The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling.

The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450-CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate-enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies.

Structural and mean level analyses of the Five-Factor Model and Locus of Control: Further evidence from Africa

The present study examines the Five-Factor Model (FFM) of personality and locus of control in French-speaking samples in Burkina Faso (N = 470) and Switzerland (Ns = 1,090, 361), using the Revised NEO Personality Inventory (NEO-PI-R) and Levenson's Internality, Powerful others, and Chance (IPC) scales. Alpha reliabilities were consistently lower in Burkina Faso, but the factor structure of the NEO-PI-R was replicated in both cultures. The intended three-factor structure of the IPC could not be replicated, although a two-factor solution was replicable across the two samples. Although scalar equivalence has not been demonstrated, mean level comparisons showed the hypothesized effects for most of the five factors and locus of control; Burkinabè scored higher in Neuroticism than anticipated. Findings from this African sample generally replicate earlier results from Asian and Western cultures, and are consistent with a biologically-based theory of personality.

Prokaryotic cells: structural organisation of the cytoskeleton and organelles

For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i) tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii) actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii) intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.

Structural components in functional data

Analyzing functional data often leads to finding common factors, for which functional principal component analysis proves to be a useful tool to summarize and characterize the random variation in a function space. The representation in terms of eigenfunctions is optimal in the sense of L-2 approximation. However, the eigenfunctions are not always directed towards an interesting and interpretable direction in the context of functional data and thus could obscure the underlying structure. To overcome such difficulty, an alternative to functional principal component analysis is proposed that produces directed components which may be more informative and easier to interpret. These structural components are similar to principal components, but are adapted to situations in which the domain of the function may be decomposed into disjoint intervals such that there is effectively independence between intervals and positive correlation within intervals. The approach is demonstrated with synthetic examples as well as real data. Properties for special cases are also studied.

Metabolic and structural rearrangement during dark-induced autophagy in soybean (Glycine max L.) nodules: an electron microscopy and 31P and 13C nuclear magnetic resonance study.

The effects of dark-induced stress on the evolution of the soluble metabolites present in senescent soybean (Glycine max L.) nodules were analysed in vitro using (13)C- and (31)P-NMR spectroscopy. Sucrose and trehalose were the predominant soluble storage carbons. During dark-induced stress, a decline in sugars and some key glycolytic metabolites was observed. Whereas 84% of the sucrose disappeared, only one-half of the trehalose was utilised. This decline coincides with the depletion of Gln, Asn, Ala and with an accumulation of ureides, which reflect a huge reduction of the N(2) fixation. Concomitantly, phosphodiesters and compounds like P-choline, a good marker of membrane phospholipids hydrolysis and cell autophagy, accumulated in the nodules. An autophagic process was confirmed by the decrease in cell fatty acid content. In addition, a slight increase in unsaturated fatty acids (oleic and linoleic acids) was observed, probably as a response to peroxidation reactions. Electron microscopy analysis revealed that, despite membranes dismantling, most of the bacteroids seem to be structurally intact. Taken together, our results show that the carbohydrate starvation induced in soybean by dark stress triggers a profound metabolic and structural rearrangement in the infected cells of soybean nodule which is representative of symbiotic cessation.

The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.

Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly.

Non-structural protein 2 (NS2) plays an important role in hepatitis C virus (HCV) assembly, but neither the exact contribution of this protein to the assembly process nor its complete structure are known. In this study we used a combination of genetic, biochemical and structural methods to decipher the role of NS2 in infectious virus particle formation. A large panel of NS2 mutations targeting the N-terminal membrane binding region was generated. They were selected based on a membrane topology model that we established by determining the NMR structures of N-terminal NS2 transmembrane segments. Mutants affected in virion assembly, but not RNA replication, were selected for pseudoreversion in cell culture. Rescue mutations restoring virus assembly to various degrees emerged in E2, p7, NS3 and NS2 itself arguing for an interaction between these proteins. To confirm this assumption we developed a fully functional JFH1 genome expressing an N-terminally tagged NS2 demonstrating efficient pull-down of NS2 with p7, E2 and NS3 and, to a lower extent, NS5A. Several of the mutations blocking virus assembly disrupted some of these interactions that were restored to various degrees by those pseudoreversions that also restored assembly. Immunofluorescence analyses revealed a time-dependent NS2 colocalization with E2 at sites close to lipid droplets (LDs) together with NS3 and NS5A. Importantly, NS2 of a mutant defective in assembly abrogates NS2 colocalization around LDs with E2 and NS3, which is restored by a pseudoreversion in p7, whereas NS5A is recruited to LDs in an NS2-independent manner. In conclusion, our results suggest that NS2 orchestrates HCV particle formation by participation in multiple protein-protein interactions required for their recruitment to assembly sites in close proximity of LDs.

