Structure Elucidation, Total Synthesis, Antibacterial In Vivo Efficacy and Biosynthesis Proposal of Myxobacterial Corramycin.

Herein, we describe the myxobacterial natural product Corramycin isolated from Corallococcus coralloides. The linear peptide structure contains an unprecedented (2R,3S)-γ-N-methyl-ß-hydroxy-histidine moiety. Corramycin exhibits anti-Gram-negative activity against Escherichia coli (E. coli) and is taken up via two transporter systems, SbmA and YejABEF. Furthermore, the Corramycin biosynthetic gene cluster (BGC) was identified and a biosynthesis model was proposed involving a 12-modular non-ribosomal peptide synthetase/polyketide synthase. Bioinformatic analysis of the BGC combined with the development of a total synthesis route allowed for the elucidation of the molecule's absolute configuration. Importantly, intravenous administration of 20 mg kg-1 of Corramycin in an E. coli mouse infection model resulted in 100 % survival of animals without toxic side effects. Corramycin is thus a promising starting point to develop a potent antibacterial drug against hospital-acquired infections.

In vitro and in vivo effects of inhibitors on actin and myosin.

The interaction of actin and myosin is essential for cell migration. We have identified kaempferol and pentahalogenated pseudilins as efficient inhibitors of migration of MDA-MB-231 breast adenocarcinoma cells. The compounds were studied with respect to possible effects on myosin-2-ATPase activity. The pentahalogenated pseudilins inhibited the enzyme activity in vitro. Flavonoids showed no effect on enzyme activity. The polymerization dynamics of actin was measured to test whether the integrity of F-actin is essential for the migration of MDA-MB-231 cells. Quercetin and kaempferol depolymerized F-actin with similar efficiencies as found for the pentahalogenated pseudilins, whereas epigallocatechin showed the weakest effect. As the inhibitory effect on cell migration may be caused by a toxic effect, we have performed a cytotoxicity test and, furthermore, investigated the influence of the test compounds on cardiac function in eleutheroembryos of medaka (Oryzias latipes). Compared with the pentahalogenated pseudilins, the cytotoxic and cardiotoxic effects of flavonoids on medaka embryos were found to be moderate.

Symptomatic chronic obstructive pulmonary disease in clinical trials and in a population-based study.

PURPOSE: Evidence-based medicine promotes the current best evidence from clinical trials to guide decisions for individual patients. We assessed whether chronic obstructive pulmonary disease (COPD) patients included in exercise training studies and pharmacologic trials match those from a non-selected COPD target population sample. METHODS: Exercise training studies were identified in a literature search. Towards a Revolution in COPD Health (TORCH) and Understanding Potential Long-Term Impacts on Function with Tiotropium (UPLIFT) were chosen to represent pharmacologic trials. Burden of Obstructive Lung Disease (BOLD) data were used to characterize target COPD population (BOLD target), defined as the presence of dyspnea (modified Medical Research Council ≥2) and non-reversible airway obstruction (post-bronchodilator FEV1/FVC ≤0.7 and FEV1% predicted ≤70 %). RESULTS: Overall 240 exercise training studies with 13,901, TORCH and UPLIFT with 12,105, and BOLD with 16,218 participants were evaluated. Males were overrepresented in exercise training studies (67.5%) and pharmacologic trials (TORCH 75.8%; UPLIFT 74.6%), whereas in BOLD target 55.8% were males (p < 0.001). In exercise training studies, 7.2% were never-smokers, 0.0% in TORCH and UPLIFT, but 36.0% in BOLD target (p < 0.001). Subjects with cardiac comorbidity were excluded from 75.4% of exercise training studies, entirely from TORCH and UPLIFT, but comprised 24.5% of BOLD target. CONCLUSIONS: COPD patients recruited in exercise training studies and in pharmacologic trials differ from target population of symptomatic COPD. Females, never-smokers, and patients with cardiac comorbidities are more likely excluded from the clinical trials.

Production of the Bengamide Class of Marine Natural Products in Myxobacteria: Biosynthesis and Structure-Activity Relationships.

