A germination study of herbicide-resistant field poppies in Spain

Field poppy, Papaver rhoeas L., is a very common weed in winter cereals in North-Eastern Spain. Its control is becoming difficult due to expanding herbicide resistance. To control field poppies there are alternative strategies such as non-chemical control that take into account the weed emergence period. However, there is a lack of knowledge of P. rhoeas emergence patterns in semi-arid conditions. Thus, here we conducted pot experiments on the emergence of P. rhoeas. We aimed to describe the emergence period and to quantify the emergence of a susceptible and of a herbicide-resistant P. rhoeas population at two locations in Catalonia, Spain, from 1998 to 2001 and until 2004 at one of them. Therefore, pots containing seeds of both populations were established at the two locations and emergence was recorded monthly. We studied the origin of the population, the sowing location, the effect of cultivation and the sowing year. First, we found that the main emergence peaks in our experiments occurred in autumn, accounting for between 65.7 and 98.5% of the annual emergence from October to December, and only little emergence was recorded in spring. This emergence pattern is different from those found in the literature corresponding to Northern European countries, where in some cases main flushes occur only in autumn, in spring and winter or only in spring. The emergence was mainly affected by cultivation, but the effect of light stimulus was observed several months later. As a consequence, cultivation should be done in early autumn, promoting emergence during the whole autumn and winter so that emerged seedlings can be controlled before sowing a spring crop. Second, most experiments showed that the emergence was significantly higher in the first autumn than in the following seasons, e.g. 4.1% emergence in the first year and only 2.1, 2.3, 0.5 and 0.6% new emergence at one of the locations for the second, third, fourth and fifth years. Thus, after having a severe P. rhoeas infestation causing a big seed rain, emergence should be stimulated by autumn cultivation in the following season and seedlings controlled by trying to deplete the soil seed bank as much as possible. Despite the fact that emergence will be staggered throughout several years and that there was a significant relationship between rainfall and emergence, so that dry years will cause a smaller emergence rate of the weed, these findings define a cultural management strategy to reduce P. rhoeas infestations and to contribute to integrated weed management strategies combining it with other tools.

Cultural control of herbicide-resistant Lolium rigidum Gaud. populations in winter cereal in Northeastern Spain

Lolium rigidum Gaud. is one of the most common weed species in winter cereals in Northeastern Spain. Herbicide resistance has been growing since the mid 90's and exclusive herbicide use is not enough in many cases, so that it is necessary to combine as many control tools as possible. Six field trials have been conducted during the cropping seasons 2001-02, 2002-03 and 2003-04 on winter cereal infested with herbicide resistant L. rigidum in Northeastern Spain testing different cultural control strategies. Sowing delay was conducted at five fields, mouldboard ploughing at four fields, the combination of sowing delay and ploughing at two fields, increasing the cereal sowing density and combined with sowing delay at one field. Sowing delay was confirmed to have an irregular efficacy depending on the L. rigidum emergence during the delay period. In the trials, weed emergence was reduced up to 88% in the best case but there was no effect in two cases. Ploughing had a more constant efficacy and reduced weed emergence between 50 and 80% although stoniness impeded in one occasion a correct soil inversion causing a very low efficacy. Increasing the cereal sowing rate did not reduce the weed population. The combination of the different methods did not increase significantly the individual efficacy, and one method was clearly more effective than the other, depending on the trial. In fields with high L. rigidum density, these methods are not effective enough and need to be combined with other methods, which are discussed in the text.

The death receptor antagonist FAIM promotes neurite outgrowth by a mechanism that depends on ERK and NF-κB signaling

Fas apoptosis inhibitory molecule (FAIM) is a protein identified as an antagonist of Fas-induced cell death. We show that FAIM overexpression fails to rescue neurons from trophic factor deprivation, but exerts a marked neurite growth–promoting action in different neuronal systems. Whereas FAIM overexpression greatly enhanced neurite outgrowth from PC12 cells and sympathetic neurons grown with nerve growth factor (NGF), reduction of endogenous FAIM levels by RNAi decreased neurite outgrowth in these cells. FAIM overexpression promoted NF-κB activation, and blocking this activation by using a super-repressor IκBα or by carrying out experiments using cortical neurons from mice that lack the p65 NF-κB subunit prevented FAIM-induced neurite outgrowth. The effect of FAIM on neurite outgrowth was also blocked by inhibition of the Ras–ERK pathway. Finally, we show that FAIM interacts with both Trk and p75 neurotrophin receptor NGF receptors in a ligand-dependent manner. These results reveal a new function of FAIM in promoting neurite outgrowth by a mechanism involving activation of the Ras–ERK pathway and NF-κB.

