New insights into the mechanism of electron transfer within flavohemoglobins: tunnelling pathways, packing density, thermodynamic and kinetic analyses.

Flavohemoglobins (FlavoHb) are metalloenzymes catalyzing the reaction of nitric oxide dioxygenation. The iron cation of the heme group needs to be preliminarily reduced to the ferrous state to be catalytically competent. This reduction is triggered by a flavin adenine dinucleotide (FAD) prosthetic group which is localized in a distinct domain of the protein. In this paper we obtain new insights into the internal long range electron transfer (over ca. 12 Å) using a combination of experimental and computational approaches. Employing a time-resolved pulse radiolysis technique we report the first direct measurement of the FADH˙→ HemeFe(III) electron transfer rate. A rate constant of (6.8 ± 0.5) × 10(3) s(-1) is found. A large panel of computational approaches are used to provide the first estimation of the thermodynamic characteristics of the internal electron transfer step within flavoHb: both the driving force and the reorganization energy are estimated as a function of the protonated state of the flavin semi-quinone. We also report an analysis of the electron pathways involved in the tunnelling of the electron through the aqueous interface between the globin and the flavin domains.

Structure of Ralstonia eutropha flavohemoglobin in complex with three antibiotic azole compounds.

Flavohemoglobins (flavoHbs) are enzymes that operate primarily as nitric oxide dioxygenases and shuttle thereby electrons among NAD(P)H, FAD, heme, and a ligated redox-active substrate such as O(2). They function in the bacterial defense against nitrosative stress and are therefore considered as targets for new antibiotic drugs. Recently, azole derivatives were proven to be attractive nitric oxide dioxygenase inhibitors, and to explore their binding characteristics, we determined the X-ray structure of the flavoHb from Ralstonia eutropha in a complex with miconazole (FHP(M)), econazole (FHP(E)), and ketoconazole (FHP(K)). In agreement with UV-vis spectroscopic data, one azole compound binds inside the distal heme pocket and ligates to the heme iron by its imidazole substituent. The two additional substituents, mostly chlorinated phenyl groups, form a series of van der Waals contacts with the protein matrix. Both interactions explain their high affinity for flavoHbs, the binding constants being 2.6, 1.2, and 11.6 µM for miconazole, econazole, and ketoconazole, respectively. The FHP(M) and FHP(Lip) (flavoHbs originally loaded with a phospholipid) structures share an "open" state and the FHP(E) and FHP(K) structures a "closed" state. Although the azole compounds were able to push the lipid out of its binding site, a fatty acid fragment is still bound inside the heme pocket of FHP(E) and FHP(K) and dictates the state of the protein. The ligand-induced open-to-closed transition involves a reorientation of the NADH domain accompanied by conformational changes in the C-terminal arm, helix E, and the CE loop resulting in an encapsulation of the heme-binding pocket. Implications of the observed open-to-closed process on the catalytic cycle are discussed.

Current process and future path for health economic assessment of pharmaceuticals in France.

The Social Security Funding Law for 2012 introduced the Economic and Public Health Assessment Committee (Commission Evaluation Economique et de Santé Publique, or CEESP) in the Social Security Code as a specialised committee affiliated with the Haute Autorité de Santé in charge of providing recommendations and health economic opinions. This article provides an in-depth description of the CEESP's structure and working methods, and analyses the impact of health economic assessment on market access of drugs in France. It also points out the areas of uncertainty and the conflicting rules following the introduction of the health economic assessment in France. The authors also provide their personal opinion on the likely future of health economic assessment of drugs in France, including the possible merge of the CEESP and the Transparency Committee, the implementation of a French threshold, and the extension of health economic assessment to a larger number of products.

Overview of external reference pricing systems in Europe.

