5-lipoxygenase-dependent biosynthesis of novel 20:4 n-3 metabolites with anti-inflammatory activity.

5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) into pro-inflammatory leukotrienes. N-3 PUFA like eicosapentaenoic acid are subject to a similar metabolism and are precursors of pro-resolving mediators. Stearidonic acid (18:4 n-3, SDA) is a plant source of n-3 PUFA that is elongated to 20:4 n-3, an analogue of AA. However, no 5-LO metabolites of 20:4 n-3 have been reported. In this study, control and 5-LO-expressing HEK293 cells were stimulated in the presence of 20:4 n-3. Metabolites were characterized by LC-MS/MS and their anti-inflammatory properties assessed using AA-induced autocrine neutrophil stimulation and leukotriene B4-mediated chemotaxis. 8­hydroxy­9,11,14,17-eicosatetraenoic acid (Δ17-8-HETE) and 8,15-dihydroxy-9,11,13,17-eicosatetraenoic acid (Δ17-8,15-diHETE) were identified as novel metabolites. Δ17-8,15-diHETE production was inhibited by the leukotriene A4 hydrolase inhibitor SC 57461A. Autocrine neutrophil leukotriene stimulation and neutrophil chemotaxis, both BLT1-dependent processes, were inhibited by Δ17-8,15-diHETE at low nM concentrations. These data support an anti-inflammatory role for Δ17-8,15-diHETE, a novel 5-LO product.

Cationic switchable lipids: pH-triggered molecular switch for siRNA delivery.

A pH-sensitive molecular switch able to change its conformation upon protonation at endosomal pH values is embedded into the structure of cationic lipidoid materials, thus conferring endosomal escape properties. Involvement of the conformational switch in the endosomal escape process was confirmed and leading material identified was able to induce efficient gene knockdown both in vitro and in vivo. The lipid nanoparticles reported here are promising for therapeutic applications and this work could serve as a template for future design of stimulus-responsive (ionic, redox, light) molecular switch for drug and gene delivery.

[Neuroimaging in pediatric epilepsy]. / L'imagerie par résonance magnétique dans l'exploration des épilepsies de l'enfant.

The main causes of epilepsy in children are cortical malformations (hemimegalencephaly, cortical dysplasia, lissencephaly, etc.) and phakomatosis (tuberous sclerosis, Sturge-Weber disease, neurofibromatosis type 1, etc.), perinatal ischemia, traumatisms, infections, mesial temporal sclerosis, metabolic diseases, and tumors. Imaging indications are precise, including partial seizures and a pathological electroencephalogram. Twenty-five percent of these epilepsy cases are pharmacoresistant. Indeed, MRI is essential to consider surgical treatment, allowing one to localize potential epileptogenic anatomic lesions. The protocol includes sequences in three planes of space, weighted in T1, T2, Flair, T1 inversion-recovery, and T1 after gadolinium injection. MRI findings are characteristic for some tumors, but most malformations are subtle. Consequently recent techniques (spectroscopy, diffusion, etc.) are crucial when conventional MRI is not sufficient. The aim of this article is to illustrate, with a substantive image revue, this wide diversity of etiologies in pediatric epilepsy, in order to help the attendee recognize MRI findings, also discussing the role of newer imaging modalities in this field.

Forced convection during feedback approach curve measurements in scanning electrochemical microscopy: maximal displacement velocity with a microdisk.

In scanning electrochemical microscopy (SECM), an approach curve performed in feedback mode involves the downward displacement of a microelectrode toward a substrate while applying a bias to detect dissolved electroactive species at a diffusion-limited rate. The resulting measured current is said to be at steady state. In order to reduce the required measurement time, the approach velocity can be increased. In this paper, we investigate experimentally and theoretically the combination of diffusion and convection processes related to a moving microdisk electrode during feedback approaches. Transient modeling and numerical simulations with moving boundaries are performed, and the results are compared to the experimental approach curves obtained in aqueous solution. The geometry and misalignment of the microelectrode influence the experimental approach curves recorded at high approach velocities. The effects are discussed through the decomposition of the current into transient diffusional, radial convectional, and axial convectional contributions. Finally a ready-to-use expression is provided to rapidly evaluate the maximal approach velocity for steady state measurements as a function of the microelectrode geometry and the physical properties of the media. This expression holds for the more restrictive case of negative feedback as well as other modes, such as SECM approach curves performed at substrates displaying first order kinetics.

Mating system and recombination affect molecular evolution in four Triticeae species.