Hepatitis C virus variants resistant to macrocyclic NS3-4A inhibitors subvert IFN-β induction by efficient MAVS cleavage.

BACKGROUND & AIMS: The hepatitis C virus (HCV) NS3-4A protease is essential for the HCV life cycle and a prime target of antiviral treatment strategies. Protease inhibitors, however, are limited by emergence of resistance-associated amino acid variants (RAVs). The capacity to cleave and inactivate mitochondrial antiviral-signaling protein (MAVS) in the RIG-I-signaling pathway is a cardinal feature of NS3-4A, by which HCV blocks induction of interferon-(IFN)-β, thereby promoting viral persistence. Here, we aimed to investigate the impact of NS3-4A RAVs on MAVS cleavage. METHODS: The impact of NS3-4A RAVs on MAVS cleavage was assessed using immunoblot analyses, luciferase reporter assays and molecular dynamics simulations to study the underlying molecular principles. IFN-β was quantified in serum from patients with different NS3-4A RAVs. RESULTS: We show that macrocyclic NS3-4A RAVS with substitutions at residue D168 of the protease result in an increased capacity of NS3-4A to cleave MAVS and suppress IFN-β induction compared with a comprehensive panel of RAVs and wild type HCV. Mechanistically, we show the reconstitution of a tight network of electrostatic interactions between protease and the peptide substrate that allows much stronger binding of MAVS to D168 RAVs than to the wild-type protease. Accordingly, we could show IFN-β serum levels to be lower in patients with treatment failure due to the selection of D168 variants compared to R155 RAVs. CONCLUSIONS: Our data constitutes a proof of concept that the selection of RAVs against specific classes of direct antivirals can lead to the predominance of viral variants with possibly adverse pathogenic characteristics.

Isolation and characterization of Saccharomyces cerevisiae mutants resistant to aculeacin A

Aculeacin A is a lipopeptide that inhibits ,B-glucan synthesis in yeasts. A number of Saccharomyces cerevisiae mutants resistant to this antibiotic were isolated, and four loci (ACRI, ACR2, ACR3, and ACR4) whose products are involved in the sensitivity to aculeacin A of yeast ceils were defined. Mutants containing mutations in the four loci were also resistant to echinocandin B, another member of this lipopeptide family of antibiotics. In contrast, acri, acr3, and acr4 mutants were resistant to papulacandin B (an antibiotic containing a disaccharide linked to two fatty acid chains that also inhibits P-glucan synthesis), but acr2 mutants were susceptible'to this antibiotic. This result defines common and specific steps in the entry and action of aculeacin A and papulacandin B. The analysis of double mutants revealed an epistatic effect of the acr2 mutation on the other three mutations. Cell walls of the four different mutants did not show significant alterations in composition with respect to the parental strain, and in vitro glucan synthase activity was also unaffected. However, cell surface hydrophobicity in three of the mutants was considerably decreased with respect to the parental strain.

Improvement of the antibacterial activity of daptomycin-loaded polymeric microparticles by Eudragit RL 100: An assessment by isothermal microcalorimetry.

