conversion of chemically synthesized and supplemented monopyrroles to tripyrrolic compounds. Subsequent semisynthesis provided the hydroxylated prodiginine. The prodiginines caused reduced infectiousness of H. schachtii for Arabidopsis thaliana plants resulting from impaired motility and stylet thrusting, providing the first insights on the mode of action in this context. Furthermore, the combined application with rhamnolipids was assessed for the first time and found to be more effective against nematode parasitism than the individual compounds. To obtain, for instance, 50% nematode control, it was sufficient to apply 7.8 µM hydroxylated prodiginine together with 0.7 µg/ml (~ 1.1 µM) di-rhamnolipids, which corresponded to ca. » of the individual EC50 values. In summary, a hybrid synthetic route toward a hydroxylated prodiginine was established and its effects and combinatorial activity with rhamnolipids on plant-parasitic nematode H. schachtii are presented, demonstrating potential application as antinematodal agents. Graphical Abstract.

Stabilisation of acute-on-chronic liver failure patients before liver transplantation predicts post-transplant survival.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a severe complication of liver cirrhosis associated with excess short-term mortality rates. Orthotopic liver transplantation (OLT) is a potentially life-saving therapeutic modality for acute-on-chronic liver failure patients, but selection of transplant candidates with an acceptable post-transplant outcome is difficult. AIM: To assess the risk of liver transplantation in patients with ACLF, and to determine parameters that predict post-transplant survival in this patient cohort. METHODS: We retrospectively analysed all 250 patients with cirrhosis who underwent their first liver transplantation between 2009 and 2014 at our institution, and assessed post-transplant outcomes. RESULTS: Of 250 cirrhotic liver transplant recipients, 98 patients fulfilled the diagnostic criteria for acute-on-chronic liver failure in the 3-month pre-transplant period. Compared to non-ACLF patients, ACLF was associated with significantly higher short-term morbidity and mortality after liver transplantation (90-day patient survival 96.1% non-ACLF vs 72.4% ACLF patients, P < 0.0001). Clinical improvement in the pre-transplant period, as defined by recovery of at least one previously failed organ system, was observed in 37 of 98 acute-on-chronic liver failure patients, mostly within several days after diagnosis. Most notably, clinical improvement prior to liver transplantation was associated with excellent post-transplant survival rates that approximated non-ACLF transplant recipients. Following the 90-day post-transplant period, patient survival and long-term graft functions were comparable between ACLF and non-ACLF liver transplant recipients for up to 5 years. CONCLUSIONS: Acute-on-chronic liver failure predicts adverse outcome after orthotopic liver transplantation. Given the dismal prognosis without transplantation, however, our results indicate that ACLF patients can be transplanted with comparably good outcomes, in particular patients who improve under conservative therapeutic measures.

[Inadequate ICU-admissions : A 12-month prospective cohort study at a German University Hospital]. / Nicht indizierte Aufnahmen auf der Intensivstation : 12-Monats-Observationsstudie an einem Universitätsklinikum.

BACKGROUND: Intensive care medicine (ICM) is increasingly utilized by a growing number of critically ill patients worldwide. The reasons for this are an increasingly ageing and multimorbid population and technological improvements in ICM. Inappropriate patient admissions to the intensive care unit (ICU) can be a threat to rational resource allocation and to patient autonomy. OBJECTIVES: In this study, the incidence, characteristics, and resource utilization of patients inappropriately admitted to ICUs are studied. METHODS: This prospective study included all consecutive patients admitted from 01 September 2012 to 31 August 2013 to the Department of Intensive Care Medicine of a German university hospital comprised of 10 ICUs and 120 beds. Inappropriate admission was defined according to category 4B of the recommendations of the Society of Critical Care Medicine (SCCM; "futility of ICU treatment" or "ICU declined by patient") and was determined in each suspected case by structured group discussions between the study team and all involved care givers including the referring team. RESULTS: In all, 66 of 6452 ICU admissions (1 %) were suspected to have been inappropriate on retrospective evaluation the day after admission. In 50 patients (0.8 %), an interdisciplinary consensus was reached on the inappropriateness of the ICU admission. Of these 50 patients, 41 (82 %) had previously declined ICU treatment in principle. This information was based on the patient's presumed wish as expressed by next of kin (56 %) or in a written advanced directive (26 %). In 9 patients (18 %), ICU treatment was considered futile. In all cases, a lack of information regarding a patient's wishes or clinical prognosis was the reason for inappropriate ICU admission. CONCLUSION: In this study, patients were regularly admitted to the ICU despite their contrary wish/directive or an unfavorable clinical condition. Although this was registered in only 1 % of all admissions, optimizing preICU admission information flow with regard to relevant exclusion criteria not only helps respect patient autonomy but also allows for more adequate resource allocation.

