Essential residues in the H-NS binding site of Hha, a co-regulator of horizontally acquired genes in Enterobacteria

Proteins of the Hha/YmoA family co-regulate with H-NS the expression of horizontally acquired genes in Enterobacteria. Systematic mutations of conserved acidic residues in Hha have allowed the identification of D48 as an essential residue for H-NS binding and the involvement of E25. Mutations of these residues resulted in deregulation of sensitive genes in vivo. D48 is only partially solvent accessible, yet it defines the functional binding interface between Hha and H-NS confirming that Hha has to undergo a conformational change to bind H-NS. Exposed acidic residues, such as E25, may electrostatically facilitate and direct the approach of Hha to the positively charged region of H-NS enabling the formation of the final complex when D48 becomes accessible by a conformational change of Hha.

Deposition and residues of azoxystrobin and imidacloprid on greenhouse lettuce with implications for human consumption

Lettuce greenhouse experiments were carried out from March to June 2011 in order to analyze how pesticides behave from the time of application until their intake via human consumption taking into account the primary distribution of pesticides, field dissipation, and post-harvest processing. In addition, experimental conditions were used to evaluate a new dynamic plant uptake model comparing its results with the experimentally derived residues. One application of imidacloprid and two of azoxystrobin were conducted. For evaluating primary pesticide distribution, two approaches based on leaf area index and vegetation cover were used and results were compared with those obtained from a tracer test. High influence of lettuce density, growth stage and type of sprayer was observed in primary distribution showing that low densities or early growth stages implied high losses of pesticides on soil. Washed and unwashed samples of lettuce were taken and analyzed from application to harvest to evaluate removal of pesticides by food processing. Results show that residues found on the Spanish preharvest interval days were in all cases below officially set maximum residue limits, although it was observed that time between application and harvest is as important for residues as application amounts. An overall reduction of 40–60% of pesticides residues was obtained from washing lettuce. Experimentally derived residues were compared with modeled residues and deviate from 1.2 to 1.4 for imidacloprid and azoxystrobin, respectively, presenting good model predictions. Resulting human intake fractions range from... for imidacloprid to ... for azoxystrobin.

A combined computational and functional approach identifies new residues involved in pH-dependent gating of ASIC1a.

Acid-sensing ion channels (ASICs) are key receptors for extracellular protons. These neuronal nonvoltage-gated Na(+) channels are involved in learning, the expression of fear, neurodegeneration after ischemia, and pain sensation. We have applied a systematic approach to identify potential pH sensors in ASIC1a and to elucidate the mechanisms by which pH variations govern ASIC gating. We first calculated the pK(a) value of all extracellular His, Glu, and Asp residues using a Poisson-Boltzmann continuum approach, based on the ASIC three-dimensional structure, to identify candidate pH-sensing residues. The role of these residues was then assessed by site-directed mutagenesis and chemical modification, combined with functional analysis. The localization of putative pH-sensing residues suggests that pH changes control ASIC gating by protonation/deprotonation of many residues per subunit in different channel domains. Analysis of the function of residues in the palm domain close to the central vertical axis of the channel allowed for prediction of conformational changes of this region during gating. Our study provides a basis for the intrinsic ASIC pH dependence and describes an approach that can also be applied to the investigation of the mechanisms of the pH dependence of other proteins.

N-Glycosylation and conserved cysteine residues in RAMP3 play a critical role for the functional expression of CRLR/RAMP3 adrenomedullin receptor.

The calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein-3 (RAMP3) can assemble into a CRLR/RAMP3 heterodimeric receptor that exhibits the characteristics of a high affinity adrenomedullin receptor. RAMP3 participates in adrenomedullin (AM) binding via its extracellular N-terminus characterized by the presence of six highly conserved cysteine residues and four N-glycosylation consensus sites. Here, we assessed the usage of these conserved residues in cotranslational modifications of RAMP3 and addressed their role in functional expression of the CRLR/RAMP3 receptor. Using a Xenopus oocyte expression system, we show that (i) RAMP3 is assembled with CRLR as a multiple N-glycosylated species in which two, three, or four consensus sites are used; (ii) elimination of all N-glycans in RAMP3 results in a significant inhibition of receptor [(125)I]AM binding and an increase in the EC(50) value for AM; (iii) several lines of indirect evidence indicate that each of the six cysteines is involved in disulfide bond formation; (iv) when all cysteines are mutated to serines, RAMP3 is N-glycosylated at all four consensus sites, suggesting that disulfide bond formation inhibits N-gylcosylation; and (v) elimination of all cysteines abolishes adrenomedullin binding and leads to a complete loss of receptor function. Our data demonstrate that cotranslational modifications of RAMP3 play a critical role in the function of the CRLR/RAMP3 adrenomedullin receptor.