The bengamides, sponge-derived natural products that have been characterized as inhibitors of methionine aminopeptidases (MetAPs), have been intensively investigated as anticancer compounds. We embarked on a multidisciplinary project to supply bengamides by fermentation of the terrestrial myxobacterium M. virescens, decipher their biosynthesis, and optimize their properties as drug leads. The characterization of the biosynthetic pathway revealed that bacterial resistance to bengamides is conferred by Leu 154 of the myxobacterial MetAP protein, and enabled transfer of the entire gene cluster into the more suitable production host M. xanthus DK1622. A combination of semisynthesis of microbially derived bengamides and total synthesis resulted in an optimized derivative that combined high cellular potency in the nanomolar range with high metabolic stability, which translated to an improved half-life in mice and antitumor efficacy in a melanoma mouse model.

Dissecting the effect of moxifloxacin in mice with infected necrosis in taurocholate induced necrotizing pancreatitis.

OBJECTIVES: To investigate the limited benefit of antibiotics in ameliorating the outcome of acute necrotizing pancreatitis, we analyzed antibiotic therapy in primarily infected necrotizing pancreatitis in mice with respect to the local pancreatic pathology as well as systemic, pancreatitis induced adverse events. METHODS: Sterile pancreatic necrosis (SN) was induced by retrograde injection of 4% taurocholate in the common bile duct of Balb/c mice. Primarily infected pancreatic necrosis (IN) was induced by co-injecting 10(8) CFU/ml Escherichia coli. 10 mg/kg of moxifloxacin was administered prior to pancreatitis induction (AN). After 24 h, animals were sacrificed to examine serum as well as organs for signs of SIRS. RESULTS: Moxifloxacin significantly reduced bacterial count in pancreatic lysates of animals with infected pancreatic necrosis (IN 4.1·10(7) ± 2.4·10(7) vs. AN 4.9·10(4) ± 2.6·10(4) CFU/g; p < 0.001). However, it did not alter pancreatic histology or pulmonary damage (Histology score: IN 23.8 ± 2.7 vs. AN 22.6 ± 1.7). Moxifloxacin reduced systemic immunoactivation (Serum IL-6: IN 330.5 ± 336.6 vs. 38.7 ± 25.5 pg/ml; p < 0.001), hypoglycemia (serum glucose: IN 105.8 ± 12.7 vs. AN 155.7 ± 39.5 mg/dl; p < 0.001), and serum aspartate aminotransferase (IN 606 ± 89.7 vs. AN 255 ± 52.1; p < 0.05). These parameters were significantly increased in animals with necrotizing pancreatitis. CONCLUSION: In the experimental setting, initial antibiotic therapy with moxifloxacin in acute infected necrotizing pancreatitis in mice does not have a beneficial impact on pancreatic pathology or pulmonary damage. However, other systemic complications induced by infected necrosis in acute pancreatitis are reduced by the administration of moxifloxacin.

Synthesis and biological evaluation of novel N-alkyl tetra- and decahydroisoquinolines: novel antifungals that target ergosterol biosynthesis.

A series of N-alkyl trans-decahydroisoquinoline, 1,2,3,4-tetrahydroisoquinoline, and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivatives were synthesized starting from the respective secondary amines by N-alkylation with alkyl bromides. The compounds with C11-alkyl chains showed antifungal potency comparable to clotrimazole, and inhibit enzymes of the ergosterol biosynthesis (Δ14-reductase and Δ8,7-isomerase), depending on the heterocyclic scaffold and the investigated species.

Risk factors and complications following percutaneous endoscopic gastrostomy: a case series of 1041 patients.

