Simulations of cross-amyloid aggregation of amyloid-ß and islet amyloid polypeptide fragments.

Amyloid-ß (Aß) and islet amyloid polypeptide (IAPP) are small peptides, classified as amyloids, that have the potential to self-assemble and form cytotoxic species, such as small soluble oligomers and large insoluble fibrils. The formation of Aß aggregates facilitates the progression of Alzheimer's disease (AD), while IAPP aggregates induce pancreatic ß-cell apoptosis, leading to exacerbation of type 2 diabetes (T2D). Cross-amyloid interactions between Aß and IAPP have been described both in vivo and in vitro, implying the role of Aß or IAPP as modulators of cytotoxic self-aggregation of each species, and suggesting that Aß-IAPP interactions are a potential molecular link between AD and T2D. Using molecular dynamics (MD) simulations, "hotspot" regions of the two peptides were studied to understand the formation of hexamers in a heterogeneous and homogeneous peptide-containing environment. Systems of only Aß(16-22) peptides formed antiparallel, ß-barrel-like structures, while systems of only IAPP(20-29) peptides formed stacked, parallel ß-sheets and had relatively unstable aggregation structures after 2 µs of simulation time. Systems containing both Aß and IAPP (1:1 ratio) hexamers showed antiparallel, ß-barrel-like structures, with an interdigitated arrangement of Aß(16-22) and IAPP(20-29). These ß-barrel structures have features of cytotoxic amyloid species identified in previous literature. Ultimately, this work seeks to provide atomistic insight into both the mechanism behind cross-amyloid interactions and structural morphologies of these toxic amyloid species.

Biophysical insights into OR2T7: Investigation of a potential prognostic marker for glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive and prevalent form of brain cancer, with an expected survival of 12-15 months following diagnosis. GBM affects the glial cells of the central nervous system, which impairs regular brain function including memory, hearing, and vision. GBM has virtually no long-term survival even with treatment, requiring novel strategies to understand disease progression. Here, we identified a somatic mutation in OR2T7, a G-protein-coupled receptor (GPCR), that correlates with reduced progression-free survival for glioblastoma (log rank p-value = 0.05), suggesting a possible role in tumor progression. The mutation, D125V, occurred in 10% of 396 glioblastoma samples in The Cancer Genome Atlas, but not in any of the 2504 DNA sequences in the 1000 Genomes Project, suggesting that the mutation may have a deleterious functional effect. In addition, transcriptome analysis showed that the p38α mitogen-activated protein kinase (MAPK), c-Fos, c-Jun, and JunB proto-oncogenes, and putative tumor suppressors RhoB and caspase-14 were underexpressed in glioblastoma samples with the D125V mutation (false discovery rate < 0.05). Molecular modeling and molecular dynamics simulations have provided preliminary structural insight and indicate a dynamic helical movement network that is influenced by the membrane-embedded, cytofacial-facing residue 125, demonstrating a possible obstruction of G-protein binding on the cytofacial exposed region. We show that the mutation impacts the "open" GPCR conformation, potentially affecting Gα-subunit binding and associated downstream activity. Overall, our findings suggest that the Val125 mutation in OR2T7 could affect glioblastoma progression by downregulating GPCR-p38 MAPK tumor-suppression pathways and impacting the biophysical characteristics of the structure that facilitates Gα-subunit binding. This study provides the theoretical basis for further experimental investigation required to confirm that the D125V mutation in OR2T7 is not a passenger mutation. With validation, the aforementioned mutation could represent an important prognostic marker and a potential therapeutic target for glioblastoma.

Lambda-PCR for precise DNA assembly and modification.