Identification of Wax Esters in Latent Print Residues by Gas Chromatography-Mass Spectromertry and Their Potential Use as Aging Parameters

Recent studies show that the composition of fingerprint residue varies significantly from the same donor as well as between donors. This variability is a major drawback in latent print dating issues. This study aimed, therefore, at the definition of a parameter that is less variable from print to print, using a ratio of peak area of a target compound degrading over time divided by the summed area of peaks of more stable compounds also found in latent print residues.Gas chromatography-mass spectrometry (GC/MS) analysis of the initial lipid composition of latent prints identifies four main classes of compounds that can be used in the definition of an aging parameter: fatty acids, sterols, sterol precursors, and wax esters (WEs). Although the entities composing the first three groups are quite well known, those composing WEs are poorly reported. Therefore, the first step of the present work was to identify WE compounds present in latent print residues deposited by different donors. Of 29 WEs recorded in the chromatograms, seven were observed in the majority of samples.The identified WE compounds were subsequently used in the definition of ratios in combination with squalene and cholesterol to reduce the variability of the initial composition between latent print residues from different persons and more particularly from the same person. Finally, the influence of a latent print enhancement process on the initial composition was studied by analyzing traces after treatment with magnetic powder, 1,2-indanedione, and cyanoacrylate.

The Lausanne Institutional Biobank: a new resource to catalyse research in personalised medicine and pharmaceutical sciences.

Breakthrough technologies which now enable the sequencing of individual genomes will irreversibly modify the way diseases are diagnosed, predicted, prevented and treated. For these technologies to reach their full potential requires, upstream, access to high-quality biomedical data and samples from large number of properly informed and consenting individuals and, downstream, the possibility to transform the emerging knowledge into a clinical utility. The Lausanne Institutional Biobank was designed as an integrated, highly versatile infrastructure to harness the power of these emerging technologies and catalyse the discovery and development of innovative therapeutics and biomarkers, and advance the field of personalised medicine. Described here are its rationale, design and governance, as well as parallel initiatives which have been launched locally to address the societal, ethical and technological issues associated with this new bio-resource. Since January 2013, inpatients admitted at Lausanne CHUV University Hospital have been systematically invited to provide a general consent for the use of their biomedical data and samples for research, to complete a standardised questionnaire, to donate a 10-ml sample of blood for future DNA extraction and to be re-contacted for future clinical trials. Over the first 18 months of operation, 14,459 patients were contacted, and 11,051 accepted to participate in the study. This initial 18-month experience illustrates that a systematic hospital-based biobank is feasible; it shows a strong engagement in research from the patient population in this University Hospital setting, and the need for a broad, integrated approach for the future of medicine to reach its full potential.

Development of pharmaceutical care services in nursing homes: practice and research in a Swiss canton.

OBJECTIVE: The aim of this study was to assess the implementation process and economic impact of a new pharmaceutical care service provided since 2002 by pharmacists in Swiss nursing homes. SETTING: The setting was 42 nursing homes located in the canton of Fribourg, Switzerland under the responsibility of 22 pharmacists. METHOD: We developed different facilitators, such as a monitoring system, a coaching program, and a research project, to help pharmacists change their practice and to improve implementation of this new service. We evaluated the implementation rate of the service delivered in nursing homes. We assessed the economic impact of the service since its start in 2002 using statistical evaluation (Chow test) with retrospective analysis of the annual drug costs per resident over an 8-year period (1998-2005). MAIN OUTCOME MEASURES: The description of the facilitators and their implications in implementation of the service; the economic impact of the service since its start in 2002. RESULTS: In 2005, after a 4-year implementation period supported by the introduction of facilitators of practice change, all 42 nursing homes (2,214 residents) had implemented the pharmaceutical care service. The annual drug costs per resident decreased by about 16.4% between 2002 and 2005; this change proved to be highly significant. The performance of the pharmacists continuously improved using a specific coaching program including an annual expert comparative report, working groups, interdisciplinary continuing education symposia, and individual feedback. This research project also determined priorities to develop practice guidelines to prevent drug-related problems in nursing homes, especially in relation to the use of psychotropic drugs. CONCLUSION: The pharmaceutical care service was fully and successfully implemented in Fribourg's nursing homes within a period of 4 years. These findings highlight the importance of facilitators designed to assist pharmacists in the implementation of practice changes. The economic impact was confirmed on a large scale, and priorities for clinical and pharmacoeconomic research were identified in order to continue to improve the quality of integrated care for the elderly.

Cyclosporine A delivery to the eye: a pharmaceutical challenge.