BACKGROUND: Most studies exclude patients with severe coagulation disorders or those taking anticoagulants when evaluating the outcomes of percutaneous endoscopic gastrostomy (PEG). OBJECTIVE: To investigate complications and risk factors of PEG in a large clinical series including patients undergoing antiplatelet and anticoagulant therapy. METHODS: During a six-year period, 1057 patients referred for PEG placement were prospectively audited for clinical outcome. Exclusion criteria and follow-up care were defined. Complications were defined as minor or severe. Uni- and multivariate analyses were used to evaluate 14 risk factors. No standardized antibiotic prophylaxis was given. RESULTS: A total of 1041 patients (66% male, 34% female) with the following conditions underwent PEG: neurogenic dysphagia (n=450), cancer (n=385) and others (n=206). No anticoagulants were administered to 351 patients, thrombosis prophylaxis was given to 348 while full therapeutic anticoagulation was received by 313. No increased bleeding risk was associated with patients who had above-normal international normalized ratio values (OR 0.79 [95% CI 0.08 to 7.64]; P=1.00). The total infection rate was 20.5% in patients with malignant disease, and 5.5% in those with nonmalignant disease. Severe complications occurred in 19 patients (bleeding 0.5%, peritonitis 1.3%). Cirrhosis (OR 2.91 [95% CI 1.31 to 6.54]; P=0.008), cancer (OR 2.34 [95% CI 1.33 to 4.12]; P=0.003) and radiation therapy (OR 2.34 [95% CI 1.35 to 4.05]; P=0.002) were significant predictors of post-PEG infection. The 30-day mortality rate was 5.8%. There were no procedure-related deaths. CONCLUSIONS: Cancer, cirrhosis and radiation therapy were predictors of infection. Post-PEG bleeding and other complications were rare events. Collectively, the data suggested that patients taking concurrent anticoagulants had no elevated risk of post-PEG bleeding.

The first teleautomatic low-voltage prosthesis with multiple therapeutic applications: a new version of the German artificial sphincter system.

To date, there are no artificial sphincter prostheses for urinary or fecal incontinence that may be implemented elsewhere instead, for example, in the upper gastrointestinal tract. Conventional systems are conceptually similar but are constructed specifically for distinct applications and are manual in operation. The German Artificial Sphincter System (GASS) II is the evolution of a highly integrative, modular, telemetric sphincter prosthesis with more than one application. Redesigning and integrating multilayer actuators into the pump allows us to reduce the input voltage to -10 to +20 V (V(PP) = 30 V). This provides for a flow rate of 2.23 mL/min and a counterpressure stability of 260 mbar. Furthermore, multiple applications have become feasible due to our standardized connection system, therapy-specific compression units, and application-specific software. These innovations allow us to integrate not only severe fecal and urinary incontinence, erectile dysfunction, and therapy-resistant reflux disease, but also morbid adiposity into the gamut of therapeutic GASS applications.

Inhibition of matrix metalloproteinase-9 with doxycycline reduces pancreatitis-associated lung injury.

BACKGROUND/AIMS: Severe lung injury, responsible for up to 15% of mortality in acute necrotizing pancreatitis patients, is promoted by neutrophil (PMN) migration into the lung. We have previously demonstrated that pulmonary injury in acute pancreatitis is mediated by PMN-derived matrix metalloproteinase-9 (MMP-9). This study was conducted to evaluate the ability of the broad-spectrum MMP inhibitor doxycycline to prevent secondary pulmonary injury in acute pancreatitis. METHODS: Eighteen rats were randomized into three groups: severe pancreatitis (SAP), severe pancreatitis + doxycycline (SAP+Dox) (30 mg/kg body mass) or control. Acute pancreatitis was induced by intraductal glycodesoxycholic acid and i.v. stimulation with cerulein. Lung sections were histologically graded for edema, microthrombi, atelectasis and hemorrhage. Active MMP-9 in lung tissue was measured with fluorescent assay (ELISA). Naphtol-AS-D-chloroacetate esterase staining was used to determine pulmonary PMN infiltration. The inhibitory effect of doxycycline on MMP-9-induced transmigration was confirmed in a Matrigel transmigration assay. RESULTS: Addition of doxycycline significantly reduced TNF-alpha-induced PMN transmigration across Matrigel membrane (12.6 +/- 2.6 vs. 20.1 +/- 3.9 PMNs; p < 0.05). SAP+Dox showed decreased concentration of active MMP-9 in lung tissue (37.89 +/- 1.75 vs. 46.29 +/- 3.68 ng/ml; p < 0.05) and as a result decreased pulmonary infiltration of PMNs (21.2 +/- 5.1 vs. 32.5 +/- 6.8; p < 0.05). Histological evaluation revealed decreased pulmonary edema (1.83 +/- 0.41 vs. 2.33 +/- 0.51, p < 0.05), atelectasis (1.67 +/- 0.52 vs. 2.33 +/- 0.52; p < 0.05) and pulmonary hemorrhage (2.5 +/- 0.55 vs. 1.83 +/- 0.41; p < 0.05) in SAP+Dox vs. SAP. These findings were paralleled by reduced pulmonary expression of active MMP-9. CONCLUSIONS: Inhibition of MMP-9 activity with doxycycline reduced pancreatitis-associated lung injury and expression of MMP-9 in pulmonary tissue. Doxycycline reduced PMN migration in vitro and in vivo and therefore might represent a novel strategy for the prevention of secondary pulmonary complications in acute pancreatitis.