The aim of the present study was to develop novel daptomycin-loaded acrylic microparticles with improved release profiles and antibacterial activity against two clinically relevant methicillin-susceptible and methicillin-resistant Staphylococcus aureus strains (MSSA and MRSA, respectively). Daptomycin was encapsulated into poly(methyl methacrylate) (PMMA) and PMMA-Eudragit RL 100 (EUD) microparticles by a double emulsion-solvent evaporation method. For comparison purposes similar formulations were prepared with vancomycin. Particle morphology, size distribution, encapsulation efficiency, surface charge, physicochemical properties, in vitro release and biocompatibility were assessed. Particles exhibited a micrometer size and a spherical morphology. The addition of EUD to the formulation caused a shift in the surface charge of the particles from negative zeta potential values (100% PMMA formulations) to strongly positive. It also improved daptomycin encapsulation efficiency and release, whereas vancomycin encapsulation and release were strongly hindered. Plain and antibiotic-loaded particles presented comparable biocompatibility profiles. The antibacterial activity of the particles was assessed by isothermal microcalorimetry against both MSSA and MRSA. Daptomycin-loaded PMMA-EUD particles presented the highest antibacterial activity against both strains. The addition of 30% EUD to the daptomycin-loaded PMMA particles caused a 40- and 20-fold decrease in the minimum inhibitory (MIC) and bactericidal concentration (MBC) values, respectively, when compared to the 100% PMMA formulations. On the other hand, vancomycin-loaded microparticles presented the highest antibacterial activity in PMMA particles. Unlike conventional methods, isothermal microcalorimetry proved to be a real-time, sensitive and accurate method for assessment of antibacterial activity of antibiotic-loaded polymeric microparticles. Finally, the addition of EUD to formulations proved to be a powerful strategy to improve daptomycin encapsulation efficiency and release, and consequently improving the microparticles activity against two relevant S. aureus strains.

Preventive and therapeutic strategies in critically ill patients with highly resistant bacteria.

The antibiotic pipeline continues to diminish and the majority of the public remains unaware of this critical situation. The cause of the decline of antibiotic development is multifactorial and currently most ICUs are confronted with the challenge of multidrug-resistant organisms. Antimicrobial multidrug resistance is expanding all over the world, with extreme and pandrug resistance being increasingly encountered, especially in healthcare-associated infections in large highly specialized hospitals. Antibiotic stewardship for critically ill patients translated into the implementation of specific guidelines, largely promoted by the Surviving Sepsis Campaign, targeted at education to optimize choice, dosage, and duration of antibiotics in order to improve outcomes and reduce the development of resistance. Inappropriate antimicrobial therapy, meaning the selection of an antibiotic to which the causative pathogen is resistant, is a consistent predictor of poor outcomes in septic patients. Therefore, pharmacokinetically/pharmacodynamically optimized dosing regimens should be given to all patients empirically and, once the pathogen and susceptibility are known, local stewardship practices may be employed on the basis of clinical response to redefine an appropriate regimen for the patient. This review will focus on the most severely ill patients, for whom substantial progress in organ support along with diagnostic and therapeutic strategies markedly increased the risk of nosocomial infections.

The membrane-permeable calmodulin inhibitors (W-7/W-13) bind to membranes changing the electrostatic surface potential. Dual effect of W-13 on EGFR activation

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca 2 /CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR;W-13 inhibits epidermal growth factordependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due toW-13inhibition of Ca 2 /CaM, but thelatter results could be due to binding of W-13 to the plasma membrane.

Coexistence of protease sensitive and resistant prion protein in 129VV homozygous sporadic Creutzfeldt-Jakob disease: a case report

Introduction: The coexistence of different molecular types of classical protease-resistant prion protein in the same individual have been described, however, the simultaneous finding of these with the recently described protease-sensitive variant or variably protease-sensitive prionopathy has, to the best of our knowledge, not yet been reported. Case presentation: A 74-year-old Caucasian woman showed a sporadic Creutzfeldt-Jakob disease clinical phenotype with reactive depression, followed by cognitive impairment, akinetic-rigid Parkinsonism with pseudobulbar syndrome and gait impairment with motor apraxia, visuospatial disorientation, and evident frontal dysfunction features such as grasping, palmomental reflex and brisk perioral reflexes. She died at age 77. Neuropathological findings showed: spongiform change in the patient"s cerebral cortex, striatum, thalamus and molecular layer of the cerebellum with proteinase K-sensitive synaptic-like, dot-like or target-like prion protein deposition in the cortex, thalamus and striatum; proteinase K-resistant prion protein in the same regions; and elongated plaque-like proteinase K-resistant prion protein in the molecular layer of the cerebellum. Molecular analysis of prion protein after proteinase K digestion revealed decreased signal intensity in immunoblot, a ladder-like protein pattern, and a 71% reduction of PrPSc signal relative to non-digested material. Her cerebellum showed a 2A prion protein type largely resistant to proteinase K. Genotype of polymorphism at codon 129 was valine homozygous. Conclusion: Molecular typing of prion protein along with clinical and neuropathological data revealed, to the best of our knowledge, the first case of the coexistence of different protease-sensitive prion proteins in the same patient in a rare case that did not fulfill the current clinical diagnostic criteria for either probable or possible sporadic Creutzfeldt-Jakob disease. This highlights the importance of molecular analyses of several brain regions in order to correctly diagnose rare and atypical prionopathies

Assessment of subjective and hemodynamic tolerance of different high- and low-flux dialysis membranes in patients undergoing chronic intermittent hemodialysis: a randomized controlled trial.

