Emilio Parisini

Halogen bonding in halocarbon–protein complexes: a structural survey

Halogen bonding has been extensively described in the context of a variety of self-assembled supramolecular systems and efficiently utilized in the rational design of materials with specific structural properties. However, it has so far received only little recognition for its possible role in the stabilization of small molecule-protein complexes. In this tutorial review, we provide a few examples of halogen bonds occurring between small halogen-substituted ligands and their biological substrates. Examples were drawn from a diverse set of compounds, ranging from chemical additives and possible environmental agents such as triclosan to pharmacologically active principles such as the volatile anesthetic halothane or HIV-1 reverse transcriptase inhibitors or a subset of non-steroidal anti-inflammatory drugs (NSAIDs) that are halogen-substituted. The crystal structures presented here, where iodine, bromine, or chlorine atoms function as halogen bonding donors and a variety of electron rich sites, such as oxygen, nitrogen and sulfur atoms, as well as aromatic π-electron systems, function as halogen bonding acceptors, prove how halogen bonds can occur in biological systems and provide a class of highly directional stabilizing contacts that can be exploited in the process of rational drug design.

Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d.

The crystal structure of the cyclin D-dependent kinase Cdk6 bound to the p19INK4d protein has been determined at 1.9 Å resolution. The results provide the first structural information for a cyclin D-dependent protein kinase and show how the INK4 family of CDK inhibitors bind. The structure indicates that the conformational changes induced by p19INK4d inhibit both productive binding of ATP and the cyclin-induced rearrangement of the kinase from an inactive to an active conformation. The structure also shows how binding of an INK4 inhibitor would prevent binding of p27Kip1, resulting in its redistribution to other CDKs. Identification of the critical residues involved in the interaction explains how mutations in Cdk4 and p16INK4a result in loss of kinase inhibition and cancer.