Mating systems and recombination are thought to have a deep impact on the organization and evolution of genomes. Because of the decline in effective population size and the interference between linked loci, the efficacy of selection is expected to be reduced in regions with low recombination rates and in the whole genome of self-fertilizing species. At the molecular level, relaxed selection is expected to result in changes in the rate of protein evolution and the pattern of codon bias. It is increasingly recognized that recombination also affects non-selective processes such as the biased gene conversion towards GC alleles (bGC). Like selection, this kind of meiotic drive in favour of GC over AT alleles is expected to be reduced in weakly recombining regions and genomes. Here, we investigated the effect of mating system and recombination on molecular evolution in four Triticeae species: two outcrossers (Secale cereale and Aegilops speltoides) and two selfers (Triticum urartu and Triticum monococcum). We found that GC content, possibly driven by bGC, is affected by mating system and recombination as theoretically predicted. Selection efficacy, however, is only weakly affected by mating system and recombination. We investigated the possible reasons for this discrepancy. A surprising one is that, in outcrossing lineages, selection efficacy could be reduced because of high substitution rates in favour of GC alleles. Outcrossers, but not selfers, would thus suffer from a 'GC-induced' genetic load. This result sheds new light on the evolution of mating systems.

Grinding up wheat: a massive loss of nucleotide diversity since domestication.

Several demographic and selective events occurred during the domestication of wheat from the allotetraploid wild emmer (Triticum turgidum ssp. dicoccoides). Cultivated wheat has since been affected by other historical events. We analyzed nucleotide diversity at 21 loci in a sample of 101 individuals representing 4 taxa corresponding to representative steps in the recent evolution of wheat (wild, domesticated, cultivated durum, and bread wheats) to unravel the evolutionary history of cultivated wheats and to quantify its impact on genetic diversity. Sequence relationships are consistent with a single domestication event and identify 2 genetically different groups of bread wheat. The wild group is not highly polymorphic, with only 212 polymorphic sites among the 21,720 bp sequenced, and, during domestication, diversity was further reduced in cultivated forms--by 69% in bread wheat and 84% in durum wheat--with considerable differences between loci, some retaining no polymorphism at all. Coalescent simulations were performed and compared with our data to estimate the intensity of the bottlenecks associated with domestication and subsequent selection. Based on our 21-locus analysis, the average intensity of domestication bottleneck was estimated at about 3--giving a population size for the domesticated form about one third that of wild dicoccoides. The most severe bottleneck, with an intensity of about 6, occurred in the evolution of durum wheat. We investigated whether some of the genes departed from the empirical distribution of most loci, suggesting that they might have been selected during domestication or breeding. We detected a departure from the null model of demographic bottleneck for the hypothetical gene HgA. However, the atypical pattern of polymorphism at this locus might reveal selection on the linked locus Gsp1A, which may affect grain softness--an important trait for end-use quality in wheat.

Estimation of long-term effective population sizes through the history of durum wheat using microsatellite data.

Estimation of long-term effective population size (N(e)) from polymorphism data alone requires an independent knowledge of mutation rate. Microsatellites provide the opportunity to estimate N(e) because their high mutation rate can be estimated from observed mutations. We used this property to estimate N(e) in allotetraploid wheat Triticum turgidum at four stages of its history since its domestication. We estimated the mutation rate of 30 microsatellite loci. Allele-specific mutation rates mu were predicted from the number of repeats of the alleles. Effective population sizes were calculated from the diversity parameter theta = 4N(e)mu. We demonstrated from simulations that the unbiased estimator of theta based on Nei's heterozygosity is the most appropriate for estimating N(e) because of a small variance and a relative robustness to variations in the mutation model compared to other estimators. We found a N(e) of 32,500 individuals with a 95% confidence interval of [20,739; 45,991] in the wild ancestor of wheat, 12,000 ([5790; 19,300]) in the domesticated form, 6000 ([2831; 9556]) in landraces, and 1300 ([689; 2031]) in recent improved varieties. This decrease illustrates the successive bottlenecks in durum wheat. No selective effect was detected on our loci, despite a complete loss of polymorphism for two of them.

Self-calibration of biplanar radiographs for a retrospective comparative study of the 3D correction of adolescent idiopathic scoliosis.

A novel technique for the 3D reconstruction of the spine from X-ray images is presented. The algorithm is based on the self-calibration of biplanar radiographs. It allows the 3D reconstruction of spines from old uncalibrated preoperative and postoperative radiographs. The reliability of the new self-calibration technique was investigated by validating its results against those of the Direct Linear Transform (DLT) on real images. An accuracy experiment was also performed using a dry spine specimen under controlled conditions. The results indicate that self-calibration is a viable technique, accurate enough to extract meaningful 3D clinical data for retrospective studies.

