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Leucine-rich repeat kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinson’s disease1and is also linked to its idiopathic form2. LRRK2 has been proposed to function in membrane trafficking3and colocalizes with microtubules4. Despite the fundamental importance of LRRK2 for understanding and treating Parkinson’s disease, structural information on the enzyme is limited. Here we report the structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported cryo-electron tomography in situ structure5. We propose that the conformation of the LRRK2 kinase domain regulates its interactions with microtubules, with a closed conformation favouring oligomerization on microtubules. We show that the catalytic half of LRRK2 is sufficient for filament formation and blocks the motility of the microtubule-based motors kinesin 1 and cytoplasmic dynein 1 in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce the formation of LRRK2 filaments in cells, whereas inhibitors that stabilize a closed conformation do not. Our findings suggest that LRRK2 can act as a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.

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The availability of bromodomain and extra-terminal inhibitors (BETi) has enabled translational epigenetic studies in cancer. BET proteins regulate transcription by selectively recognizing acetylated lysine residues on chromatin. BETi compete with this process leading to both downregulation and upregulation of gene expression. Hypoxia enables progression of triple negative breast cancer (TNBC), the most aggressive form of breast cancer, partly by driving metabolic adaptation, angiogenesis and metastasis through upregulation of hypoxia-regulated genes (for example, carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A). Responses to hypoxia can be mediated epigenetically, thus we investigated whether BETi JQ1 could impair the TNBC response induced by hypoxia and exert anti-tumour effects. JQ1 significantly modulated 44% of hypoxia-induced genes, of which two-thirds were downregulated including CA9 and VEGF-A. JQ1 prevented HIF binding to the hypoxia response element in CA9 promoter, but did not alter HIF expression or activity, suggesting some HIF targets are BET-dependent. JQ1 reduced TNBC growth in vitro and in vivo and inhibited xenograft vascularization. These findings identify that BETi dually targets angiogenesis and the hypoxic response, an effective combination at reducing tumour growth in preclinical studies.

Disruption of the intricate gene expression program represents one of major driving factors for the development, progression and maintenance of human cancer, and is often associated with acquired therapeutic resistance. At the molecular level, cancerous phenotypes are the outcome of cellular functions of critical genes, regulatory interactions of histones and chromatin remodeling complexes in response to dynamic and persistent upstream signals. A large body of genetic and biochemical evidence suggests that the chromatin remodelers integrate the extracellular and cytoplasmic signals to control gene activity. Consequently, widespread dysregulation of chromatin remodelers and the resulting inappropriate expression of regulatory genes, together, lead to oncogenesis. We summarize the recent developments and current state of the dysregulation of the chromatin remodeling components as the driving mechanism underlying the growth and progression of human tumors. Because chromatin remodelers, modifying enzymes and protein–protein interactions participate in interpreting the epigenetic code, selective chromatin remodelers and bromodomains have emerged as new frontiers for pharmacological intervention to develop future anti-cancer strategies to be used either as single-agent or in combination therapies with chemotherapeutics or radiotherapy.

SolanumL. section PetotaDumort., which includes the cultivated potato (S. tuberosumL.) and its wild relatives, has been the subject of intensive taxonomic research in the last 25 years. The last comprehensive taxonomic treatment by Hawkes (1990) recognized seven cultivated and 225 wild species, yet the latest estimate is four cultivated and about 100 wild species. These changes have all involved synonymy of preexisting names, but our intensive nomenclatural research in the mega-diverse Solanum(1000+ species) revealed a name, Solanum stipuloideumRusby, that is the earlier valid name for the species long known as Solanum circaeifoliumBitter. Because this latter name is frequently used in the literature we here present a taxonomic treatment of S. stipuloideumwith complete synonymy in order to clarify nomenclature and species circumscription for this common species; this study also highlights the value of genus-wide taxonomic treatments.