BACKGROUND AND OBJECTIVES: External reference pricing (ERP) is a price regulation tool widely used by policy makers in the European Union (EU) Member States (MS) to contain drug cost, although in theory, it may contribute to modulate prices up and down. The objective of this article was to summarise and discuss the main findings of part of a large project conducted for the European Commission ('External reference pricing of medicinal products: simulation-based considerations for cross-country coordination'; see www.ec.europa.eu/health/healthcare/docs/erp_reimbursement_medicinal_products_en.pdf) that aimed to provide an overview of ERP systems, both on processes and potential issues in 31 European countries (28 EU MS, Iceland, Norway, and Switzerland). METHODS: A systematic structured literature review was conducted to identify and characterise the use of ERP in the selected countries, to describe its impact on the prices of pharmaceuticals, and to discuss the possible cross-country coordination issues in EU MS. This research was complemented with a consultation of competent authorities' and international organisations' representatives to address the main issues or uncertainties identified through the literature review. RESULTS: All selected countries applied ERP, except the United Kingdom and Sweden. Twenty-three countries used ERP as the main systematic criterion for pricing. In the majority of European countries, ERP was based on legislated pricing rules with different levels of accuracy. ERP was applied either for all marketed drugs or for specific categories of medicines; it was mainly used for publicly reimbursed medicines. The number of reference countries included in the basket varied from 1 to 31. There was a great variation in the calculation methods used to compute the price; 15 countries used the average price, 7 countries used the lowest price, and 7 countries used other calculation methods. Reported limitations of ERP application included the lack of reliable sources of price information, price heterogeneity, exchange rate volatility, and hidden discounts. Spill-over effect and downward price convergence have often been mentioned as ERP's consequences leading to pricing strategies from pharmaceutical companies. CONCLUSION: While ERP is widely used in Europe, processes and availability of price information vary from one country to another, thus limiting ERP implementation. Furthermore, ERP spill-over effect is a major concern of pharmaceutical firms leading to implementation of the so-called 'launch sequence strategies'.

Antimicrobial agents act differently on Staphyloccocus aureus and Ralstonia eutropha flavohemoglobins.

Flavohemoglobins (FlavoHb) play a key role in bacterial resistance to nitrosative stress and NO signaling modulation. In this study, we cloned, expressed, and characterized the flavoHb from the opportunistic pathogen, Staphylococcus aureus. The higher amino-acid sequence homology is shared with that from Saccharomyces cerevisiae which was therefore used to build a model structure by homology modeling. Interestingly, the high sequence homology with S. cerevisiae did not correlate with the enzymatic and kinetic properties which are much similar to those of Escherichia coli. In vitro and aerobically, we showed that S. aureus and Ralstonia eutropha flavoHbs accept cytochrome c and oxygen as substrates. Based on this feature, we investigated the preferences for both substrates depending on miconazole or econazole addition and found that the inhibitor chemical composition is determinant.

Systematic review of appropriate cognitive assessment instruments used in clinical trials of schizophrenia, major depressive disorder and bipolar disorder.

Cognitive dysfunction is increasingly recognized as a symptom in mental conditions including schizophrenia, major depressive disorder (MDD), and bipolar disorder (BPD). Despite the many available cognitive assessment instruments, consensus is lacking on their appropriate use in clinical trials. We conducted a systematic literature review in Embase, PubMed/Medline and PsychINFO to identify appropriate cognitive function instruments for use in clinical trials of schizophrenia, MDD, and BPD. Instruments were identified from the articles. Instruments and articles were excluded if they did not address schizophrenia, MDD, or BPD. Instrument appropriateness was further assessed by the criteria of the Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) initiative: test-retest reliability, utility, relationship to functional status, potential changeability to pharmacological agents, and tolerability and practicality for clinical trials. The database search yielded 173 articles describing 150 instruments used to assess cognitive function. Seventeen additional instruments were identified through Google and clinicaltrials.gov. Among all these, only 30 (18%) were deemed appropriate for use in the diseases of interest. Of these, 27 were studied in schizophrenia, one in MDD and two in BPD. These findings suggest the need for careful selection of appropriate cognitive assessment instruments, as not all may be valid in these disorders.

Routine clinical assessment of cognitive functioning in schizophrenia, major depressive disorder, and bipolar disorder.