Meta-analysis: Travel and Risk for Venous Thromboembolism

Context The body of evidence on the epidemiology of long-distance travel and venous thromboembolism (VTE) is heterogeneous and inconclusive. Contribution The reviewers found 14 eligible studies, which had significant between-study heterogeneity, and the pooled relative risk for VTE was 2.0 (95% CI, 1.5 to 2.7). The reviewers eliminated the heterogeneity by excluding 6 casecontrol studies in which control participants had been referred for VTE testing. The relative risk was 2.8 (CI, 2.2 to 3.7) in the remaining included studies and 1.2 (CI, 0.9 to 1.6) in the excluded studies. Implication By excluding studies with control participants who had a different risk for VTE than the source population for the case patients, the authors clarified a confusing body of evidence. The Editors Because of the rapid increase in air and other modes of travel in recent years, the potential risk for travel-related venous thromboembolism (VTE) is a growing public health concern (1). Worldwide, 2.5 billion passengers will travel by air in 2010 (2), which underscores the large global population at risk for this serious condition. In addition to the direct and indirect costs of evaluation and treatment, mortality risk is high: In ambulatory population-based cohorts, the estimated 28-day mortality for a first episode of VTE is 11% (3). Although a positive relationship between travel and VTE is often discussed and assumed to exist, the results of previous studies are surprisingly conflicting. Several epidemiologic studies have investigated this relationship over the past decade; approximately half have found no relationship between travel and VTE (48), whereas the rest have identified elevated risk (916). Demonstrating the presence and magnitude of such potential risk is crucial to determine the appropriateness of additional controlled trials or policy measures to prevent travel-related VTE. In addition, if a relationship exists, quantifying the doseresponse relationshiphow duration of travel relates to VTEis central to determining the travel circumstances under which sufficient risk could be present to justify preventive interventions. To determine whether travel is associated with risk for VTE, to quantify the doseresponse relationship, and to identify reasons for the contradictory findings reported by previous studies, we performed a systematic review and meta-analysis of studies of travel and risk for VTE. We hypothesized that travel would be associated with the risk for VTE and that this risk would increase with greater duration of travel. Methods We followed the MOOSE (Meta-analysis of Observational Studies in Epidemiology) guidelines during all stages of design, implementation, and reporting of this meta-analysis (17). Search Strategy and Data Sources We searched for all studies that provided an effect estimate for a potential association between travel and VTE. Inclusion criteria were observational studies or clinical trials that included patients who traveled by any mode of transportation; that used nontraveling persons for comparison; and that diagnosed VTE by appropriate radiologic investigation (ultrasonography or venography for deep venous thrombosis [DVT] and computed tomography or ventilationperfusion scan for pulmonary embolism [PE]), autopsy findings, or documentation of hospitalization for VTE. We excluded studies if they only evaluated treatments for VTE, rather than travel as a risk factor for VTE; if VTE was diagnosed by less rigorous criteria (such as by using only administrative codes in outpatients); or if no comparison group of nontravelers was included (which would preclude estimation of relative risks [RRs] associated with travel). We performed our search by using MEDLINE, EMBASE, BIOSIS, CINAHL, the Cochrane library, grey-literature sources (a system for information on grey literature in Europe, a British library inside database, and dissertation abstracts online); one of the investigators also hand-searched the reference lists of identified studies. For each database, the years searched included the earliest available online year of indexing up to March 2008, without language restrictions. We exploded each search term. Our MEDLINE search terms were ((travel[MeSH Terms] OR travel[Text Word]) OR (transportation[MeSH Terms] OR transportation[Text Word]) OR journey[All Fields] OR flying[All Fields]) AND ((thrombosis[MeSH Terms] OR thrombosis[Text Word]) OR (embolism[MeSH Terms] OR embolism[Text Word]) OR DVT[All Fields] OR PE[All Fields] OR clot[All Fields]) NOT (review[Publication Type] OR review literature as topic[MeSH Terms] OR review[Text Word]) NOT (case reports[Publication Type]). Study Selection Of 1560 identified abstracts, we excluded 1518 on screening because they were commentaries, general reviews, or case reports (Figure 1). Two investigators independently examined the full text of the remaining 42 studies to confirm eligibility for inclusion. The second reviewer was blinded to the study investigators and journal of publication. Interobserver agreement between the 2 reviewers for initial study inclusion was high (= 0.89). We resolved disagreement by mutual discussion and, if required, by consultation with a third investigator. Of the 42 studies, we excluded 15 because they lacked a nontraveling comparison group, 11 because they evaluated preventive measures for travel-related VTE, 1 because it did not use the required criteria for VTE diagnosis (18), and 1 because no events occurred and it also lacked a nontraveling comparison group (19). This resulted in the final selection of 14 studies, including 1 prospective cohort study (15), 1 retrospective cohort study (16), 1 casecrossover study (13), and 11 casecontrol studies (412, 14, 20). The investigators can provide a complete list of abstracts searched with reasons for exclusion on request. Figure 1. Study flow diagram. VTE = venous thromboembolism. Data Extraction and Quality Assessment We collected data on the year the study was performed, study design, study location, inclusion and exclusion criteria, number of participants, duration and mode of travel, duration of follow up, and adjusted relative risks and odds ratios with CIs. Two investigators performed independent data extraction by using a standardized data collection form. We resolved disagreement by mutual discussion and, if required, by consulting a third investigator. All prespecified data points were available from the published manuscripts, except for data on dose-response (duration of travel and risk for VTE) in 4 studies (1113, 15); we contacted the investigators to request the missing data but received no responses. Because no standardized criteria have been established for judging the quality of observational studies, quality scores can differ depending on the scale chosen, and interpretation of such scores is difficult (21), we selected a priori several important design characteristics that may affect study quality to evaluate as sources of heterogeneity, including inclusion and exclusion criteria, method of travel history assessment, selection criteria for control participants, matching criteria, and control for confounding. We were particularly interested in the selection criteria for control participants in casecontrol studies because of the potential for selection bias that could substantially alter the validity of the obtained results. We assessed potential recall bias by the method (self-report vs. travel records) and timing (before vs. after VTE evaluation) of travel history assessment. Statistical Analysis We obtained pooled risk estimates for risk for VTE with travel by using random-effects models, according to the method of DerSimonian and Laird (22), with inverse-variance weighting. We used the most fully multivariable-adjusted effect estimate and recorded the included covariates. Because travel-related VTE is an uncommon outcome, odds ratios from casecontrol studies approximate risk ratios or relative risks (RRs) from cohort studies, and we pooled them to generate 1 common RR. Henceforth, we will refer to odds ratios from casecontrol studies as RRs. We assessed heterogeneity among studies by using Cochrane Q and I 2 statistics. We evaluated the following predefined sources of potential heterogeneity: study design (cohort vs. casecrossover vs. casecontrol), selection criteria for control participants in casecontrol studies (referred for VTE evaluation vs. nonreferred), study location (Europe vs. North America vs. Australia or New Zealand), minimum duration of travel required for inclusion (<8 vs. 8 hours), duration of follow-up after travel completed (3 vs. >3 weeks), type of VTE studied (DVT vs. PE), mode of travel (air vs. surface [land or sea]), exposure assessment (self-report vs. travel records), and number of included covariates. We obtained effect estimates by mode of travel from the individual studies or, when such estimates were unavailable, from previously reported pooled estimates (5). We used meta-regression to examine sources of heterogeneity by using the Wald test in random-effects meta-regression models. We also used meta-regression to test for a doseresponse relationship between duration of travel and risk for VTE. We assessed potential publication bias by visually examining a funnel plot with the Begg test (23), a statistical analogue of the visual funnel graph, and the Egger test (24). We used STATA, version 9.0 (StataCorp, College Station, Texas), for all analyses. We defined statistical significance as a 2-tailed value less than 0.05. Role of the Funding Source Our study received no outside funding. Results Table 1 summarizes the characteristics of the 14 included studies. We identified 4055 cases of VTE, including 3980 cases in the 11 casecontrol studies (with 5413 control participants), 29 in the 2 cohort studies (10932 participants), and 46 in the casecrossover study (5408 participants). Seven of the studies reported statistically significant asso