ABSTRACTThe identification of molecular markers for economically significant traits should greatly improve the speed and efficiency of all peanut (Arachis hypogaeaL.) breeding programs. Development and phenotypic evaluation of recombinant inbred line (RIL) populations of peanut, along with molecular genotyping, will be essential for association of markers with traits. The primary objectives of this research were to develop 16 structured RIL populations that can be used by the peanut research community, and to begin high-resolution phenotyping of these populations. Crosses were made using a 2 by 8 (common by unique) factorial nested association mapping design. Parents were selected to attempt to maximize genetic diversity while meeting practical breeding objectives. First, two modern runner cultivars (Tifrunner and Florida-07) were selected as common parents because runner cultivars account for about 80 of the production in the U.S. Second, the eight unique parents were selected to supply diversity across market classes and botanical varieties and are donors of favorable alleles for enhancing drought tolerance and resistance to most important disease of peanut in the U.S. The eight unique parents are N08082olJCT (a Bailey derived high oleic breeding line), C76-16, NC 3033, SPT 06-06, SSD 6 (PI 576638), OLin, New Mexico Valencia A, and Florunner. The 16 populations were advanced using summer and winter nurseries. Input from multiple disciplines has resulted in a prioritized list of populations and traits that should be examined, and seed increase has begun to provide the community with material for extensive phenotyping. In-depth phenotyping and genotyping of these populations should result in markers that can be deployed by breeding programs for the development of improved cultivars.

Abstract: Nowadays, wire-arc spraying of chromium steel has gained an important market share for corrosion and wear protection applications. However, detailed studies are the basis for further process optimization. In order to optimize the process parameters and to evaluate the effects of the spray parameters DoE-based experiments had been carried out with high-speed camera shoots. In this article, the effects of spray current, voltage, and atomizing gas pressure on the particle jet properties, mean particle velocity and mean particle temperature and plume width on X46Cr13 wire are presented using an online process monitoring device. Moreover, the properties of the coatings concerning the morphology, composition and phase formation were subject of the investigations using SEM, EDX, and XRD-analysis. These deep investigations allow a defined verification of the influence of process parameters on spray plume and coating properties and are the basis for further process optimization.

The kinase PIM-1 plays a pivotal role in cytokine signaling and is implicated in the development of a number of tumors. The three-dimensional structure of PIM-1 is characterized by an unique hinge region which lacks a second hydrogen bond donor and makes it particularly important to determine how inhibitors bind to this kinase. We determined the structures of PIM-1 in complex with bisindolylmaleimide (BIM-1) and established the structure−activity relationship (SAR) for this inhibitor class. In addition, we screened a kinase targeted library and identified a number of high affinity inhibitors of PIM-1 such as imidazo[1,2-b]pyridazines, pyrazolo[1,5-a]pyrimidines, and members of the flavonoid family. In this paper we present an initial SAR of the identified scaffolds determined on the basis of a thermostability shift assay, calorimetric binding data, and biochemical assays which may find applications for the treatment of PIM-1 dependent cancer types.

By treatment with Woollins' reagent in toluene solution, carboxylic acids are converted to selenocarboxylic acids. The latter react in situ to provide new products of acid- or base-promoted substitution, addition, and amidation.

As we enter the twenty–first century, the demand for taxonomy is greater than ever before. The global imperative for the conservation of biodiversity has brought into sharp focus both the need for and the needs of taxonomic research. Although commentators today frequently, and rightly, stress how comparatively little we know about the diversity of organisms with which we share our planet, we should not lose sight of the enormous progress that has been made in cataloguing, describing and understanding the Earth's plants, animals and microbes. This research programme was probably born among our hunter–gatherer ancestors, baptized by the Greeks, and then reached maturity in post–Enlightenment Europe with the Linnaean revolution. Its success has been due to the legion of naturalists and taxonomists who have explored every nook and cranny of the living world, and to a series of rules and protocols that have allowed this massive corpus of knowledge to be organized and arranged in an age of paper and post. Any student of biology in the nineteenth century, and for perhaps the first three–quarters of the twentieth century, would have spent a large fraction of his or her time learning systematic biology, and the tools and techniques needed to identify and describe various elements of the flora and fauna. Systematic biology and taxonomy was the fertile soil in which emerging sciences such as physiology, genetics, ecology and evolutionary biology had their roots and drew nourishment.