The Tertiary structural and thermal evolution of the central Alps - Compressional and extensional structures in an orogenic belt

The Western Alpine Are has been created during the Cretaceous and the Tertiary orogenies. The interference patterns of the Tertiary structures suggest their formation during continental collision of the European and the Adriatic Plates, with an accompanying anticlockwise rotation of the Adriatic indenter. Extensional structures are mainly related to ductile deformation by simple shear. These structures developed at a deep tectonic level, in granitic crustal rocks, at depths in excess of 10 km. In the early Palaeogene period of the Tertiary Orogeny, the main Tertiary nappe emplacement resulted from a NW-thrusting of the Austroalpine, Penninic and Helvetic nappes. Heating of the deep zone of the Upper Cretaceous and Tertiary nappe stack by geothermal heat flow is responsible for the Tertiary regional metamorphism, reaching amphibolite-facies conditions in the Lepontine Gneiss Dome (geothermal gradient 25 degrees C/ km). The Tertiary thrusting occurred mainly during prograde metamorphic conditions with creation of a penetrative NW-SE-oriented stretching lineation, X(1) (finite extension), parallel to the direction of simple shear. Earliest cooling after the culmination of the Tertiary metamorphism, some 38 Ma ago, is recorded by the cooling curves of the Monte Rosa and Mischabel nappes to the west and the Suretta Nappe to the east of the Lepontine Gneiss Dome. The onset of dextral transpression, with a strong extension parallel to the mountain belt, and the oldest S-vergent `'backfolding'' took place some 35 to 30 Ma ago during retrograde amphibolite-facies conditions and before the intrusion of the Oligocene dikes north of the Periadriatic Line. The main updoming of the Lepontine Gneiss Dome started some 32-30 Ma ago with the intrusion of the Bergell tonalites and granodiorites, concomitant with S-vergent backfolding and backthrusting and dextral strike-slip movements along the Tonale and Canavese Lines (Argand's Insubric phase). Subsequently, the center of main updoming migrated slowly to the west, reaching the Simplon region some 20 Ma ago. This was contemporaneous with the westward migration of the Adriatic indenter. Between 20 Ma and the present, the Western Aar Massif-Toce culmination was the center of strong uplift. The youngest S-vergent backfolds, the Glishorn anticline and the Berisal syncline fold the 12 Ma Rb/Sr biotite isochron and are cut by the 11 Ma old Rhone-Simplon Line. The discrete Rhone-Simplon Line represents a late retrograde manifestation in the preexisting ductile Simplon Shear Zone. This fault zone is still active today. The Oligocene-Neogene dextral transpression and extension in the Simplon area were concurrent with thrusting to the northwest of the Helvetic nappes, the Prealpes (35-15 Ma) and with the Jura thin-skinned thrust (11-3 Ma). It was also contemporaneous with thrusting to the south of the Bergamasc (> 35-5 Ma) and Milan thrusts (16-5 Ma).

Structural and functional brain changes in middle-aged type 2 diabetic patients: a cross-sectional study.

BACKGROUND Type 2 diabetes mellitus (T2DM) is an emerging risk factor for cognitive impairment. Whether this impairment is a direct effect of this metabolic disorder on brain function, a consequence of vascular disease, or both, remains unknown. Structural and functional neuroimaging studies in patients with T2DM could help to elucidate this question. OBJECTIVE We designed a cross-sectional study comparing 25 T2DM patients with 25 age- and gender-matched healthy control participants. Clinical information, APOE genotype, lipid and glucose analysis, structural cerebral magnetic resonance imaging including voxel-based morphometry, and F-18 fluorodeoxyglucose positron emission tomography were obtained in all subjects. METHODS Gray matter densities and metabolic differences between groups were analyzed using statistical parametric mapping. In addition to comparing the neuroimaging profiles of both groups, we correlated neuroimaging findings with HbA1c levels, duration of T2DM, and insulin resistance measurement (HOMA-IR) in the diabetic patients group. Results: Patients with T2DM presented reduced gray matter densities and reduced cerebral glucose metabolism in several fronto-temporal brain regions after controlling for various vascular risk factors. Furthermore, within the T2DM group, longer disease duration, and higher HbA1c levels and HOMA-IR were associated with lower gray matter density and reduced cerebral glucose metabolism in fronto-temporal regions. CONCLUSION In agreement with previous reports, our findings indicate that T2DM leads to structural and metabolic abnormalities in fronto-temporal areas. Furthermore, they suggest that these abnormalities are not entirely explained by the role of T2DM as a cardiovascular risk factor.

Identification of structural variation in mouse genomes.