Lambda-polymerase chain reaction (λ-PCR) is a novel and open-source method for DNA assembly and cloning projects. λ-PCR uses overlap extension to ultimately assemble linear and circular DNA fragments, but it allows the single-stranded DNA (ssDNA) primers of the PCR extension to first exist as double-stranded DNA (dsDNA). Having dsDNA at this step is advantageous for the stability of large insertion products, to avoid inhibitory secondary structures during direct synthesis, and to reduce costs. Three variations of λ-PCR were created to convert an initial dsDNA product into an ssDNA "megaprimer" to be used in overlap extension: (i) complete digestion by λ-exonuclease, (ii) asymmetric PCR, and (iii) partial digestion by λ-exonuclease. Four case studies are presented that demonstrate the use of λ-PCR in simple gene cloning, simultaneous multipart assemblies, gene cloning not achievable with commercial kits, and the use of thermodynamic simulations to guide λ-PCR assembly strategies. High DNA assembly and cloning efficiencies have been achieved with λ-PCR for a fraction of the cost and time associated with conventional methods and some commercial kits.

Lipophilic tail modifications of 2-(hydroxymethyl)pyrrolidine scaffold reveal dual sphingosine kinase 1 and 2 inhibitors.

The sphingosine 1-phosphate (S1P) signaling pathway is an attractive target for pharmacological manipulation due to its involvement in cancer progression and immune cell chemotaxis. The synthesis of S1P is catalyzed by the action of sphingosine kinase 1 or 2 (SphK1 or SphK2) on sphingosine and ATP. While potent and selective inhibitors of SphK1 or SphK2 have been reported, development of potent dual SphK1/SphK2 inhibitors are still needed. Towards this end, we report the structure-activity relationship profiling of 2-(hydroxymethyl)pyrrolidine-based inhibitors with 22d being the most potent dual SphK1/SphK2 inhibitor (SphK1 Ki = 0.679 µM, SphK2 Ki = 0.951 µM) reported in this series. 22d inhibited the growth of engineered Saccharomyces cerevisiae and decreased S1P levels in histiocytic lymphoma myeloid cell line (U937 cells), demonstrating inhibition of SphK1 and 2 in vitro. Molecular modeling studies of 22d docked inside the Sph binding pocket of both SphK1 and SphK2 indicate essential hydrogen bond between the 2-(hydroxymethyl)pyrrolidine head to interact with aspartic acid and serine residues near the ATP binding pocket, which provide the basis for dual inhibition. In addition, the dodecyl tail adopts a "J-shape" conformation found in crystal structure of sphingosine bound to SphK1. Collectively, these studies provide insight into the intermolecular interactions in the SphK1 and 2 active sites to achieve maximal dual inhibitory activity.

Targeting of Antithrombin in Hemophilia A or B with RNAi Therapy.

BACKGROUND: Current hemophilia treatment involves frequent intravenous infusions of clotting factors, which is associated with variable hemostatic protection, a high treatment burden, and a risk of the development of inhibitory alloantibodies. Fitusiran, an investigational RNA interference (RNAi) therapy that targets antithrombin (encoded by SERPINC1), is in development to address these and other limitations. METHODS: In this phase 1 dose-escalation study, we enrolled 4 healthy volunteers and 25 participants with moderate or severe hemophilia A or B who did not have inhibitory alloantibodies. Healthy volunteers received a single subcutaneous injection of fitusiran (at a dose of 0.03 mg per kilogram of body weight) or placebo. The participants with hemophilia received three injections of fitusiran administered either once weekly (at a dose of 0.015, 0.045, or 0.075 mg per kilogram) or once monthly (at a dose of 0.225, 0.45, 0.9, or 1.8 mg per kilogram or a fixed dose of 80 mg). The study objectives were to assess the pharmacokinetic and pharmacodynamic characteristics and safety of fitusiran. RESULTS: No thromboembolic events were observed during the study. The most common adverse events were mild injection-site reactions. Plasma levels of fitusiran increased in a dose-dependent manner and showed no accumulation with repeated administration. The monthly regimen induced a dose-dependent mean maximum antithrombin reduction of 70 to 89% from baseline. A reduction in the antithrombin level of more than 75% from baseline resulted in median peak thrombin values at the lower end of the range observed in healthy participants. CONCLUSIONS: Once-monthly subcutaneous administration of fitusiran resulted in dose-dependent lowering of the antithrombin level and increased thrombin generation in participants with hemophilia A or B who did not have inhibitory alloantibodies. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov number, NCT02035605 .).