Hepatitis C infection is characterised by three key features, which are the consequence of a complex interaction between genetic determinants of immune and other host factors and viral characteristics:1A high rate of viral persistence after acute infection resulting from a combination of weak T cell responsiveness and specific viral mechanisms of immune escape.2Marked interindividual variability in end-organ damage (fibrosis and cirrhosis), probably due to host genetic polymorphisms in genes governing the immune response and fibrosis pathways in addition to viral pathogenicity factors.3Significant resistance to antiviral therapies. Viral mechanisms of antiviral resistance parallel those of viral persistence, and include the intriguing possibility that hepatitis C may infect immunologically privileged sites such as the central nervous system.

Water−ligand observed via gradient spectroscopy (WaterLOGSY) represents a powerful method for primary NMR screening in the identification of compounds interacting with macromolecules, including proteins and DNA or RNA fragments. The method is useful for the detection of compounds binding to a receptor with binding affinity in the micromolar range. The Achille's heel of the method, as with all the techniques that detect the ligand resonances, is its inability to identify strong ligands with slow dissociation rates. We show here that the use of a reference compound with a known KD in the micromolar range together with properly designed competition binding experiments (c-WaterLOGSY) permits the detection of strong binders. A derived mathematical expression is used for the selection of the appropriate setup NMR experimental conditions and for an approximate determination of the binding constant. The experiment requires low ligand concentration, therefore allowing its application in the identification of potential strong inhibitors that are only marginally soluble. The technique is particularly suitable for rapid screening of chemical mixtures and plant or fungi extracts.

The synthesis of the liposidomycin degradation product 4 from d-glucose establishes its stereochemistry as 5‘S,6‘S and, by incorporation of the earlier diazepanone relative stereochemical assignment, establishes the absolute stereochemistry of the liposidomycins 1 and 2 as 5‘S,6‘S,2‘ ‘‘S,3‘‘‘S.

The formation of a complex between β-catenin and members of the TCF/LEF family of high-mobility group proteins is a key regulatory event in the wnt-signaling pathway, essential for embryonal development as well as the growth of normal and malignant colon epithelium. We have characterized the binding of TCF4 to human β-catenin by steady-state intrinsic fluorescence quenching experiments, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding studies in solution and in heterogeneous phase showed that TCF4 binds reversibly to β-catenin with an affinity (K_B) of 3(±1) 10⁸ M⁻¹. Site-directed mutagenesis, together with calorimetric measurements, revealed that residue D16 in TCF4 plays a crucial role in high-affinity binding. Mutation of this residue to alanine resulted in a decrease of K_B by two orders of magnitude as well as a significant reduction in binding enthalpy. Binding of TCF4 to β-catenin gave rise to a large negative enthalpy change at 25 °C (−29.7 kcal/mol). Binding enthalpies were strongly temperature dependent, which resulted in the determination of a large heat capacity change upon binding of −1.5 kcal/(mol K). The molecular events that take place upon complex formation are discussed using the measured thermodynamic data together with the crystal structure of the β-catenin arm repeat region/TCF complex. Copyright 2001 Academic Press

The resolved piperidinecarboxylate (R)-7 was converted to siastatin B (1) by an efficient and stereoselective sequence that includes a bromo-β-lactonization and an N-acyliminium azidation. Two analogues (3 and 4) of siastatin were also prepared.