As more evidence points to the association of cognitive dysfunction with mental health disorders, the assessment of cognitive function in routine clinical care of these disorders is increasingly important. Despite this, it remains unknown how cognitive function is measured in routine clinical practice. The objective of this study was to assess psychiatrists' awareness of cognitive dysfunction in mental health disorders and their methods of cognitive assessment. An online survey was disseminated to psychiatrists in Europe, Asia, Australia and the United States. The survey asked about their perceptions of cognitive dysfunction in several mental health disorders, knowledge of cognitive assessment, method of cognitive assessment, and instruments used to measure cognitive function. Among the 61 respondents, most perceived that schizophrenia was associated with the greatest cognitive dysfunction. Many were unaware whether guidelines were available on cognitive assessment. In schizophrenia, 59% of psychiatrists reportedly used cognitive instruments, while the remainder relied solely on patient history interviews. The use of instruments to assess cognition in major depressive disorder (MDD) and bipolar disorder (BPD) was lower, 38% and 37% respectively. Of the reported instruments used, only a few were actually appropriate for use in the diseases of interest (12% in schizophrenia, 3% in MDD and 0% in BPD). Other instruments reported were clinical measures that did not assess cognition. These findings reveal some inconsistencies in psychiatrists' routine clinical evaluation of cognitive function. There appeared to be low use of true cognitive assessment instruments in clinical practice and confusion regarding what constituted a cognitive assessment instrument.

Difference of perceptions and evaluation of cognitive dysfunction in major depressive disorder patients across psychiatrists internationally.

BACKGROUND: Many studies have suggested that major depressive disorder (MDD) is often associated with cognitive dysfunction. Despite this, guidance addressing assessment of cognitive dysfunction in MDD is lacking. The aim of this study was to examine psychiatrists' perceptions and evaluation of cognitive dysfunction in routine practice in MDD patients across different countries. METHOD: A total of 61 psychiatrists in the US, Germany, France, Spain, Hong Kong, and Australia participated in an online survey about perceptions of cognitive dysfunction in MDD patients, evaluation of cognition and instruments used in cognitive evaluation. RESULTS: Most psychiatrists reportedly relied on patient history interviews for cognitive evaluation (83% in France and approximately 60% in the USA, Germany, Australia and Hong Kong). The remainder used a cognitive instrument or a combination of cognitive instrument and patient history interview for assessment. Of those using instruments for cognitive assessment, only nine named instruments that were appropriate for cognitive evaluation. The remainder reported other clinical measures not intended for cognitive evaluation. CONCLUSIONS: Overall, psychiatrists in routine clinical practice value the assessment of cognitive in MDD. However, there is a lack of standardization in these assessments and misconceptions regarding proper assessment.

Active site analysis of yeast flavohemoglobin based on its structure with a small ligand or econazole.

UNLABELLED: Flavohemoglobins (flavoHbs) serve various microorganisms as the major protective enzymes against NO˙-mediated toxicity. FlavoHbs dominantly function as an NO˙ dioxygenase (O2+ NO→ NO3 -), the required electron being shuttled from NAD(P)H via FAD to the heme iron. The X-ray structures of the flavoHb from Saccharomyces cerevisae presented in complex with an unknown small ligand (Yhb) and with econazole (Yhb(E) ) at 2.1 and 3.0 Å resolutions, respectively, reveal a high architectural accordance between prokaryotic and eukaryotic family members. The active site is characterized by a proximal heme side with a strictly conserved histidine, glutamate and tyrosine triad and a highly variable distal heme side with helix shifts up to 10 Å mainly dependent on the presence/absence and size of the bound ligand. In yeast flavoHb, the small heme iron ligand adjusts a catalytically productive active site geometry that reliably suggests the NO and O(2) binding site. O(2) is activated by its ligation to an electron-rich heme iron and a hydrogen bond to Tyr29 and Gln53. High active site similarities between eukaryotic Yhb and bacterial single-domain globins argue for identical biochemical reactions. Binding of the bulky econazole implies a large-scale induced-fit process concerning, in particular, an outwards shift of helices B and E to increase the active site pocket. Yeast Yhb and Ralstonia eutropha flavoHb both structurally studied in complex with econazole indicate conformational differences between the inhibitors and the polypeptide primarily caused by stable binding of a phospholipid to the latter and by distinct loop D structures. DATABASE: Structural data and final coordinates of Yhb and Yhb-econazole are available in the Protein Data Bank under the accession numbers 4G1V and 4G1B.