In Silico Characterization of Structural Distinctions between Isoforms of Human and Mouse Sphingosine Kinases for Accelerating Drug Discovery.

Alterations in cellular signaling pathways are associated with multiple disease states including cancers and fibrosis. Current research efforts to attenuate cancers, specifically lymphatic cancer, focus on inhibition of two sphingosine kinase isoforms, sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2). Determining differences in structural and physicochemical binding site properties of SphKs is attractive to refine inhibitor potency and isoform selectivity. This study utilizes a predictive in silico approach to determine key differences in binding sites in SphK isoforms in human and mouse species. Homology modeling, molecular docking of inhibitors, analysis of binding pocket residue positions, development of pharmacophore models, and analysis of binding cavity volume were performed to determine isoform- and species-selective characteristics of the binding site and generate a system to rank potential inhibitors. Interestingly, docking studies showed compounds bound to mouse SphK1 in a manner more similar to human SphK2 than to human SphK1, indicating that SphKs in mice have structural properties distinct from humans that confounds prediction of ligand selectivity in mice. Our studies aid in the development and production of new compound classes by highlighting structural distinctions and identifying the role of key residues that cause observable, functional differences in isoforms and between orthologues.

HIV-1 Env gp41 Transmembrane Domain Dynamics Are Modulated by Lipid, Water, and Ion Interactions.

The gp41 transmembrane domain (TMD) of the envelope glycoprotein of the human immunodeficiency virus modulates the conformation of the viral envelope spike, the only druggable target on the surface of the virion. Targeting the envelope glycoprotein with small-molecule and antibody therapies requires an understanding of gp41 TMD dynamics, which is often challenging given the difficulties in describing native membrane properties. Here, atomistic molecular dynamics simulations of a trimeric, prefusion gp41 TMD in a model, asymmetric viral membrane that mimics the native viral envelope were performed. Water and chloride ions were observed to permeate the membrane and interact with the highly conserved arginine bundle, (R696)3, at the center of the membrane and influenced TMD stability by creating a network of hydrogen bonds and electrostatic interactions. We propose that this (R696)3 - water - anion network plays an important role in viral fusion with the host cell by modulating protein conformational changes within the membrane. Additionally, R683 and R707 at the exofacial and cytofacial membrane-water interfaces, respectively, are anchored in the lipid headgroup region and serve as a junction point for stabilization of the termini. The membrane thins as a result of the tilting of the gp41 trimer with nearby lipids increasing in volume, leading to an entropic driving force for TMD conformational change. These results provide additional detail and perspective on the influence of certain lipid types on TMD dynamics and a rationale for targeting key residues of the TMD for therapeutic design. These insights into the molecular details of TMD membrane anchoring will build toward a greater understanding of the dynamics that lead to viral fusion with the host cell.

Curare: from laboratory to law court.

Dr. David Bevan held the Wesley-Bourne Chair of Anesthesia at McGill University, Chair of Anesthesia at UBC, Anesthetist-in-Chief at the University Health Network/Mount Sinai Hospital and subsequently Chair of the Department of Anesthesia at University of Toronto until his retirement in 2006. Dr. Bevan's research contributions included seminal work in neuromuscular blockade and this work, in addition to his expertise as a reviewer, led to several editorial appointments, including Editor-in-Chief for CIM (2003-2010). Dr. Bevan played a role in the introduction of the Anesthesia Care Team concept in Ontario. He published widely and was awarded multiple international pro-fessional honors.

1978: Forty years later.

Think back; think wayyy back: before laptops, internet and smartphones. Bank machines didn't exist, tele-phones were permanently plugged into the wall, airport security meant only checking that you paid for your ticket and medical journals came in the mail (as did the bills for the journals). As it turned out, the 1970s was not a kind decade for medical journals, and several were struggling financially. Even the New England Journal of Medicine (NEJM), desperate for cash, was forced to offer a lifetime subscription to anyone who could pay the princely sum of $350! (A colleague of ours, now retired, still receives the weekly NEJM by mail, 45 years later!).

Long-term safety and efficacy of factor IX gene therapy in hemophilia B.