Leukocyte interactions with endothelial cells play an important role during inflammatory processes. Leukocytes pass a monolayer of endothelial cells (ECM) to migrate into the extravascular space. The aim of the current study was to investigate whether prostaglandin E1(PGE1) influences the process of leukocyte migration. In a prospective controlled study, the influence of prostaglandin E1(50–5000 ng/mL) on leukocyte migration through endothelial cell monolayers (n= 7) was investigated. Human umbilical endothelial cells (HUVEC) and/or leukocytes were preincubated with clinically relevant, higher, and lower concentrations of prostaglandin E1and the amount of leukocyte migration after 3 h was measured. HUVEC were cultured on microporous membrane filters until achievement of a monolayer for investigation of leukocyte migration. Polymorphonuclear leukocytes (PMNL) were isolated from healthy volunteers and PMNL migration was studied under the influence of PGE1. In clinically relevant concentrations, PGE1was able to increase significantly leukocyte migration through endothelial cell monolayers (205 ± 7.8%, P< 0.05 compared to control; when treating PMNL alone, migration rate was 120 ± 9.2% compared to control, ns; only endothelial cell monolayers treated up to 145 ± 10.2%, P< 0.05 compared to control) showing a dose-dependent effect. In this assay, both cell types (PMNL and ECM) could be treated simultaneously, simulating the clinical situation after an iv administration. In conclusion, PGE1is able to increase leukocyte migration through endothelial cell monolayers when both cell types are pretreated. The treatment of either leukocytes or endothelial cell monolayers in the cell coculture showed no significant increase. These findings support the theory that prostaglandins may play a major role during inflammation. Future clinical studies are warranted to confirm this hypothesis.

Under basic conditions, phenyl 1,2-dithio-α-d-mannopyranoside forms a glycal-1,2-episulfide, which undergoes controlled oligomerization to afford a family of thio-oligo-α-d-mannopyranosides in a single reaction. The episulfide can also be intercepted by added thiolates, which leads to other sorts of thioglycosides. These α-(1→2)-linked thio-mannopyranosides might have application as mimics of natural structures such as viral high-mannose glycoproteins or ManLAM.

Objective:To evaluate thrombogenicity of prothrombin complex concentrates (PCCs) in critically ill patients.¶Design:Prospective clinical study.¶Setting:Medical intensive care unit at a university hospital.¶Patients:16 consecutive patients suffering from acquired deficiencies of coagulation factors and with either overt bleeding from any site or a planned invasive procedure.¶Interventions:2000 factor IX units of PCCs intravenously.¶Measurements and results:Prothrombin time (PT), activated partial prothrombin time, fibrinogen, platelet count, plasma levels of coagulation factors II, V, VII, VIII, IX, X, antithrombin, protein C, thrombin-antithrombin complex (TAT), prothrombin fragment F1+2, and the fibrin degradation product D-dimer were measured prior to and 1, 3, and 24 h after administration of PCCs. PT as well as coagulation factors II, VII, IX, and X, TAT, and F1+2showed a significant increase after administration of PCCs. All other parameters remained unchanged.¶Conclusions:Administration of PCCs induces thrombin generation. No evidence for induction of disseminated intravascular coagulation in biochemical terms could be found. When rapid correction of acquired coagulation factor disturbances is warranted, the use of PCCs seems reasonable, but the elevated risk of intravascular thrombus formation should be kept in mind.

Abstract:  Clinically apparent involvement of the central nervous system is a rare event in cases of disseminated histoplasmosis, even in HIV-infected persons. Despite therapy with amphotericin B, mortality remains very high. Reported here is the case of an HIV-infected patient with a 3-month history of fever, cough, weight loss and miliary lung infiltrates. Four weeks after initiation of tuberculostatic therapy, high-grade fever, neurological symptoms, personality changes and respiratory deterioration occurred. Magnetic resonance imaging of the brain showed multiple mass lesions, and a chest radiograph revealed worsening of pulmonary infiltrates. Methenamine silver staining of a lung biopsy specimen demonstrated Histoplasma capsulatum. Subsequently, this pathogen was cultured from lavage fluid. Following high-dose intravenous fluconazole therapy (800 mg once daily), the patient's condition improved markedly within 10 days, followed by an almost complete resolution of pulmonary and cerebral mass lesions. This is believed to be the first documented case of rapid improvement of disseminated histoplasmosis with central nervous system involvement in an HIV-infected patient upon induction of therapy with fluconazole.