The immunoadhesin glycoprotein VI-Fc regulates arterial remodelling after mechanical injury in ApoE-/- mice.

AIMS: Rupture of advanced atherosclerotic plaques initiates platelet activation and aggregation as subendothelial collagen is exposed. Platelet collagen receptor glycoprotein VI (GPVI) was found to bind preferentially to the core region of human plaques. Consequently, platelets contribute to inflammatory processes and trigger atherosclerotic lesion progression. In this study, we examined binding of soluble platelet collagen receptor GPVI-Fc to atherosclerotic lesions and its effect on platelet-triggered athero-progression and neointima formation after wire-induced carotid injury. METHODS AND RESULTS: For binding studies after ligation-induced arterial injury, the left common carotid artery of C57BL/6J mice was ligated. For binding studies at spontaneously formed atherosclerotic lesion sites, Apolipoprotein E-deficient (ApoE(-/-)) mice were fed a 0.25% cholesterol diet over 16 weeks. Binding of [(124)I]GPVI-Fc was monitored by autoradiography 48 h after intravenous injection and by immunostaining. To study the effect of GPVI-Fc on neointima formation vs. control-Fc, a wire-induced injury of the left A. carotis communis of ApoE(-/-)-mice was performed. Mice were treated intraperitoneally with GPVI-Fc for 8 days and neointima formation was assessed 4 weeks after intervention. [(124)I]GPVI-Fc preferentially bound to injury sites after carotid ligation in C57BL/6J mice and to lipid-rich atherosclerotic lesions of the carotid artery and aortic arch in uninjured ApoE(-/-)-mice. Histological examinations of wire-injured carotid arteries showed that neointima formation was significantly reduced in GPVI-Fc-treated ApoE(-/-) mice compared to ApoE(-/-) mice receiving control-Fc (P < 0.05). CONCLUSION: GPVI-Fc preferentially bound to sites of vascular injury and was able to inhibit neointima formation after wire-induced vascular injury in ApoE(-/-) mice. Thus, soluble GPVI-Fc might be also a promising compound to attenuate lesion progression after plaque rupture.

Distinct regional atrophy in the corpus callosum of patients with temporal lobe epilepsy.

We analysed the influence of mesial temporal lobe epilepsy on the thickness of the corpus callosum (CC) in a large sample of well-characterized patients (n = 96) and healthy controls (n = 28). In particular, we investigated whether callosal structures are differentially affected depending on the affected hemisphere and age of epilepsy onset. Overall, we observed that epilepsy is associated with a decreased thickness in posterior callosal regions. Patients with an early onset, especially patients with left onset, additionally exhibited a smaller callosal thickness in more anterior and midbody regions. These findings may reflect non-specific as well as specific effects of temporal lobe epilepsy on CC development and interhemispheric connectivity.

Murine genotype impacts pancreatitis severity and systemic inflammation: An experimental study.

BACKGROUND: Little is known regarding the impact of host response in acute pancreatitis. Here, we induce murine necrotizing pancreatitis in 9 different mouse strains. MATERIALS AND METHODS: We examined 9 different mouse strains: Balb/CB4J, C3H/HEJ, NOD/SHILT, A/J, AKR/J, C57BI/6J, DBA/2J, FVB/NJ, 129S1/SvlmJ. 10 animals per strain were randomly allotted to two groups. Sterile necrotizing pancreatitis was induced by injection of taurocholate into the common bile duct. Control animals were injected with saline. Every 6 h, clinical parameters were examined and scored. After 24 h, animals were sacrificed to examine and compare serum enzymes, histology, bronchoalveolar lavage fluid, and serum IL-6. RESULTS: Histologically, taurocholate treated animals scored significantly higher than control animals. Concordantly, serum lipase and amylase were significantly elevated in pancreatitis animals in all strains. NOD/SHILT and AKR/J mice had the highest enzyme activity. 24 h after induction, there were no signs of increased pulmonary vascular leak in taurocholate animals. Remarkably, interleukin 6 was not increased at all in C57BL/6J, C3H/HeJ, and 129S1/SvlmJ mice compared to all other strains. CONCLUSION: The genetic strain has an impact on pancreatitis severity and systemic inflammatory response in a murine taurocholate induction model. Analogous differences in humans may partially account for the disparity in post-ERCP pancreatitis.