BACKGROUND: In patients with severe hemophilia B, gene therapy that is mediated by a novel self-complementary adeno-associated virus serotype 8 (AAV8) vector has been shown to raise factor IX levels for periods of up to 16 months. We wanted to determine the durability of transgene expression, the vector dose-response relationship, and the level of persistent or late toxicity. METHODS: We evaluated the stability of transgene expression and long-term safety in 10 patients with severe hemophilia B: 6 patients who had been enrolled in an initial phase 1 dose-escalation trial, with 2 patients each receiving a low, intermediate, or high dose, and 4 additional patients who received the high dose (2×10(12) vector genomes per kilogram of body weight). The patients subsequently underwent extensive clinical and laboratory monitoring. RESULTS: A single intravenous infusion of vector in all 10 patients with severe hemophilia B resulted in a dose-dependent increase in circulating factor IX to a level that was 1 to 6% of the normal value over a median period of 3.2 years, with observation ongoing. In the high-dose group, a consistent increase in the factor IX level to a mean (±SD) of 5.1±1.7% was observed in all 6 patients, which resulted in a reduction of more than 90% in both bleeding episodes and the use of prophylactic factor IX concentrate. A transient increase in the mean alanine aminotransferase level to 86 IU per liter (range, 36 to 202) occurred between week 7 and week 10 in 4 of the 6 patients in the high-dose group but resolved over a median of 5 days (range, 2 to 35) after prednisolone treatment. CONCLUSIONS: In 10 patients with severe hemophilia B, the infusion of a single dose of AAV8 vector resulted in long-term therapeutic factor IX expression associated with clinical improvement. With a follow-up period of up to 3 years, no late toxic effects from the therapy were reported. (Funded by the National Heart, Lung, and Blood Institute and others; ClinicalTrials.gov number, NCT00979238.).

Pegylated, full-length, recombinant factor VIII for prophylactic and on-demand treatment of severe hemophilia A.

Current management of hemophilia A includes prophylaxis with factor VIII (FVIII) replacement every 2 to 3 days. BAX 855, Baxalta's pegylated full-length recombinant FVIII (rFVIII), was designed to increase half-life and, thus, reduce the frequency of prophylactic infusions while maintaining hemostatic efficacy. BAX 855 was evaluated in previously treated patients with severe hemophilia A who were aged 12 to 65 years. A phase 1 study compared the pharmacokinetic (PK) profile of BAX 855 with that of licensed rFVIII (Advate). In a pivotal study, the annualized bleeding rate (ABR), PK parameters, and efficacy of bleeding treatment were assessed. In the phase 1 study, the mean half-life (T1/2) and the mean residence time of BAX 855 compared with Advate were 1.4- to 1.5-fold higher. These results were confirmed in the pivotal study. The pivotal study met its primary endpoint: Prophylaxis with BAX 855 resulted in an ABR that was significantly lower than half the ABR of on-demand treatment (P < .0001). The median ABR was 1.9, and 39.6% of compliant subjects had no bleeding episodes during prophylaxis, whereas subjects treated on-demand had a median ABR of 41.5. BAX 855 was also efficacious for the treatment of bleeding episodes, with 95.9% of bleeding episodes treated with 1 to 2 infusions and 96.1% having efficacy ratings of excellent/good. No FVIII inhibitory antibodies or safety signals were identified. These studies provide evidence that BAX 855 was safe and efficacious for on-demand treatment and prophylaxis administered twice weekly in patients with hemophilia A. The trials were registered at www.clinicaltrials.gov as #NCT01736475 and #NCT01599819.

Influence of sequence and lipid type on membrane perturbation by human and rat amyloid ß-peptide (1-42).