Abstract: Thermodynamic aspects of protein stabilization by two widespread naturally occurring osmolytes, β-hydroxyectoine and betaine, were studied using differential scanning calorimetry (DSC) and bovine ribonuclease A (RNase A) as a model protein. The osmolyte β-hydroxyectoine purified from Marinococcus was found to be a very efficient stabilizer. At a concentration of 3 M it increased the melting temperature of RNase A (T m ) by more than 12 K and gave rise to a stability increase of 10.6 kJ/mol at room temperature. The heat capacity difference between the folded and unfolded state (ΔC p ) was found to be significantly increased. Betaine stabilized RNase A only at concentrations less than 3 M. Also, here ΔC p was found to be increased. Calculation of the number of water molecules that additionally bind to unfolded RNase A resulted in surprisingly low numbers for both osmolytes. The significant stabilization of RNase A by β-hydroxyectoine makes this osmolyte an interesting stabilizer in biotechnological processes in which enzymes are applied in the presence of denaturants or at high temperature.

SH2 domains are phosphotyrosine specific interaction modules with largely overlapping sequence specificities. A recent structure by Bae et al. revealed that SH2 domain specificity can be mediated by secondary binding sites located outside the phosphotyrosine binding pocket.

Recessive genetic male‐sterility has been widely used to facilitate crossing in recurrent selection programs in autogamous crop species. Dominant genetic male‐sterility, on the other hand, has not been widely used. Mass and half‐sib family selection schemes have been described for the dominant male‐sterile program. Our objective was to describe S1family recurrent selection schemes and expected selection responses for the dominant male‐sterile program. There is no mechanism for intermating selected S1families in a dominant male‐sterile program, other than manual emasculation and pollination, because S1families are completely fertile. We proposed various S1family recurrent selection schemes where intermating is done between selected S1families and a male‐sterile allele source as a means for overcoming this problem. These schemes include S1family recurrent selection using the reference population as the male‐sterile allele source and combined S1and half‐sib or S1and full‐sib family recurrent selection using half‐sib or full‐sib families, respectively, as the male‐sterile allele source. Expected selection responses were described for selection of the same or different traits in different family types for combined family type selection schemes. The strengths of S1family selection in a dominant genetic male‐sterile program are that complete S1family fertility eliminates the problem of male‐sterile phenotype pleiotropy and selection response per year is often greater for S1compared to mass or half‐sib family recurrent selection. The weakness of S1family selection in a dominant compared to a recessive male‐sterile program is that expected selection response is less than that for S1family selection in allogamous species where intermating is done strictly between S1selected families.

A new subtype (MVP-5180) of human immunodeficiency virus type 1 (HIV-1) was isolated from a Cameroonian AIDS patient. MVP-5180 was grown in several human T-cell lines and the monocytic U937 line. MVP-5180 DNA could not be amplified by nested primer PCR with conventional env primers and could be only very faintly amplified with gag and pol primers. Most German, Ivoirian, and Malawian anti-HIV-1 sera reacted faintly or moderately with Env proteins in an MVP-5180 immunoblot, whereas some Cameroonian sera reacted strongly. Of HIV-1-infected Cameroonians, 8% were identified by serological methods as infected with MVP-5180; 7% were positive when MVP-5180-specific PCR env primers were used. DNA sequence analysis of MVP-5180 showed that its genetic organization was that of HIV-1, with 65% similarity to HIV-1 and 56% similarity to HIV-2 consensus sequences. The env gene of MVP-5180 had similarities to HIV-1 and HIV-2 of 53 and of 49%, respectively. V3 loop analysis identified a crown of Gly-Pro-Met-Arg by using cloned DNA and Gly-Pro-Leu-Arg by using PCR-amplified DNA, neither of which configuration has been described for other HIV strains. In an analysis of relationships, MVP-5180 occupied a position distant to all other HIV-1 strains, including the chimpanzee simian immunodeficiency virus type 1 SIVcpz and the Uganda virus U455, and closer to the HIV-1/HIV-2 divergence node. MVP-5180, together with another Cameroonian isolate, ANT-70, constitutes a group subtype O of the most divergent HIV-1 isolates yet identified. Characterization of MVP-5180 is important for understanding the natural history of the primate immunodeficiency viruses and for the development of vaccines and diagnostics.