An unexpected link between fatty acid synthase and cholesterol synthesis in proinflammatory macrophage activation

Different immune activation states require distinct metabolic features and activities in immune cells. For instance, inhibition of fatty acid synthase (FASN), which catalyzes the synthesis of long-chain fatty acids, prevents the proinflammatory response in macrophages; however, the precise role of this enzyme in this response remains poorly defined. Consistent with previous studies, we found here that FASN is essential for lipopolysaccharide-induced, Toll-like receptor (TLR)-mediated macrophage activation. Interestingly, only agents that block FASN upstream of acetoacetyl-CoA synthesis, including the well-characterized FASN inhibitor C75, inhibited TLR4 signaling, while those acting downstream had no effect. We found that acetoacetyl-CoA could overcome C75's inhibitory effect, whereas other FASN metabolites, including palmitate, did not prevent C75-mediated inhibition. This suggested an unexpected role for acetoacetyl-CoA in inflammation that is independent of its role in palmitate synthesis. Our evidence further suggested that acetoacetyl-CoA arising from FASN activity promotes cholesterol production, indicating a surprising link between fatty acid synthesis and cholesterol synthesis. We further demonstrate that this process is required for TLR4 to enter lipid rafts and facilitate TLR4 signaling. In conclusion, we have uncovered an unexpected link between FASN and cholesterol synthesis that appears to be required for TLR signal transduction and proinflammatory macrophage activation

Application of geostatistical simulation models in the charac- terization of complex geological structures

Η χρήση μεθόδων προσομοίωσης στη γεωστατιστική μπορεί να οδηγήσει στην ανάπτυξη αριθμητικών μοντέλων χωρικής κατανομής συνεχών γεωλογικών μεταβλητών (περιεκτικότητα, πάχος, πυκνότητα, κλπ) ή κατηγορικών μεταβλητών (γεωλογικοί σχηματισμοί και λιθολογικές φάσεις ή τύποι πετρωμάτων). Στην παρούσα εργασία, η ανασκόπηση των κλασικών μεθόδων προσομοίωσης, όπως η Sequential Indicator Simulation (SIS), αναδεικνύει ένα σημαντικό μειονέκτημα που προκύπτει από τις θεωρητικές δυσκολίες στην ανάπτυξη ενός έγκυρου μοντέλου συνδιασποράς. Αντιθέτως, ένα παρόμοιο μοντέλο μπορεί να οριστεί αυτόματα στο πλαίσιο της Truncated Gaussian Method (TGS). Η μέθοδος αυτή βασίζεται στη δημιουργία κατηγορικών μεταβλητών μέσω της αποκοπής μίας πολλαπλά κανονικής τυχαίας μεταβλητής σε διάφορα όρια. Η Plurigaussian Simulation Method (PGS) αποτελεί επέκταση της προηγούμενης με τη διαφορά στην ταυτόχρονη αποκοπή περισσότερων της μίας τυχαίων μεταβλητών. Στη συνέχεια της εργασίας παρουσιάζεται μία εφαρμογή αυτής της μεθόδου στην πεδιάδα της Δυτικής Θεσσαλίας. Τα αποτελέσματα δείχνουν ότι η μέθοδος είναι αποτελεσματική στην αναπαραγωγή των χωρικών χαρακτηριστικών των διαφόρων λιθολογικών σχηματισμών και της κατανομής τους στο χώρο.Geostatistical simulation methods are able to generate numerical models or relations of the spatial distribution of a continuous geologic variable (grade, thickness, density, etc.) or a categorical variable (geological units and lithofacies or rock types). In this work, a review of traditional simulation techniques, as the Sequential Indicator Simulation (SIS), reveals a major pitfall that comes from theoretical difficulties in the development of a valid cross covariance model. On the contrary, a valid indicator cross covariance model is automatically defined in the framework of the Truncated Gaussian Simulation Method (TGS). This method is based on the concept that the categorical variables are obtained by truncating one standard multigaussian random variable at different thresholds. Plurigaussian Simulation Method (PGS) is an extension of the TGS Method but based on the simultaneous truncation of several multigaussian variables. An application of Plurigaussian method to simulate the lithofacies in the alluvial formations of the West Thessaly Basin is finally presented. This method was shown to be effective in reproducing the spatial characteristics of the different lithofacies and their distribution across the studied area

Application of geostatistical simulation models in the charac- terization of complex geological structures