NSAID Exposure and Risk of Nonunion: A Meta-Analysis of Case–Control and Cohort Studies

Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used for postoperative pain control. However, concerns regarding the potential deleterious effects of NSAIDs on bone healing have compelled many physicians to avoid NSAIDs in patients with healing fractures, osteotomies, and fusions. We systematically reviewed and analyzed the best clinical evidence regarding the effects of NSAID exposure on bone healing. Medline, Embase, and Cochrane electronic databases were searched for prospective and retrospective clinical studies of fracture, osteotomy, and fusion studies of patients with NSAID exposure and nonunion as an outcome. Study quality was assessed using the Newcastle–Ottawa Scale. Data on study design, patient characteristics, and risk estimates were extracted. Pooled effect estimates were calculated. Subanalyses were performed by bone type and by NSAID dose, duration, and route of administration. In the initial analysis of 11 cohort and case-control studies, the pooled odds ratio for nonunion with NSAID exposure was 3.0 (95% confidence interval 1.6–5.6). A significant association between lower-quality studies and higher reported odds ratios for nonunion was observed. When only higher-quality studies were considered, seven spine fusion studies were analyzed, and no statistically significant association between NSAID exposure and nonunion was identified (odds ratio = 2.2, 95% confidence interval 0.8–6.3). There was no increased risk of nonunion with NSAID exposure when only the highest-quality studies were assessed. Randomized controlled trials assessing NSAID exposure in fracture, fusion, and osteotomy populations are warranted to confirm or refute the findings of this meta-analysis of observational studies.

Ly6 family proteins in neutrophil biology

The murine Ly6 complex was identified 35 years ago using antisera to lymphocytes. With advances in mAb development, molecular cloning, and genome sequencing, >20 structurally related genes have been identified within this complex on chromosome 15. All members of the Ly6 family and their human homologues share the highly conserved LU domain and most also possess a GPI anchor. Interestingly, many Ly6 proteins are expressed in a lineage‐specific fashion, and their expression often correlates with stages of differentiation. As a result, Ly6 proteins are frequently used as surface markers for leukocyte subset identification and targets for antibody‐mediated depletion. Murine neutrophils display prominent surface expression of several Ly6 proteins, including Ly6B, Ly6C, and Ly6G. Although the physiology of most Ly6 proteins is not well understood, a role in neutrophil functions, such as migration, is recognized increasingly. In this review, we will provide an overview of the Ly6 complex and discuss, in detail, the specific Ly6 proteins implicated in neutrophil biology.

A New Strategy for the Stereoselective Synthesis of 1,2,3-Trisubstituted Cyclopropanes

The stereoselective synthesis of highly functionalized 1,2,3-trisubstituted cyclopropanes 1 and 2, starting from readily available furans 3 or N-protected pyrrole 4, is described. Furthermore, exceptionally high diastereocontrol in agreement with the Felkin-Anh model was observed for the addition of nucleophiles to the title compounds.

Crystal structure determination at 1.4 Å resolution of ferredoxin from the green alga Chlorella fusca

BACKGROUND [2Fe-2S] ferredoxins, also called plant-type ferredoxins, are low-potential redox proteins that are widely distributed in biological systems. In photosynthesis, the plant-type ferredoxins function as the central molecule for distributing electrons from the photolysis of water to a number of ferredox-independent enzymes, as well as to cyclic photophosphorylation electron transfer. This paper reports only the second structure of a [2Fe-2S] ferredoxin from a eukaryotic organism in its native form. RESULTS Ferredoxin from the green algae Chlorella fusca has been purified, characterised, crystallised and its structure determined to 1.4 A resolution - the highest resolution structure published to date for a plant-type ferredoxin. The structure has the general features of the plant-type ferredoxins already described, with conformational differences corresponding to regions of higher mobility. Immunological data indicate that a serine residue within the protein is partially phosphorylated. A slightly electropositive shift in the measured redox potential value, -325 mV, is observed in comparison with other ferredoxins. CONCLUSIONS This high-resolution structure provides a detailed picture of the hydrogen-bonding pattern around the [2Fe-2S] cluster of a plant-type ferredoxin; for the first time, it was possible to obtain reliable error estimates for the geometrical parameters. The presence of phosphoserine in the protein indicates a possible mechanism for the regulation of the distribution of reducing power from the photosynthetic electron-transfer chain.