Cholestasis and regulation of genes related to drug metabolism and biliary transport in rat liver following treatment with cyclosporine A and sirolimus (Rapamycin).

Cyclosporine A and sirolimus are used alone or in combination as immunosuppressants in organ transplantation. To elucidate hepatic side effects, we examined hepatic mRNA of proteins involved in biliary and hepatocellular transport of drugs, formation of glutathione (GSH) and drug metabolising cytochrome P-450 enzymes (CYPs) in rats treated orally for 2 weeks with cyclosporine A (15 mg/kg/day), sirolimus (0.4 mg/kg/day), their combination (same doses), or vehicle. Liver function tests (alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase and bilirubin) in blood were then analysed as were hepatic mRNA levels of canalicular transport proteins (Mrp2, Bsep, Mdr1b and Mdr2), sinusoidal transport proteins (Ntcp, Oatp1 and Oatp2), GSH related enzymes (gamma-glutamylcysteine synthetase light (GCSlc) and heavy (GCShc) chain subunits and glutathione-S-transferase) and CYPs (CYP3A9, CYP1A2, CYP2E1 and CYP2BI/II). Cyclosporine A caused moderate cholestatic changes in liver enzymes, which was synergistically exacerbated by sirolimus. The data suggest that the underlying mechanisms behind cholestasis were not totally identical in the different treatment regimens. Cholestasis secondary to cyclosporine A could be related to reduction in mRNA expression of GSH synthesising enzymes and Mrp2, leading to reduced protection against oxidative stress and reduced bile acid-independent bile flow. After sirolimus treatment, Mrp2 mRNA was also reduced together with reduced levels of most CYPs and increased Oatp2, possibly leading to accumulation of toxic metabolites in the hepatocytes. The enhanced cholestatic effect of the combination treatment could be related to reduced GSH synthesising enzymes and even more pronounced reduction in Mrp2 mRNA and increase of Oatp2 mRNA.

[Transesophageal echocardiography as intraoperative monitoring for the resection of an intra-atrial tumor]. / TEE bei fibrolamellärem Leberzellkarzinom. Transösophageale Echokardiographie als intraoperatives Monitoring bei Resektion eines intraatrialen Tumors.

Fibrolamellar hepatocellular carcinomas (FLHCC) can grow into the inferior vena cava and the right atrium thus rendering it impossible to insert a pulmonary artery catheter or increasing the risk of tumor embolisation. We report of a young patient with intraatrial growth of a FLHCC where intraoperative transesophageal echocardiography (TEE) instead of a pulmonary artery catheter for continuous monitoring of hemodynamics was used. Hemodynamic parameters as well as the surgical result could easily be assessed.

Autoprotection in acetaminophen intoxication in rats: the role of liver regeneration.

Autoprotection by acetaminophen, i.e. increased resistance to toxic effects caused by pretreatment, is a well-known phenomenon. The purpose of the present work was to identify mechanisms for increased acetaminophen tolerance induced by pretreatment of rats. One group of female Wistar rats (pretreated rats) received acetaminophen orally in increasing doses (1 to 4.3 g/kg) twice a week for 3 weeks, one group (naïve rats) received the vehicle. At time zero pretreated rats received a toxic dose of 7.5 g/kg (100% lethal in naïve rats), and naïve rats received a toxic dose of 4.3 g/kg. Blood and liver tissue were collected before and 12, 24, 36, and 48 hr after the toxic dose and were analysed for hepatic glutathione and cysteine contents, hepatic glutathione-S-transferase and blood alanine aminotransferase activity, as well as acetaminophen concentration in plasma. Steady-state mRNA levels of proteins involved in acetaminophen detoxification, cell division and acute phase response were measured, liver tissue was examined for proliferating cell nuclear antigen and degree of hepatocyte necrosis. Six naïve rats not receiving acetaminophen served as controls. The mortality was the same in pre-treated and naïve rats (33 percent). Thus, pretreatment increased the tolerance twice. Before the toxic dose pretreated rats compared to control rats had higher activity of glutathione-S-transferase (liver) and alanine aminotransferase (serum), higher hepatic mRNA level of glutathione-S-transferase and gamma-glutamylcysteine synthetase heavy and light chain subunits, and lower hepatic concentration of glutathione, cysteine and mRNA of CYP1A2 than control rats. After the toxic dose, the mRNA levels of glutathione-S-transferase, gamma-glutamylcysteine synthetase heavy and light chain subunits, and CYP1A2 in naïve rats rose, approaching those of pretreated rats. Proliferating cell nuclear antigen labelling was high in pretreated rats, while only slightly increased in a few of the naïve rats. Necrotic hepatocytes were found at all time intervals in pretreated rats, and in naïve rats they appeared after 12 hr, peaking after 36 hr. Pretreatment increased the tolerance to acetaminophen toxicity twice, as estimated by mortality. The data indicate that pretreatment may reduce the relative production of toxic metabolites, but it primarily enhances the protection against these metabolites by regenerating hepatocytes.