Η χρήση μεθόδων προσομοίωσης στη γεωστατιστική μπορεί να οδηγήσει στην ανάπτυξη αριθμητικών μοντέλων χωρικής κατανομής συνεχών γεωλογικών μεταβλητών (περιεκτικότητα, πάχος, πυκνότητα, κλπ) ή κατηγορικών μεταβλητών (γεωλογικοί σχηματισμοί και λιθολογικές φάσεις ή τύποι πετρωμάτων). Στην παρούσα εργασία, η ανασκόπηση των κλασικών μεθόδων προσομοίωσης, όπως η Sequential Indicator Simulation (SIS), αναδεικνύει ένα σημαντικό μειονέκτημα που προκύπτει από τις θεωρητικές δυσκολίες στην ανάπτυξη ενός έγκυρου μοντέλου συνδιασποράς. Αντιθέτως, ένα παρόμοιο μοντέλο μπορεί να οριστεί αυτόματα στο πλαίσιο της Truncated Gaussian Method (TGS). Η μέθοδος αυτή βασίζεται στη δημιουργία κατηγορικών μεταβλητών μέσω της αποκοπής μίας πολλαπλά κανονικής τυχαίας μεταβλητής σε διάφορα όρια. Η Plurigaussian Simulation Method (PGS) αποτελεί επέκταση της προηγούμενης με τη διαφορά στην ταυτόχρονη αποκοπή περισσότερων της μίας τυχαίων μεταβλητών. Στη συνέχεια της εργασίας παρουσιάζεται μία εφαρμογή αυτής της μεθόδου στην πεδιάδα της Δυτικής Θεσσαλίας. Τα αποτελέσματα δείχνουν ότι η μέθοδος είναι αποτελεσματική στην αναπαραγωγή των χωρικών χαρακτηριστικών των διαφόρων λιθολογικών σχηματισμών και της κατανομής τους στο χώρο.Geostatistical simulation methods are able to generate numerical models or relations of the spatial distribution of a continuous geologic variable (grade, thickness, density, etc.) or a categorical variable (geological units and lithofacies or rock types). In this work, a review of traditional simulation techniques, as the Sequential Indicator Simulation (SIS), reveals a major pitfall that comes from theoretical difficulties in the development of a valid cross covariance model. On the contrary, a valid indicator cross covariance model is automatically defined in the framework of the Truncated Gaussian Simulation Method (TGS). This method is based on the concept that the categorical variables are obtained by truncating one standard multigaussian random variable at different thresholds. Plurigaussian Simulation Method (PGS) is an extension of the TGS Method but based on the simultaneous truncation of several multigaussian variables. An application of Plurigaussian method to simulate the lithofacies in the alluvial formations of the West Thessaly Basin is finally presented. This method was shown to be effective in reproducing the spatial characteristics of the different lithofacies and their distribution across the studied area

Circulation of Different Lineages of Dengue Virus 2, Genotype American/Asian in Brazil: Dynamics and Molecular and Phylogenetic Characterization

The American/Asian genotype of Dengue virus type 2 (DENV-2) was introduced into the Americas in the 80′s. Although there is no data showing when this genotype was first introduced into Brazil, it was first detected in Brazil in 1990. After which the virus spread throughout the country and major epidemics occurred in 1998, 2007/08 and 2010. In this study we sequenced 12 DENV-2 genomes obtained from serum samples of patients with dengue fever residing in São José do Rio Preto, São Paulo (SJRP/SP), Brazil, in 2008. The whole open reading frame or envelope sequences were used to perform phylogenetic, phylogeographic and evolutionary analyses. Isolates from SJRP/SP were grouped within one lineage (BR3) close to isolates from Rio de Janeiro, Brazil. Isolates from SJRP were probably introduced there at least in 2007, prior to its detection in the 2008 outbreak. DENV-2 circulation in Brazil is characterized by the introduction, displacement and circulation of three well-defined lineages in different times, most probably from the Caribbean. Thirty-seven unique amino acid substitutions were observed among the lineages, including seven amino acid differences in domains I to III of the envelope protein. Moreover, we dated here, for the first time, the introduction of American/Asian genotype into Brazil (lineage BR1) to 1988/89, followed by the introduction of lineages BR2 (1998–2000) and BR3 (2003–05). Our results show a delay between the introduction and detection of DENV-2 lineages in Brazil, reinforcing the importance and need for surveillance programs to detect and trace the evolution of these viruses. Additionally, Brazilian DENV-2 differed in genetic diversity, date of introduction and geographic origin and distribution in Brazil, and these are important factors for the evolution, dynamics and control of dengue.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Grant )Fundação de Amparo à Pesquisa do Estado de São PauloFundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG grant

Sulfated Polysaccharide, Curdlan Sulfate, Efficiently Prevents Entry/Fusion and Restricts Antibody-Dependent Enhancement of Dengue Virus Infection In Vitro: A Possible Candidate for Clinical Application