Protein-protein interactions in receptor activation and intracellular signalling.

Abstract We review here signalling complexes that we have defined using X-ray analysis in our laboratory. They include growth factors and their receptors: nerve growth factor (NGF) and its hetero-hexameric 7S NGF storage complex, hepatocyte growth factor/scatter factor (HGF/SF) NK1 dimers and fibroblast growth factor (FGF1) in complex with its receptor (FGFR2) ectodomain and heparin. We also review our recent structural studies on intracellular signalling complexes, focusing on phosducin transducin Gβγ, CK2 protein kinase and its complexes, and the cyclin D-dependent kinase, Cdk6, bound to the cell cycle inhibitor p19INK4d. Comparing the structures of these complexes with others we show that the surface area buried in signalling interactions does not always give a good indication of the strength of the interactions. We show that conformational changes are often important in complexes with intermediate buried surface areas of 1500 to 2000 Å2, such as Cdk6 INK4 interactions. Some interactions involve recognition of continuous epitopes, where there is no necessity for a tertiary structure and very often the binding conformation is induced during the process of interaction, for example phosducin binding to the βγ subunits (Gtβγ) of the heterotrimeric G protein transducin.

Ab initio solution and refinement of two high-potential iron protein structures at atomic ­resolution

The crystal structure of the reduced high-potential iron protein (HiPIP) from Chromatium vinosum has been redetermined in a new orthorhombic crystal modification, and the structure of its H42Q mutant has been determined in orthorhombic (H42Q-1) and cubic (H42Q-2) modifications. The first two were solved by ab initio direct methods using data collected to atomic resolution (1.20 and 0. 93 A, respectively). The recombinant wild type (rc-WT) with two HiPIP molecules in the asymmetric unit has 1264 protein atoms and 335 solvent sites, and is the second largest structure reported so far that has been solved by pure direct methods. The solutions were obtained in a fully automated way and included more than 80% of the protein atoms. Restrained anisotropic refinement for rc-WT and H42Q-1 converged to R(1) = summation operator||F(o)| - |F(c)|| / summation operator|F(o)| of 12.0 and 13.6%, respectively [data with I > 2sigma(I)], and 12.8 and 15.5% (all data). H42Q-2 contains two molecules in the asymmetric unit and diffracted only to 2.6 A. In both molecules of rc-WT and in the single unique molecule of H42Q-1 the [Fe(4)S(4)](2+) cluster dimensions are very similar and show a characteristic tetragonal distortion with four short Fe-S bonds along four approximately parallel cube edges, and eight long Fe-S bonds. The unique protein molecules in H42Q-2 and rc-WT are also very similar in other respects, except for the hydrogen bonding around the mutated residue that is at the surface of the protein, supporting the hypothesis that the difference in redox potentials at lower pH values is caused primarily by differences in the charge distribution near the surface of the protein rather than by structural differences in the cluster region.

Synthesis of [M3H(CO)9(µ3-σ:η2:η2-C6H7)](M = Ru or Os). Molecular and crystal structure of the ruthenium cluster

The dienyl cluster compounds [M3H(CO)9(µ3-σ:η2:η2-C6H7)](M = Ru or Os) have been synthesised from [Os3H2(CO)10] or [M3(CO)10(MeCN)2](M = Ru or Os) with cyclohexa-1,3-diene. The molecular and crystal structure of [Ru3H(CO)9(C6H7)] has been established by single-crystal X-ray diffraction analysis: monoclinic, space group P21, a= 8.487(6), b= 12.031(3), c= 9.073(2)A, β= 92.43(4)° and Z= 2. The cyclohexadienyl ligand is involved in one σ and two π interactions with the metal triangle, while the H(hydride) ligand bridges the long Ru–Ru bond [3.052(1)A]. The other two Ru–Ru bond lengths are the same [2.837(1)A] and comparable to those observed in [Ru3(CO)9(µ3-η2:η2:η2-C6H6)]. The H(hyride) position afforded by the diffraction experiment has been compared with the result of potential-energy minimization procedures. The molecular organization within the lattice has been explored by means of atom–atom packing potential-energy calculations showing the presence of a network of C–H ⋯ O intermolecular hydrogen-bonding interactions.