Thiol-reactive agents biphasically regulate inositol 1,4,5-trisphosphate binding and Ca(2+) release activities in bovine adrenal cortex microsomes.

Within all endocrine cells, the inositol 1,4,5-trisphosphate (InsP(3)) receptor plays an important role in regulation of the intracellular Ca(2+) concentration. In the present study we showed that a single short-term treatment with either N-ethylmaleimide (known to decrease InsP(3) receptor activity) or thimerosal (known to increase InsP(3) receptor activity) caused time-dependent biphasic effects on the InsP(3) binding activity of bovine adrenal cortex microsomes. The early potentiating effect of thiol-reactive agents translated into a 2-fold increase in binding affinity and Ca(2+) release efficiency. The late dampening effect of thiol-reactive agents translated into a continuous reduction of the maximal binding capacity of the microsomes with a concomitant decrease in Ca(2+) release efficiency. Under these conditions, Western blot analyses demonstrated that the level of InsP(3) receptor protein was not modified. Sequential treatments with thimerosal and the reducing agent dithiothreitol showed that the InsP(3) receptor can readily oscillate between high and low affinity states that are related to its alkylation state. Our results suggest a common mode of action of thiol-reactive agents on the InsP(3) receptor. These results also support the contention that cellular mechanisms of thiol group modification could play important roles in regulation of the intracellular Ca(2+) concentration.

FK506 blocks intracellular Ca2+ oscillations in bovine adrenal glomerulosa cells.

The inositol 1,4,5-trisphosphate (InsP(3)) receptor is a ligand-gated Ca(2+) channel playing an important role in the control of intracellular Ca(2+). In the study presented here, we demonstrate that angiotensin (AngII), phorbol ester (PMA), and FK506 significantly increase the level of InsP(3) receptor phosphorylation in intact bovine adrenal glomerulosa cells. With a back-phosphorylation approach, we showed that the InsP(3) receptor is a good substrate for protein kinase C (PKC) and that FK506 increases the level of PKC-mediated InsP(3) receptor phosphorylation. With a microsomal preparation from bovine adrenal cortex, we showed that PKC enhances the release of Ca(2+) induced by a submaximal dose of InsP(3). We also showed that FK506 blocks intracellular Ca(2+) oscillations in isolated adrenal glomerulosa cells by progressively increasing the intracellular Ca(2+) concentration to a high plateau level. This effect is consistent with an inhibitory role of FK506 on calcineurin dephosphorylation of the InsP(3) receptor, thus keeping the receptor in a phosphorylated, high-conductance state. Our results provide further evidence for the crucial role of the InsP(3) receptor in the regulation of intracellular Ca(2+) oscillations and show that FK506, by maintaining the phosphorylated state of the InsP(3) receptor, causes important changes in the Ca(2+) oscillatory process.

Optimizing adherence to pharmaceutical care plans.

Adherence to pharmacotherapy is essential to optimal therapeutic outcomes. The pivotal role of the pharmacist in optimizing adherence encompasses many actions: assessing the adherence problem, identifying predisposing factors, providing comprehensive counseling, and recommending specific adherence strategies targeted to the patient's needs. Patients who have chronic conditions, physical or cognitive impairments, or cultural backgrounds outside the mainstream may have special needs that should be addressed in the adherence plan. Pharmaceutical care plans also should take into account the patient's age, stage of life, and literacy level. Although a wide range of adherence aids and strategies are available, the key to success is to tailor the intervention to the individual patient and, when necessary, to combine interventions to optimize adherence.

Different populations of inositol 1,4,5-trisphosphate receptors expressed in the bovine adrenal cortex.

The inositol 1,4,5-trisphosphate (InsP3) receptor forms a tetrameric channel responsible for the release of Ca2+ from intracellular stores. In the present study we showed that the experimental approach used to separate bound and free ligands may discriminate between two populations of InsP3 binding sites in bovine adrenal cortex microsomes. A large population of low affinity sites and a small population of high affinity sites were detected with centrifugation and filtration approaches, respectively. Both populations were found in the supernatant and the cytoskeleton fractions of Triton X-100 solubilized microsomes. After treatment of microsomes with thimerosal, an alkylating reagent known to increase InsP3 receptor affinity, the filtration and the centrifugation approaches yielded identical results. With selective anti-InsP3 receptor antibodies, we showed that types 1, 2 and 3 InsP3 receptors are present in intact microsomes and in the cytoskeleton fraction. Binding studies on immunoprecipitated receptors revealed that anti-type 1 antibody recognizes a large population of low affinity sites whereas anti-type 2 antibody recognizes a small population of high affinity sites. Our results indicate that the three types of InsP3 receptors are expressed at different levels in the bovine adrenal cortex. The presence of different types of InsP3 receptors with different ligand binding affinities and their association with the cytoskeleton offer a convenient way for the cell to simultaneously regulate its intracellular Ca2+ concentration and reorganize the spatial distribution of its Ca2+ stores.