Assay of functional plasminogen in rat plasma applicable to experimental studies of thrombolysis.

An improved sensitive, specific, precise and accurate assay of plasminogen in rat plasma was developed. It is performed in 96-well microtiter plates and can be completed within one hour. The assay is based on activation of plasminogen by human urokinase-type plasminogen activator (uPA) and simultaneous measurement of generated plasmin with the specific plasmin substrate H-D-Val-Phe-Lys-4-nitroanilide (S-2390), using purified native rat plasminogen for calibration. The concentration of S-2390 in the final reaction mixture during the whole reaction period is much greater than the Km value (approximately 20 microM) for rat plasmin-cleavage of S-2390 ensuring that hydrolysis of substrate follows zero order kinetics and that the substrate produces a 20-35 fold decrease in rate of inhibition of plasmin by its target inhibitors in plasma. Analogous to the human system the target plasma inhibitors of rat plasmin are shown to be plasmin inhibitor and alpha-macroglobulins. Tranexamic acid (0.8 mM) is incorporated in the reaction mixture resulting in a 19-fold increase in the rate of plasminogen activation and presumably an about 50-fold decrease in the rate of inhibition of generated plasmin by plasmin inhibitor. The assay is suitable for accurate measurement of plasminogen in samples obtained from animals containing pharmacological concentrations of uPA or tissue-type plasminogen activator (tPA) in their plasma when in vitro plasminogen activation is blocked at pH 5 by collecting blood in acidic anticoagulant. Judged from in vitro experiments formation of catalytic active plasmin-alpha-macroglobulin complexes during massive activation of plasminogen in vivo does not interfere with the assay.

[Treatment of anesthesia-induced atelactasis]. / Prävention und Therapie anästhesiebedingter Atelektasen.

During general anaesthesia even healthy lungs tend to collapse. Thus, up to 20% of previously functional lung tissue may be lost for gas exchange. It should be advantageous to treat this pathologic condition. After explaining the clinical problem of lung collapse, the concept of opening these lungs and keeping them open will be discussed. Some results of the first randomized clinical trials on intraoperative lung recruitment will be presented. Finally, a systematic description of all treatment steps tries to provide the anaesthesiologist with a useful practical guide for applying the "alveolar recruitment strategy" in their daily care of patients undergoing general anaesthesia.

Reaction of human alpha2-antiplasmin and plasmin stopped-flow fluorescence kinetics.

The interaction of human plasmin with human alpha2-antiplasmin was measured in the presence and absence of lysine-binding ligands using the corresponding active site fluorescence changes. The stopped-flow method allows for direct determination of reliable values of the second order rate constant for the fast association step of plasmin and alpha2-antiplasmin in the absence of another interacting compound, e.g. a plasmin substrate. At pH 7.4, 25 degrees C, k1 = 2.2 x 10(7) M(-1)s(-1) was obtained. Substantial reductions in k1 were seen in the presence of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid at concentrations corresponding to lysine-binding site interactions at kringle 4 of plasmin; at saturation the rate constant is reduced 20-fold, whereas the effect of saturation of kringle 1 is only a 2-fold reduction. It is thus found that the interaction of alpha2-antiplasmin with the lysine-binding site of kringle 1 is of little importance compared with that of kringle 4 in regulating the inhibition reaction of plasmin with alpha2-antiplasmin. Similar results were recently obtained for the bovine plasmin-bovine alpha2-antiplasmin reaction (Christensen et al. (1995) Biochem. J. 305, 97-102).

Different N-terminal forms of alpha 2-plasmin inhibitor in human plasma.

Mature alpha 2-plasmin inhibitor in human plasma has 12 more N-terminal residues than hitherto anticipated. The first residue is the methionine at position 28, downstream from the N-terminus of the pre-protein. The cDNA sequence predicts that the site cleaved upon formation of the mature inhibitor is a typical signal-peptidase recognition site. The mature inhibitor (464 residues) and the previously reported, and presumably degraded, form with N-terminal asparagine (452 residues), are present in plasma in about equal amounts. They both form a stable complex with plasmin. Recent studies on a recombinant alpha 2-plasmin inhibitor suggest that the 12 additional residues have functional implications [Sumi, Ichikawa, Nakamura, Miura and Aoki (1989) J. Biochem. 106, 703-707].