Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

1-Thio-beta-D-galactofuranosides: synthesis and evaluation as beta-D- galactofuranosidase inhibitors

Beta-D-galactofuranosidase is a good chemotherapeutic target for the design of inhibitors, since beta-D-galactofuranose is a constituent of important parasite glycoconjugates but is not present in the host mammals. With this aim, we have synthesized for the first time alkyl, benzyl and aryl 1-thio-beta-D-galactofuranosides by condensation of penta-O-benzoyl-alpha,beta-D-galactofuranose with the corresponding thiols, in the presence of SnCl4as catalyst. The complete chemical and spectroscopical characterization of these compounds showed that the reaction was stereoselective. Debenzoylation with sodium methoxide afforded the beta-S-galactofuranosides in high yield. The thioglycosides were tested as inhibitors of the beta-D- galactofuranosidase of Penicillium fellutanum, using for the first time 4-nitrophenyl-beta-D-galactofuranoside as chromogenic substrate. The 4- aminophenyl-1-thio-beta-D-galactofuranoside, obtained by catalytic hydrogenation of the nitrophenyl derivative, was the best inhibitor being then an adequate ligand for the preparation of an affinity phase aimed at the isolation of beta-d-galactofuranosidases from different sources. Also the inhibitory activity of d-galactono-1, 4-lactone was shown.      

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Antibiotic dosing in the 'at risk' critically ill patient: Linking pathophysiology with pharmacokinetics/pharmacodynamics in sepsis and trauma patients

Background

Critical illness, mediated by trauma or sepsis, can lead to physiological changes that alter the pharmacokinetics of antibiotics and may result in sub-therapeutic concentrations at the sites of infection. The first aim of this project is to identify the clinical characteristics of critically ill patients with significant trauma that have been recently admitted to ICU that may predict the dosing requirements for the antibiotic, cefazolin. The second aim of this is to identify the clinical characteristics of critically ill patients with sepsis that may predict the dosing requirements for the combination antibiotic, piperacillin-tazobactam.

Methods/Design

This is an observational pharmacokinetic study of patients with trauma (cefazolin) or with sepsis (piperacillin-tazobactam). Participants will have samples from blood and urine, collected at different intervals. Patients will also have a microdialysis catheter inserted into subcutaneous tissue to measure interstitial fluid penetration of the antibiotic. Participants will be administered sinistrin, indocyanine green and sodium bromide as well as have cardiac output monitoring performed and tetrapolar bioimpedance to determine physiological changes resulting from pathology. Analysis of samples will be performed using validated liquid chromatography tandem mass-spectrometry. Pharmacokinetic analysis will be performed using non-linear mixed effects modeling to determine individual and population pharmacokinetic parameters of antibiotics.

Discussion

The study will describe cefazolin and piperacillin-tazobactam concentrations in plasma and the interstitial fluid of tissues in trauma and sepsis patients respectively. The results of this study will guide clinicians to effectively dose these antibiotics in order to maximize the concentration of antibiotics in the interstitial fluid of tissues.     

Growth and Cadmium Phytoextraction by Swiss Chard,Maize, Rice,Noccaea caerulescens,and Alyssum murale in pH Adjusted Biosolids Amended Soils

Past applications of biosolids to soils at some locations added higher Cd levels than presently permitted. Cadmium phytoextraction would alleviate current land use constraints. Unamended farm soil, and biosolids amended farm and mine soils were obtained from a Fulton Co., IL biosolids management facility. Soils contained 0.16, 22.8, 45.3 mg Cd kg^–1 and 43.1, 482, 812 mg Zn kg^–1 respectively with initial pH 6.0, 6.1, 6.4. In greenhouse studies, Swiss chard (Beta vulgaris var. cicla), a Cd-accumulator maize (inbred B37 Zea mays) and a southern France Cd-hyperaccumulator genotype of Noccaea caerulescens were tested for Cd accumulation and phytoextraction. Soil pH was adjusted from ～5.5–7.0. Additionally 100 rice (Oryza sativa) genotypes and the Ni-hyperaccumulator Alyssum murale were screened for potential phytoextraction use.

Chard suffered phytotoxicity at low pH and accumulated up to 90 mg Cd kg^–1 on the biosolids amended mine soil. The maize inbred accumulated up to 45 mg Cd kg^–1 with only mild phytotoxicity symptoms during early growth at pH > 6.0. N. caerulescens did not exhibit phytotoxicity symptoms at any pH, and accumulated up to 235 mg Cd kg^–1 in 3 months. Reharvested N. caerulescens accumulated up to 900 mg Cd kg^–1 after 10 months. Neither Alyssum nor 90% of rice genotypes survived acceptably.