Differential response of normal and transformed mammary epithelial cells to combined treatment of anti-miR-21 and radiation.

PURPOSE: MicroRNA miR-21 has emerged as a therapeutic target in the treatment of breast cancer. This study was designed to compare the responses of breast cancer cells and non-transformed breast epithelial cells to a combined regimen of miR-21 inhibition and radiation. MATERIALS AND METHODS: The MDA-MB-361 (breast cancer) and MCF-10A (non-transformed mammary epithelial) cell lines were used for the comparison in this in vitro study. The stable knockdown of miR-21 was performed by using lentiviral approach. The response of the cells was monitored 4, 24 and 48 h after the irradiation with 0.25 and 2.5 Gy, using sham-irradiated cells as controls. The response of the cells was established by performing various functional assays - cell viability and cell attachment, clonogenic survival, cell cycle analysis and 3D microtissue formation. RESULTS: The knockdown of miR-21 induced significant increase in apoptosis and growth delay in MDA-MB-361 cancer cells compared to non-transformed MCF-10A cells. After combined radiation and anti-miR-21 treatment, MDA-MB-361 cells show reduced cell growth and viability what is presented in their inability to form colonies. MCF-10A cells were not as sensitive to the combined treatment and that has also been confirmed with colony forming assay. CONCLUSIONS: Cellular response to a combined treatment of anti-miR-21 and radiation is different between cancer and non-cancer cells which highly support the idea of linking miR-21 inhibitor and radiation treatment in the future therapeutic approaches for breast cancer.

Three-dimensional microtissues essentially contribute to preclinical validations of therapeutic targets in breast cancer.

A 3D microtissues using T47D and JIMT-1 cells were generated to analyze tissue-like response of breast cancer cells after combined human epidermal growth factor receptor 2 (HER2)-targeted treatment and radiation. Following lentiviral knockdown of HER2, we compared growth rate alterations using 2D monolayers, 3D microtissues, and mouse xenografts. Additionally, to model combined therapeutic strategies, we treated HER2-depleted T47D cells and 3D microtissues using trastuzumab (anti-HER2 antibody) in combination with irradiation. Comparison of HER2 knockdown with corresponding controls revealed growth impairment due to HER2 knockdown in T47D 2D monolayers, 3D microtissues, and xenografts (after 2, 12, and ≥40 days, respectively). In contrast, HER2 knockdown was less effective in inhibiting growth of trastuzumab-resistant JIMT-1 cells in vitro and in vivo. Combined administration of trastuzumab and radiation treatment was also analyzed using T47D 3D microtissues. Administration of both, radiation (5 Gy) and trastuzumab, significantly enhanced the growth inhibiting effect in 3D microtissues. To improve the predictive power of potential drugs--as single agents or in combination--here, we show that regarding tumor growth analyses, 3D microtissues are highly comparable to outcomes derived from xenografts. Considering increased limitations for animal experiments on the one hand and strong need of novel drugs on the other hand, it is indispensable to include highly reproducible 3D microtissue platform in preclinical analyses to validate more accurately the capacity of future drug-combined radiotherapy.

Maternal and fetal side effects of tocolysis using transdermal nitroglycerin or intravenous fenoterol combined with magnesium sulfate.