10.1371/journal.pntd.0002188PLoS Neglected Tropical Diseases74

Redox-switch regulatory mechanism of thiolase from Clostridium acetobutylicum

Thiolase is the first enzyme catalysing the condensation of two acetyl-coenzyme A (CoA) molecules to form acetoacetyl-CoA in a dedicated pathway towards the biosynthesis of n-butanol, an important solvent and biofuel. Here we elucidate the crystal structure of Clostridium acetobutylicum thiolase (CaTHL) in its reduced/oxidized states. CaTHL, unlike those from other aerobic bacteria such as Escherichia coli and Zoogloea ramegera, is regulated by the redox-switch modulation through reversible disulfide bond formation between two catalytic cysteine residues, Cys88 and Cys378. When CaTHL is overexpressed in wild-type C. acetobutylicum, butanol production is reduced due to the disturbance of acidogenic to solventogenic shift. The CaTHLV77Q/N153Y/A286K mutant, which is not able to form disulfide bonds, exhibits higher activity than wild-type CaTHL, and enhances butanol production upon overexpression. On the basis of these results, we suggest that CaTHL functions as a key enzyme in the regulation of the main metabolism of C. acetobutylicum through a redox-switch regulatory mechanism.close0

Visualization of membrane protein domains by cryo-electron microscopy of dengue virus

Improved technology for reconstructing cryo-electron microscopy (cryo-EM) images has now made it possible to determine secondary structural features of membrane proteins in enveloped viruses. The structure of mature dengue virus particles was determined to a resolution of 9.5 Å by cryo-EM and image reconstruction techniques, establishing the secondary structural disposition of the 180 envelope (E) and 180 membrane (M) proteins in the lipid envelope. The ɑ-helical 'stem' regions of the E molecules, as well as part of the N-terminal section of the M proteins, are buried in the outer leaflet of the viral membrane. The 'anchor' regions of E and the M proteins each form antiparallel E-E and M-M transmembrane alpha-helices, leaving their C termini on the exterior of the viral membrane, consistent with the predicted topology of the unprocessed polyprotein. This is one of only a few determinations of the disposition of transmembrane proteins in situ and shows that the nucleocapsid core and envelope proteins do not have a direct interaction in the mature virus

Structural studies of viperin, an antiviral radical SAM enzyme.

Viperin is an IFN-inducible radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S-adenosylhomocysteine (SAH) or 5'-deoxyadenosine (5'-dAdo) and l-methionine (l-Met). Viperin contains a partial (βα)6-barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first β-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5'-dAdo and l-Met (SAM cleavage products) is consistent with the canonical mechanism of 5'-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTP-binding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.Wellcome Trust NIH (USA

Characterization of a dengue type-specific epitope on dengue 3 virus envelope protein domain III

Dengue virus (DENV) is a mosquito-borne disease caused by four genetically and serologically related viruses termed DENV-1, -2, -3 and -4. The DENV envelope (E) protein ectodomain can be divided into three structural domains designated ED1, ED2 and ED3. The ED3 domain contains DENV type-specific and DENV complex-reactive antigenic sites. To date, nearly all antigenic studies on the E protein have focused on DENV-2. In this study, the epitope recognized by a DENV-3 type-specific monoclonal antibody (mAb 14A4-8) was mapped to the DENV-3 ED3 domain using a combination of physical and biological techniques. Epitope mapping revealed that amino acid residues V305, L306, K308, E309, V310, K325, A329, G381 and I387 were critical for the binding of mAb 14A4-8 and amino acid residues T303, K307, K386, W389 and R391 were peripheral residues for this epitope. The location of the mAb 14A4-8 epitope overlaps with the DENV complex-reactive antigenic site in the DENV-3 ED3 domain

Capturing a Flavivirus Pre-Fusion Intermediate

During cell entry of flaviviruses, low endosomal pH triggers the rearrangement of the viral surface glycoproteins to a fusion-active state that allows the release of the infectious RNA into the cytoplasm. In this work, West Nile virus was complexed with Fab fragments of the neutralizing mAb E16 and was subsequently exposed to low pH, trapping the virions in a pre-fusion intermediate state. The structure of the complex was studied by cryo-electron microscopy and provides the first structural glimpse of a flavivirus fusion intermediate near physiological conditions. A radial expansion of the outer protein layer of the virion was observed compared to the structure at pH 8. The resulting ∼60 Å-wide shell of low density between lipid bilayer and outer protein layer is likely traversed by the stem region of the E glycoprotein. By using antibody fragments, we have captured a structural intermediate of a virus that likely occurs during cell entry. The trapping of structural transition states by antibody fragments will be applicable for other processes in the flavivirus life cycle and delineating other cellular events that involve conformational rearrangements