Structural variation is variation in structure of DNA regions affecting DNA sequence length and/or orientation. It generally includes deletions, insertions, copy-number gains, inversions, and transposable elements. Traditionally, the identification of structural variation in genomes has been challenging. However, with the recent advances in high-throughput DNA sequencing and paired-end mapping (PEM) methods, the ability to identify structural variation and their respective association to human diseases has improved considerably. In this review, we describe our current knowledge of structural variation in the mouse, one of the prime model systems for studying human diseases and mammalian biology. We further present the evolutionary implications of structural variation on transposable elements. We conclude with future directions on the study of structural variation in mouse genomes that will increase our understanding of molecular architecture and functional consequences of structural variation.

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex.

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane alpha-helix that may be involved in intramembrane protein-protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix alpha(0), formed by NS3 residues 12-23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design.

Synorogenic extension in the Tethyan Himalaya documented by structural studies and the Kubler index, Lachung La area, NW India

Tectonic observations in the Tethyan Himalaya reveal an important extensional event that succeeds the emplacement of SW-verging nappes. A major thrust, called the Kum Tso Thrust, has been backfolded and reactivated by normal faulting associated with this event. Measurements of the Kubler index, coupled with characterization of clay-size paragenesis show the effect of normal faulting on the regional metamorphic zonation and indicate that important extension zones, like the Sarchu-Lachung La Normal Fault Zone (SLFZ), exist within the Tethyan Himalaya. Diagenetic limestones from within the SLFZ are characterized by the occurrence of mixed-layered clay phases, kaolinite and an illite with a 001 peak >0.4 Delta degrees2 theta. This zone is bordered by two anchizonal-to-epizonal zones, where illite peaks become narrower. Further to the NE the successive appearance of biotite, chloritoid, garnet and garnet-staurolite-kyanite assemblapes testifies to an increase in metamorphic grade. The cataclastic samples from the normal faults contain kaolinite, smectite and a `broad' illite, indicating that extension occurs under diagenetic conditions.

A functional and structural study of the major metalloprotease secreted by the pathogenic fungus Aspergillus fumigatus.

Fungalysins are secreted fungal peptidases with the ability to degrade the extracellular matrix proteins elastin and collagen and are thought to act as virulence factors in diseases caused by fungi. Fungalysins constitute a unique family among zinc-dependent peptidases that bears low sequence similarity to known bacterial peptidases of the thermolysin family. The crystal structure of the archetype of the fungalysin family, Aspergillus fumigatus metalloprotease (AfuMep), has been obtained for the first time. The 1.8 Å resolution structure of AfuMep corresponds to that of an autoproteolyzed proenzyme with separate polypeptide chains corresponding to the N-terminal prodomain in a binary complex with the C-terminal zinc-bound catalytic domain. The prodomain consists of a tandem of cystatin-like folds whose C-terminal end is buried into the active-site cleft of the catalytic domain. The catalytic domain harbouring the key catalytic zinc ion and its ligands, two histidines and one glutamic acid, undergoes a conspicuous rearrangement of its N-terminal end during maturation. One key positively charged amino-acid residue and the C-terminal disulfide bridge appear to contribute to its structural-functional properties. Thus, structural, biophysical and biochemical analysis were combined to provide a deeper comprehension of the underlying properties of A. fumigatus fungalysin, serving as a framework for the as yet poorly known metallopeptidases from pathogenic fungi.

X-ray structures of Sap1 and Sap5: structural comparison of the secreted aspartic proteinases from Candida albicans.

Proteolytic activity is an important virulence factor for Candida albicans (C. albicans). It is attributed to the family of the secreted aspartic proteinases (Saps) from C. albicans with a minimum of 10 members. Saps show controlled expression and regulation for the individual stages of the infection process. Distinct isoenzymes can be responsible for adherence and tissue damage of local infections, while others cause systemic diseases. Earlier, only the structures of Sap2 and Sap3 were known. In our research, we have now succeeded in solving the X-ray crystal structures of the apoenzyme of Sap1 and Sap5 in complex with pepstatin A at 2.05 and 2.5 A resolution, respectively. With the structure of Sap1, we have completed the set of structures of isoenzyme subgroup Sap1-3. Of subgroup Sap4-6, the structure of the enzyme Sap5 is the first structure that has been described up to now. This facilitates comparison of structural details as well as inhibitor binding modes among the different subgroup members. Structural analysis reveals a highly conserved overall secondary structure of Sap1-3 and Sap5. However, Sap5 clearly differs from Sap1-3 by its electrostatic overall charge as well as through structural conformation of its entrance to the active site cleft. Design of inhibitors specific for Sap5 should concentrate on the S4 and S3 pockets, which significantly differ from Sap1-3 in size and electrostatic charge. Both Sap1 and Sap5 seem to play a major part in superficial Candida infections. Determination of the isoenzymes' structures can contribute to the development of new Sap-specific inhibitors for the treatment of superficial infections with a structure-based drug design program.