The hallmark characteristics of plaque formation and neuronal cell death in Alzheimer's disease (AD) are caused principally by the amyloid ß-peptide (Aß). Aß sequence and lipid composition are essential variables to consider when elucidating the impact of biological membranes on Aß structure and the effect of Aß on membrane integrity. Atomistic molecular dynamics simulations testing two Aß sequences, human and rat Aß (HAß and RAß, respectively), and five lipid types were performed to assess the effect of these variables on membrane perturbation and potential link to AD phenotype differences based on differences in sequence. All metrics agree insomuch that monomeric HAß and RAß contribute to membrane perturbation by causing a more rigid, gel-like lipid phase. Differences between HAß and RAß binding on degree of membrane perturbation were based on lipid headgroup properties. Cholesterol was found to moderate the amount of perturbation caused by HAß and RAß in a model raft membrane. The difference in position of an arginine residue between HAß and RAß influenced peptide-membrane interactions and was determined to be the mediating factor in observed differences in lipid affinity and degree of membrane disruption. Overall, this work increases our understanding of the influence of sequence and lipid type on Aß-membrane interactions and their relationship to AD.

Molecular Dynamics Simulations of Amyloid ß-Peptide (1-42): Tetramer Formation and Membrane Interactions.

The aggregation cascade and peptide-membrane interactions of the amyloid ß-peptide (Aß) have been implicated as toxic events in the development and progression of Alzheimer's disease. Aß42 forms oligomers and ultimately plaques, and it has been hypothesized that these oligomeric species are the main toxic species contributing to neuronal cell death. To better understand oligomerization events and subsequent oligomer-membrane interactions of Aß42, we performed atomistic molecular-dynamics (MD) simulations to characterize both interpeptide interactions and perturbation of model membranes by the peptides. MD simulations were utilized to first show the formation of a tetramer unit by four separate Aß42 peptides. Aß42 tetramers adopted an oblate ellipsoid shape and showed a significant increase in ß-strand formation in the final tetramer unit relative to the monomers, indicative of on-pathway events for fibril formation. The Aß42 tetramer unit that formed in the initial simulations was used in subsequent MD simulations in the presence of a pure POPC or cholesterol-rich raft model membrane. Tetramer-membrane simulations resulted in elongation of the tetramer in the presence of both model membranes, with tetramer-raft interactions giving rise to the rearrangement of key hydrophobic regions in the tetramer and the formation of a more rod-like structure indicative of a fibril-seeding aggregate. Membrane perturbation by the tetramer was manifested in the form of more ordered, rigid membranes, with the pure POPC being affected to a greater extent than the raft membrane. These results provide critical atomistic insight into the aggregation pathway of Aß42 and a putative toxic mechanism in the pathogenesis of Alzheimer's disease.

Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase.

Adenine deaminases (Ade) and hypoxanthine/guanine phosphoribosyltransferases (Hpt) are widely distributed enzymes involved in purine salvage. Characterization of the previously uncharacterized Ade (MJ1459 gene product) and Hpt (MJ1655 gene product) are discussed here and provide insight into purine salvage in Methanocaldococcus jannaschii. Ade was demonstrated to use either Fe(II) and/or Mn(II) as the catalytic metal. Hpt demonstrated no detectable activity with adenine, but was equally specific for hypoxanthine and guanine with a kcat /KM of 3.2 × 10(7) and 3.0 × 10(7) s(- 1) M(- 1) , respectively. These results demonstrate that hypoxanthine and IMP are the central metabolites in purine salvage in M. jannaschii for AMP and GMP production. A conserved cysteine (C127, M. jannaschii numbering) was examined due to its high conservation in bacterial and archaeal homologues. To assess the role of this highly conserved cysteine in M. jannaschii Ade, site-directed mutagenesis was performed. It was determined that mutation to serine (C127S) completely abolished Ade activity and mutation to alanine (C127A) exhibited 10-fold decrease in kcat over the wild type Ade. To further investigate the role of C127, detailed molecular docking and dynamics studies were performed and revealed adenine was unable to properly orient in the active site in the C127A and C127S Ade model structures due to distinct differences in active site conformation and rotation of D261. Together this work illuminates purine salvage in M. jannaschii and the critical role of a cysteine residue in maintaining active site conformation of Ade. Proteins 2016; 84:828-840. © 2016 Wiley Periodicals, Inc.

Pharmacophore modeling improves virtual screening for novel peroxisome proliferator-activated receptor-gamma ligands.

Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear hormone receptor involved in regulating various metabolic and immune processes. The PPAR family of receptors possesses a large binding cavity that imparts promiscuity of ligand binding not common to other nuclear receptors. This feature increases the challenge of using computational methods to identify PPAR ligands that will dock favorably into a structural model. Utilizing both ligand- and structure-based pharmacophore methods, we sought to improve agonist prediction by grouping ligands according to pharmacophore features, and pairing models derived from these features with receptor structures for docking. For 22 of the 33 receptor structures evaluated we observed an increase in true positive rate (TPR) when screening was restricted to compounds sharing molecular features found in rosiglitazone. A combination of structure models used for docking resulted in a higher TPR (40 %) when compared to docking with a single structure model (<20 %). Prediction was also improved when specific protein-ligand interactions between the docked ligands and structure models were given greater weight than the calculated free energy of binding. A large-scale screen of compounds using a marketed drug database verified the predictive ability of the selected structure models. This study highlights the steps necessary to improve screening for PPARγ ligands using multiple structure models, ligand-based pharmacophore data, evaluation of protein-ligand interactions, and comparison of docking datasets. The unique combination of methods presented here holds potential for more efficient screening of compounds with unknown affinity for PPARγ that could serve as candidates for therapeutic development.

Biophysical and molecular-dynamics studies of phosphatidic acid binding by the Dvl-2 DEP domain.

The Wnt-dependent, ß-catenin-independent pathway modulates cell movement and behavior. A downstream regulator of this signaling pathway is Dishevelled (Dvl), which, among other multiple interactions, binds to the Frizzled receptor and the plasma membrane via phosphatidic acid (PA) in a mechanism proposed to be pH-dependent. While the Dvl DEP domain is central to the ß-catenin-independent Wnt signaling function, the mechanism underlying its physical interaction with the membrane remains elusive. In this report, we elucidate the structural and functional basis of PA association to the Dvl2 DEP domain. Nuclear magnetic resonance, molecular-dynamics simulations, and mutagenesis data indicated that the domain interacted with the phospholipid through the basic helix 3 and a contiguous loop with moderate affinity. The association suggested that PA binding promoted local conformational changes in helix 2 and ß-strand 4, both of which are compromised to maintain a stable hydrophobic core in the DEP domain. We also show that the Dvl2 DEP domain bound PA in a pH-dependent manner in a mechanism that resembles deprotonation of PA. Collectively, our results structurally define the PA-binding properties of the Dvl2 DEP domain, which can be exploited for the investigation of binding mechanisms of other DEP domain-interacting proteins.

Simulations of monomeric amyloid ß-peptide (1-40) with varying solution conditions and oxidation state of Met35: implications for aggregation.

The amyloid ß-peptide (Aß) is a 40-42 residue peptide that is the principal toxic species in Alzheimer's disease (AD). The oxidation of methionine-35 (Met35) to the sulfoxide form (Met35(ox)) has been identified as potential modulator of Aß aggregation. The role Met35(ox) plays in Aß neurotoxicity differs among experimental studies, which may be due to inconsistent solution conditions (pH, buffer, temperature). We applied atomistic molecular dynamics (MD) simulations as a means to probe the dynamics of the monomeric 40-residue alloform of Aß (Aß40) containing Met35 or Met35(ox) in an effort to resolve the conflicting experimental results. We found that Met35 oxidation decreases the ß-strand content of the C-terminal hydrophobic region (residues 29-40), with a specific effect on the secondary structure of residues 33-35, thus potentially impeding aggregation. Further, there is an important interplay between oxidation state and solution conditions, with pH and salt concentration augmenting the effects of oxidation. The results presented here serve to rationalize the conflicting results seen in experimental studies and provide a fundamental biophysical characterization of monomeric Aß40 dynamics in both reduced and oxidized forms, providing insight into the biochemical mechanism of Aß40 and oxidative stress related to AD.

An in-vitro assessment of tranexamic acid as an adjunct to rFVIII or rFVIIa treatment in haemophilia A.