OBJECTIVE: To compare the maternal and fetal side effects of transdermal nitroglycerin and intravenous fenoterol combined with magnesium sulfate in a prospective randomised study. STUDY DESIGN: Fifty pregnant women between 27 and 35 weeks of gestation with preterm labour were treated with either nitroglycerin (0.4-0.8 mg/h) or fenoterol (60 - 120 microg/h). Outcome parameters were (1) the effects on fetal and maternal heart frequency (FHF/MHF) and blood pressure, and (2) subjective experiences of adverse effects assessed by utilising a questionnaire. RESULTS: In the fenoterol group, elevated mean MHF, FHF and systolic blood pressure were recorded compared to nitroglycerin. Fewer maternal side effects were reported in the nitroglycerin group. Palpitations (82%), tremor (68%) and restlessness (64%) were most common in the fenoterol group (two drop-outs), whereas nitroglycerin caused headaches in 71% of the cases (four drop-outs). CONCLUSION: Transdermal nitroglycerin appears to be a safe therapy for the mother and fetus and is a promising new option for the treatment of preterm labour.

Simulator Network project report: a tool for improvement of teaching materials and targeted resource usage in Skills Labs.

During the last decade, medical education in the German-speaking world has been striving to become more practice-oriented. This is currently being achieved in many schools through the implementation of simulation-based instruction in Skills Labs. Simulators are thus an essential part of this type of medical training, and their acquisition and operation by a Skills Lab require a large outlay of resources. Therefore, the Practical Skills Committee of the Medical Education Society (GMA) introduced a new project, which aims to improve the flow of information between the Skills Labs and enable a transparent assessment of the simulators via an online database (the Simulator Network).

Infected pancreatic necrosis increases the severity of experimental necrotizing pancreatitis in mice.

OBJECTIVE: Infection of pancreatic necrosis in necrotizing pancreatitis increases the lethality of patients with acute pancreatitis. To examine mechanisms underlying this clinical observation, we developed and tested a model, in which primary infection of necrosis is achieved in taurocholate-induced pancreatitis in mice. METHODS: Sterile necrosis of acute necrotizing pancreatitis was induced by retrograde injection of 4% taurocholate into the common bile duct of Balb/c mice. Primary infection of pancreatic necrosis was induced by coinjecting 10 colony-forming units of Escherichia coli. Animals were killed after 6, 12, 24, 48, and 120 hours, and pancreatic damage and pancreatitis-associated systemic inflammatory response were assessed. RESULTS: Mice with pancreatic acinar cell necrosis had an increased bacterial concentration in all tissues and showed sustained bacteremia. Acute pancreatitis was induced only by coinjection of taurocholate and not by bacterial infection alone. Infection of pancreatic necrosis increased pancreatic damage and the pulmonary vascular leak. Serum glucose concentrations serving as a parameter of hepatic function were reduced in mice with infected pancreatic necrosis. CONCLUSIONS: Primary infection of pancreatic necrosis with E. coli increases both pancreatic damage and pulmonary and hepatic complications in acute necrotizing pancreatitis in mice.

Differential effects of temporal pole resection with amygdalohippocampectomy versus selective amygdalohippocampectomy on material-specific memory in patients with mesial temporal lobe epilepsy.

PURPOSE: In the surgical treatment of mesial temporal lobe epilepsy, there is converging evidence that individually tailored or selective approaches have a favorable cognitive outcome compared to standard resections. There is, however, also evidence that due to collateral damage, selective surgery can be less selective than suggested. As part of a prospective transregional research project the present study evaluated the outcome in memory and nonmemory functions, following two selective approaches: a combined temporal pole resection with amygdalohippocampectomy (TPR+) and transsylvian selective amygdalohippocampectomy (SAH). METHODS: One year after surgery, cognitive outcomes of postoperatively seizure-free patients with mesial TLE and hippocampal sclerosis, who underwent either TPR+ (N = 35) or SAH (N = 62) in two German epilepsy centers (Bonn/Berlin), were compared. RESULTS: Repeated measurement MANOVA and separate post hoc testing indicated a double dissociation of verbal/figural memory outcome as dependent on side and type of surgery. Verbal memory outcome was worse after left-sided operation, but especially for SAH, whereas figural memory outcome was worse after right-sided operation, preferentially for TPR+. Attention improved independent of side or type of surgery, and language functions showed some improvement after right-sided surgeries. DISCUSSION: The results indicate a differential effect of left/right SAH versus TPR+ on material-specific memory insofar as transsylvian SAH appears to be favorable in right and TPR+ in left MTLE. The different outcomes are discussed in terms of a different surgical affection of the temporal pole and stem, and different roles of these structures for verbal and figural memory.