Systemic administration of cyclosporine A (CsA) is commonly used in the treatment of local ophthalmic conditions involving cytokines, such as corneal graft rejection, autoimmune uveitis and dry eye syndrome. Local administration is expected to avoid the various side effects associated with systemic delivery. However, the currently available systems using oils to deliver CsA topically are poorly tolerated and provide a low bioavailability. These difficulties may be overcome through formulations aimed at improving CsA water solubility (e.g. cyclodextrins), or those designed to facilitate tissue drug penetration using penetration enhancers. The use of colloidal carriers (micelles, emulsions, liposomes and nanoparticles) as well as the approach using hydrosoluble prodrugs of CsA have shown promising results. Solid devices such as shields and particles of collagen have been investigated to enhance retention time on the eye surface. Some of these topical formulations have shown efficacy in the treatment of extraocular diseases but were inefficient at reaching intraocular targets. Microspheres, implants and liposomes have been developed to be directly administered subconjunctivally or intravitreally in order to enhance CsA concentration in the vitreous. Although progress has been made, there is still room for improvement in CsA ocular application, as none of these formulations is ideal.

Identification of promising antigenic components in latent fingermark residues

An analysis of latent fingermark residues by Sodium-Dodecyl-Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) followed by silver staining allowed the detection of different proteins, from which two major bands, corresponding to proteins of 56 and 64 kDa molecular weight, could be identified. Two other bands, corresponding to proteins of 52 and 48 kDa were also visualizable along with some other weaker bands of lower molecular weights. In order to identify these proteins, three antibodies directed against human proteins were tested on western blots of fingermarks residues: anti-keratin 1 and 10 (K1/10), anti-cathepsin-D (Cat.D) and anti-dermcidin (Derm.). The corresponding antigens are known to be present in the stratum corneum of desquamating stratified epithelium (K1/10, Cat.D) and/or in eccrine sweat (Cat.D, Derm.). The two major bands were identified as consistent with keratin 1 and 10. The pro-form and the active form of the cathepsin-D have also been identified from two other bands. Dermcidin could not be detected in the western blot. In addition, these antibodies have been tested on latent fingermarks left on polyvinylidene fluoride (PVDF) membrane, as well as on whitened and non-whitened paper. The detection of fingermarks was successful with all three antibodies.

From geriatric service to primary care: a retrospective pharmaceutical analysis of the medical prescription

Introduction The population of elderly persons is increasing andnegative outcomes due to polymedication are frequent. Discrepanciesin information about medication are frequent when older persons aretransitioning from hospital to home, increasing the risk of hospitalreadmission. The aims of this study were a) to determine discrepanciesin medical regimen indicated in two official discharge documents(DS = discharge summary, DP=discharge prescription); b) to characterizethe pharmacotherapy prescribed in older patients dischargedfrom a geriatric service.Materials & Methods Elderly patients (N=230) discharged from thegeriatric service (CHUV, Lausanne) over a 6-month period (January toJune 2009) were selected. Community pharmacists compared DS andDP to identify discrepancies including (a) drugs' name; (b) schedule ofadministration, dosage, frequency, prn prescription, treatment durationand galenic formulation. Beers' criteria were applied to identifypotentially inappropriate drugs and a descriptive analysis of drug costs,prescription profiles and generics were also performed.Results On average, patients were 82 ± 7 years old and stayed23.0 ± 11.6 days in the geriatric service. The delay between the datesof patient's discharge with the DP and the sending of the DS to hisgeneral physician averaged 14.0 ± 7.5 days (range 1-55). The DPhad an average of 10.0 ± 3.3 drugs (range 2-19). 77% of patients hadat least one discrepancy. A drug was missing on the DS in 57.8% ofpatients and 19.6% had a missing prn prescription. Among the 2312drugs prescribed, 3% belonged to Beers' list. They were prescribed to61 patients (26.5%), with 6 patients cumulating two Beers' potentiallyinappropriate drugs in their treatment. Analgesics (85% of thepatients), anticoagulants (80%), mineral supplements (77%), laxatives(52%) and antihypertensives (46%) were the drug classes most frequentlyprescribed. Mean costs of treatment as per DP was160.4 ± 179.4 Euros. Generic prescription represented more than 5%of the costs for 3 therapeutic classes (cholesterol-lowering agents(64%), antihypertensives (50%) and antidepressants (47%)).Discussion & Conclusion The high discrepancy rate between medicationlisted in the DP and the DS highlights a need for safetyimprovement. Potential benefits are expected from reinforced pharmacist-physician collaboration in transition from hospital to primarycare. In addition, even though Beers' criteria are questionable, thedrugs prescribed in this already fragile population, and the potentialopportunities of economical optimizations, are advocating thedevelopment and the scientific evaluation of a structured advancedcollaborative pharmacy practice service. This foresees improvedeffectiveness, safety and efficiency in the medication management ofelderly persons.