Clinical experience and experimental data suggest that intradialytic hemodynamic profiles could be influenced by the characteristics of the dialysis membranes. Even within the worldwide used polysulfone family, intolerance to specific membranes was occasionally evoked. The aim of this study was to compare hemodynamically some of the commonly used polysulfone dialyzers in Switzerland. We performed an open-label, randomized, cross-over trial, including 25 hemodialysis patients. Four polysulfone dialyzers, A (Revaclear high-flux, Gambro, Stockholm, Sweden), B (Helixone high-flux, Fresenius), C (Xevonta high-flux, BBraun, Melsungen, Germany), and D (Helixone low-flux, Fresenius, Bad Homburg vor der Höhe, Germany), were compared. The hemodynamic profile was assessed and patients were asked to provide tolerance feedback. The mean score (±SD) subjectively assigned to dialysis quality on a 1-10 scale was A 8.4 ± 1.3, B 8.6 ± 1.3, C 8.5 ± 1.6, D 8.5 ± 1.5. Kt/V was A 1.58 ± 0.30, B 1.67 ± 0.33, C 1.62 ± 0.32, D 1.45 ± 0.31. The low- compared with the high-flux membranes, correlated to higher systolic (128.1 ± 13.1 vs. 125.6 ± 12.1 mmHg, P < 0.01) and diastolic (76.8 ± 8.7 vs. 75.3 ± 9.0 mmHg; P < 0.05) pressures, higher peripheral resistance (1.44 ± 0.19 vs. 1.40 ± 0.18 s × mmHg/mL; P < 0.05) and lower cardiac output (3.76 ± 0.62 vs. 3.82 ± 0.59 L/min; P < 0.05). Hypotension events (decrease in systolic blood pressure by >20 mmHg) were 70 with A, 87 with B, 73 with C, and 75 with D (P < 0.01 B vs. A, 0.05 B vs. C and 0.07 B vs. D). The low-flux membrane correlated to higher blood pressure levels compared with the high-flux ones. The Helixone high-flux membrane ensured the best efficiency. Unfortunately, the very same dialyzer correlated to a higher incidence of hypotensive episodes.

Antibiotic susceptibility and molecular epidemiology of Panton-Valentine leukocidin-positive meticillin-resistant Staphylococcus aureus: An international survey

The antibiotic susceptibility and molecular epidemiology of Panton-Valentine leukocidin (PVL)-positive meticillin-resistant Staphylococcus aureus (MRSA) isolates reported from 17 countries in the Americas, Europe and, Australia-Asia were analysed. Among a total of 3236 non-duplicate isolates, the lowest susceptibility was observed to erythromycin in all regions. Susceptibility to ciprofloxacin showed large variation (25%, 75% and 84% in the Americas, Europe and Australia-Asia, respectively). Two vancomycin-intermediate PVL-positive MRSA isolates were reported, one from Hong Kong and the other from The Netherlands. Resistance to trimethoprim/sulfamethoxazole and linezolid was <1%. Among 1798 MRSA isolates from 13 countries that were tested for the requested 10 non-β-lactam antibiotics, 49.4% were multisusceptible. However, multiresistant isolates (resistant to at least three classes of non-β-lactam antibiotics) were reported from all regions. Sequence type 30 (ST30) was reported worldwide, whereas ST80 and ST93 were exclusive to Europe and Australia, respectively. USA300 and related clones (ST8) are progressively replacing the ST80 clone in several European countries. Eight major clusters were discriminated by multilocus variable-number tandem repeat assay (MLVA), showing a certain geographic specificity. PVL-positive MRSA isolates frequently remain multisusceptible to non-β-lactam agents, but multiresistance is already prevalent in all regions. Surveillance of MRSA susceptibility patterns should be monitored to provide clinicians with the most current information regarding changes in resistance patterns.