Haemophilia is characterised by defective thrombin generation, reduced clot stability and spontaneous bleeding. Treatment with factor VIII (FVIII) concentrate or bypassing agents (e.g. recombinant factor VIIa (rFVIIa)) is generally effective. Occasionally, haemostasis is not achieved, which may reflect a failure of factor concentrate to normalise clot stability. Tranexamic acid (TXA) is often used to aid haemostasis in surgery (e.g. joint replacements and dental procedures). Used routinely as an adjunct, it may enhance clot stability and allow effective, reliable, and cost-effective treatment at lower doses of factor concentrate. This study hypothesised that clot stabilising adjunct TXA is required in addition to factor substitution to normalise clot stability in whole blood from patients with severe haemophilia A. The in vitro effect of varying concentrations of recombinant FVIII or recombinant FVIIa and adjunct TXA on whole blood clot stability was measured by thromboelastometry. Coagulation was triggered by tissue factor and clots were challenged with tissue plasminogen activator. The area under the elasticity curve was the primary endpoint. High concentrations of FVIII and rFVIIa increased clot stability to levels that were not significantly different from controls (Mean ± SD: control 112,694 ± 84,115; FVIII 78,662 ± 74,126; rFVIIa 95,918 ± 88,492). However, the response was highly variable between individuals and demonstrates why some patients show clinical resistance to treatment. Addition of TXA resulted in normalised clot stability in all individuals, even when combined with the lowest doses of factor concentrate. The results support the concept that a more efficient, reliable and cost effective treatment may be obtained if TXA is combined with factor concentrates to treat individuals with haemophilia.

Aggregation of Alzheimer's amyloid ß-peptide in biological membranes: a molecular dynamics study.

Numerous studies have concluded that the interaction of the amyloid ß-peptide (Aß) and cellular membranes contributes to the toxicity and cell death observed in the progression of Alzheimer's disease. Aggregated Aß species disrupt membranes, leading to physical instability and ion leakage. Further, the presence of Aß on the membrane surface increases the aggregation rate of the peptide, as diffusion occurs in two dimensions, increasing the probability of interpeptide interactions. Molecular dynamics (MD) simulations have been used to investigate Aß in a number of environments, including aqueous solution and membranes. We previously showed that monomeric Aß40 remains embedded in membranes composed of the most common lipids found in the cell membrane, but that the presence of ganglioside GM1 promotes release of the peptide into the extracellular medium. Here, we explore the interactions of two Aß40 peptides in model membranes to understand whether aggregation can occur prior to the release of the peptide into the aqueous environment. We found that aggregation occurred, to different extents, in each of the model membranes and that the aggregates, once formed, did not exit the membrane environment. This information may have important implications for understanding the affinity of Aß for membranes and the mechanism of Aß toxicity in Alzheimer's disease.

Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis.

Terpene volatiles play important roles in plant-organism interactions as attractants of pollinators or as defense compounds against herbivores. Among the most common plant volatiles are homoterpenes, which are often emitted from night-scented flowers and from aerial tissues upon herbivore attack. Homoterpene volatiles released from herbivore-damaged tissue are thought to contribute to indirect plant defense by attracting natural enemies of pests. Moreover, homoterpenes have been demonstrated to induce defensive responses in plant-plant interaction. Although early steps in the biosynthesis of homoterpenes have been elucidated, the identity of the enzyme responsible for the direct formation of these volatiles has remained unknown. Here, we demonstrate that CYP82G1 (At3g25180), a cytochrome P450 monooxygenase of the Arabidopsis CYP82 family, is responsible for the breakdown of the C(20)-precursor (E,E)-geranyllinalool to the insect-induced C(16)-homoterpene (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). Recombinant CYP82G1 shows narrow substrate specificity for (E,E)-geranyllinalool and its C(15)-analog (E)-nerolidol, which is converted to the respective C(11)-homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). Homology-based modeling and substrate docking support an oxidative bond cleavage of the alcohol substrate via syn-elimination of the polar head, together with an allylic C-5 hydrogen atom. CYP82G1 is constitutively expressed in Arabidopsis stems and inflorescences and shows highly coordinated herbivore-induced expression with geranyllinalool synthase in leaves depending on the F-box protein COI-1. CYP82G1 represents a unique characterized enzyme in the plant CYP82 family with a function as a DMNT/TMTT homoterpene synthase.