Both N. caerulescens and B37 maize show promise for Cd phytoextraction in IL and require field evaluation; both plants could be utilized for nearly continuous Cd removal. Other maize inbreds may offer higher Cd phytoextraction at lower pH, and mono-cross hybrids higher shoot biomass yields. Further, maize grown only for biomass Cd maximum removal could be double-cropped.     

Characterization of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Beijing isolates from the Mediterranean area

Background  

The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases.     

Release method and anatomical hook location: effects on short-term mortality of angler-caught Acanthopagrus australis and Argyrosomus japonicus

One field and 3 aquaria experiments were done to quantify the short-term mortality of yellowfin bream Acanthopagrus australis and mulloway Argyrosomus japonicus after being angled and subjected to 3 general handling treatments. Anglers were supplied with identical J-type hooks and asked to handle hooked fish by either (1) physically removing the hook or (2) cutting the line (5 cm from the mouth of the fish) and leaving the hook in. Some hooked A. japonicus were subjected to a third handling treatment where the line was cut underwater without exposing the fish to air. Technical and biological data were collected before all fish were released into sea cages and monitored for 5 d. Control fish were seined and similarly caged and monitored. Concentrations of plasma glucose and cortisol were collected from a sample of fish on the first and last day of the experiments. Significant predictors of mortality for both species involved the presence of blood at the mouth and an interaction between anatomical hook location and hook removal. A. australis and A. japonicus that had their ingested hooks removed experienced the greatest mortalities (87.5 and 72.7%, respectively). Typically, these fish suffered damage to their oesophagus, stomach wall and vital organs. Mortality rates of A. australis and A. japonicus were significantly decreased to 1.7 and 16%, respectively, when they were released with their lines cut, with some of these fish free of hooks after 5 d. In contrast, few mortalities occurred in either species when the hooks were removed or the lines cut on mouth-hooked fish or in A. japonicus when it was released with no air exposure. For A. australis, the field- and aquaria-based experiments provided comparable results in terms of identifying treatment-specific effects, but there were potential biases in rates of hook ingestion. Irrespective of the treatment of fish, all experiments caused physiological changes measured as elevations in either plasma cortisol or glucose. We concluded that anglers should cut the line from hook-ingested A. australis and A. japonicus, but remove the hook from mouth-hooked individuals to prevent subsequent ingestion. Further research is required to examine the longer-term consequences of these handling practices on fish health.     

Damage and partitioned mortality of teleosts discarded from two Australian penaeid fishing gears

Six field experiments were perfromed to (1) quantify the scale loss and partitioned (immediate and short-term) mortality of key teleosts discarded from a penaeid seiner and trawler operating in 2 estuaries in southeastern Australia, and (2) assess the utility of modified operational and/or onboard handling procedures for maximising survival. For both gears, several non-target species were caught, handled and discarded according to 2 general categories of treatment ('mild' and 'extreme') representing the plausible limits of severity of commercial operations. The mild treatments involved the shortest conventional deployments of the gears followed by the immediate sorting of catches in water-filled trays, while the extreme treatments comprised the longest conventional deployments and sorting in dry trays. Discards were examined for immediate mortalities and scale loss before sorting onboard, while live individuals of key species, along with appropriate numbers of controls, were released into sea cages and monitored for mortalities up to 5 d. For both gears, there was a trend of higher percentages of immediate mortalities in the extreme treatments, and more scale loss from dead than live seined and trawled silver biddy Gerres subfasciatus, seined tarwhine Rhabdosargus sarba and trawled yellowfin bream Acanthopagrus australis. Despite considerable interspecific variabilities, few intraspecific differences were detected between treatments for the short-term mortalities of live discards; however, for all species, these deaths were greater than those incurred by the controls (most of which survived). The partitioned mortality estimates were combined to provide a range of total mortalities for seined (95.97 and 99.07%, respectively) and trawled (71.74 and 97.64%) G. subfasciatus, seined R. sarba (23.95 and 100%) and trawled southern herring Herklotsichthys castelnaui (100%) and A. australis (3.40 and 35.01%). Because most deaths occurred irrespective of onboard handling procedures, we conclude that simply reducing the duration of gear deployments would provide a first step toward mitigating discard mortality in these fisheries.