A Krüppel-like zinc finger protein is involved in nitrogen-fixing root nodule organogenesis.

Mechanisms regulating plant host differentiation of the nitrogen-fixing root nodules remain mostly unknown. Sinorhizobium meliloti induces this process in Medicago sativa in which the Mszpt2-1 gene is expressed in vascular bundles of roots and nodules. This gene codes for a Krüppel-like zinc finger protein, a class of transcription factors involved in many animal developmental processes. Expression of Mszpt2-1 in yeast cells conferred osmotic tolerance. Antisense plants grew normally but developed nonfunctional nodules, in which differentiation of the nitrogen-fixing zone and bacterial invasion were arrested. Hence, a vascular bundle-associated Krüppel-like gene is required for the formation of the central nitrogen-fixing zone of the root nodule.

Synthesis, acid-base behavior, and binding properties of 6-modified myo-inositol 1,4,5-tris(phosphate)s.

myo-Inositol 1,4,5-tris(phosphate) was modified at position 6. The analogues synthesized are reported in this publication are 6-deoxy-myo-inositol 1,4,5-tris(phosphate), 6-fluoro-6-deoxy-myo-inositol 1,4,5-tris(phosphate), epi-inositol 1, 4,5-tris(phosphate), and 6-amino-6-deoxy-myo-inositol 1,4, 5-tris(phosphate). These derivatives showed poor affinity for the Ins(1,4,5)P(3) receptors. The inframolecular acid-base behavior and the cooperative effects between the phosphate groups could help explain the loss of affinity of these 6-modified analogues.

Billing third party payers for pharmaceutical care services.

OBJECTIVE: To describe the steps pharmacists must complete when seeking compensation from third party payers for pharmaceutical care services. DATA SOURCES: Government publications; professional publications, including manuals and newsletters; authors' personal experience. DATA SYNTHESIS: Pharmacists in increasing numbers are meeting with success in getting reimbursed by third party payers for patient care activities. However, many pharmacists remain reluctant to seek compensation because they do not understand the steps involved. Preparatory steps include obtaining a provider/supplier number, procuring appropriate claim forms, developing data collection and documentation systems, establishing professional fees, creating a marketing plan, and developing an accounting system. To bill for specific patient care services, pharmacists need to collect the patient's insurance information, obtain a statement of medical necessity from the patient's physician, complete the appropriate claim form accurately, and submit the claim with supporting documentation to the insurer. Although many claims from pharmacists are rejected initially, pharmacists who work with third party payers to understand the reasons for denial of payment often receive compensation when claims are resubmitted. CONCLUSION: Pharmacists who follow these guidelines for billing third party payers for pharmaceutical care services should notice an increase in the number of paid claims.

General and self-consistent method for the calibration of polarization modulators, polarimeters, and mueller-matrix ellipsometers.

Calibration of polarization-state generators (PSG's), polarimeters, and Mueller-matrix ellipsometers (MME's) is an important factor in the practical use of these instruments. A new general procedure, the eigenvalue calibration method (ECM), is presented. It can calibrate any complete MME consisting of a PSG and a polarimeter that generate and measure, respectively, all the states of polarization of light. In the ECM, the PSG and the polarimeter are described by two 4 x 4 matrices W and A, and their 32 coefficients are determined from three or four measurements performed on reference samples. Those references are smooth isotropic samples and perfect linear polarizers. Their optical characteristics are unambiguously determined during the calibration from the eigenvalues of the measured matrices. The ECM does not require accurate alignment of the various optical elements and does not involve any first-order approximation. The ECM also displays an efficient error control capability that can be used to improve the MME behavior. The ECM is illustrated by an experimental calibration, at two wavelengths (458 and 633 nm), of a MME consisting of a coupled phase modulator associated with a prism division-of-amplitude polarimeter.

Songs of innocence and experience: student's poems about their medical education.

Personal creative writing is increasingly used in medical schools to foster self-reflection. This article looks particularly at poetry as a vehicle for expressing personal experiences of professional development. The authors present a series of poems written by students at their medical school. In them, the students reflect on embryology, gross anatomy, telling (or not telling) bad news to trusting patients (and family members), encountering death, and encountering their own anger and frustration with the demands of medicine. These poems not only capture individual students' feelings and imaginations but also demonstrate the students' constant struggle to sustain their idealism about medicine throughout the four years of their education.