Biosynthesis-Assisted Structural Elucidation of the Bartolosides, Chlorinated Aromatic Glycolipids from Cyanobacteria

The isolation of the bartolosides, unprecedented cyanobacterial glycolipids featuring aliphatic chains with chlorine substituents and C-glycosyl moieties, is reported. Their chlorinated dialkylresorcinol (DAR) core presented a major structural-elucidation challenge. To overcome this, we discovered the bartoloside (brt) biosynthetic gene cluster and linked it to the natural products through in vitro characterization of the DAR-forming ketosynthase and aromatase. Bioinformatic analysis also revealed a novel potential halogenase. Knowledge of the bartoloside biosynthesis constrained the DAR core structure by defining key pathway intermediates, ultimately allowing us to determine the full structures of the bartolosides. This work illustrates the power of genomics to enable the use of biosynthetic information for structure elucidation.

The fine-scale architecture of structural variants in 17 mouse genomes.

BACKGROUND: Accurate catalogs of structural variants (SVs) in mammalian genomes are necessary to elucidate the potential mechanisms that drive SV formation and to assess their functional impact. Next generation sequencing methods for SV detection are an advance on array-based methods, but are almost exclusively limited to four basic types: deletions, insertions, inversions and copy number gains. RESULTS: By visual inspection of 100 Mbp of genome to which next generation sequence data from 17 inbred mouse strains had been aligned, we identify and interpret 21 paired-end mapping patterns, which we validate by PCR. These paired-end mapping patterns reveal a greater diversity and complexity in SVs than previously recognized. In addition, Sanger-based sequence analysis of 4,176 breakpoints at 261 SV sites reveal additional complexity at approximately a quarter of structural variants analyzed. We find micro-deletions and micro-insertions at SV breakpoints, ranging from 1 to 107 bp, and SNPs that extend breakpoint micro-homology and may catalyze SV formation. CONCLUSIONS: An integrative approach using experimental analyses to train computational SV calling is essential for the accurate resolution of the architecture of SVs. We find considerable complexity in SV formation; about a quarter of SVs in the mouse are composed of a complex mixture of deletion, insertion, inversion and copy number gain. Computational methods can be adapted to identify most paired-end mapping patterns.

Functional chromatin features are associated with structural mutations in cancer.

BACKGROUND: Structural mutations (SMs) play a major role in cancer development. In some cancers, such as breast and ovarian, DNA double-strand breaks (DSBs) occur more frequently in transcribed regions, while in other cancer types such as prostate, there is a consistent depletion of breakpoints in transcribed regions. Despite such regularity, little is understood about the mechanisms driving these effects. A few works have suggested that protein binding may be relevant, e.g. in studies of androgen receptor binding and active chromatin in specific cell types. We hypothesized that this behavior might be general, i.e. that correlation between protein-DNA binding (and open chromatin) and breakpoint locations is common across divergent cancers. RESULTS: We investigated this hypothesis by comprehensively analyzing the relationship among 457 ENCODE protein binding ChIP-seq experiments, 125 DnaseI and 24 FAIRE experiments, and 14,600 SMs from 8 diverse cancer datasets covering 147 samples. In most cancers, including breast and ovarian, we found enrichment of protein binding and open chromatin in the vicinity of SM breakpoints at distances up to 200 kb. Furthermore, for all cancer types we observed an enhanced enrichment in regions distant from genes when compared to regions proximal to genes, suggesting that the SM-induction mechanism is independent from the bias of DSBs to occur near transcribed regions. We also observed a stronger effect for sites with more than one protein bound. CONCLUSIONS: Protein binding and open chromatin state are associated with nearby SM breakpoints in many cancer datasets. These observations suggest a consistent mechanism underlying SM locations across different cancers.

Losing identity: structural diversity of transposable elements belonging to different classes in the genome of Anopheles gambiae

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide 0 side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Plant ion channels: gene families, physiology, and functional genomics analyses

Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization- and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide-gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport.

Hox cluster genomics in the horn shark, Heterodontus francisci

Reconstructing the evolutionary history of Hox cluster origins will lead to insights into the developmental and evolutionary significance of Hox gene clusters in vertebrate phylogeny and to their role in the origins of various vertebrate body plans. We have isolated two Hox clusters from the horn shark, Heterodontus francisci. These have been sequenced and compared with one another and with other chordate Hox clusters. The results show that one of the horn shark clusters (HoxM) is orthologous to the mammalian HoxA cluster and shows a structural similarity to the amphioxus cluster, whereas the other shark cluster (HoxN) is orthologous to the mammalian HoxD cluster based on cluster organization and a comparison with noncoding and Hox gene-coding sequences. The persistence of an identifiable HoxA cluster over an 800-million-year divergence time demonstrates that the Hox gene clusters are highly integrated and structured genetic entities. The data presented herein identify many noncoding sequence motifs conserved over 800 million years that may function as genetic control motifs essential to the developmental process.

Lung Age Is Related To Carotid Structural Alterations In Hypertensive Subjects.

Hypertensive patients exhibit higher cardiovascular risk and reduced lung function compared with the general population. Whether this association stems from the coexistence of two highly prevalent diseases or from direct or indirect links of pathophysiological mechanisms is presently unclear. This study investigated the association between lung function and carotid features in non-smoking hypertensive subjects with supposed normal lung function. Hypertensive patients (n = 67) were cross-sectionally evaluated by clinical, hemodynamic, laboratory, and carotid ultrasound analysis. Forced vital capacity, forced expired volume in 1 second and in 6 seconds, and lung age were estimated by spirometry. Subjects with ventilatory abnormalities according to current guidelines were excluded. Regression analysis adjusted for age and prior smoking history showed that lung age and the percentage of predicted spirometric parameters associated with common carotid intima-media thickness, diameter, and stiffness. Further analyses, adjusted for additional potential confounders, revealed that lung age was the spirometric parameter exhibiting the most significant regression coefficients with carotid features. Conversely, plasma C-reactive protein and matrix-metalloproteinases-2/9 levels did not influence this relationship. The present findings point toward lung age as a potential marker of vascular remodeling and indicate that lung and vascular remodeling might share common pathophysiological mechanisms in hypertensive subjects.

Structural Connectivity Of The Default Mode Network And Cognition In Alzheimer׳s Disease.

Disconnectivity between the Default Mode Network (DMN) nodes can cause clinical symptoms and cognitive deficits in Alzheimer׳s disease (AD). We aimed to examine the structural connectivity between DMN nodes, to verify the extent in which white matter disconnection affects cognitive performance. MRI data of 76 subjects (25 mild AD, 21 amnestic Mild Cognitive Impairment subjects and 30 controls) were acquired on a 3.0T scanner. ExploreDTI software (fractional Anisotropy threshold=0.25 and the angular threshold=60°) calculated axial, radial, and mean diffusivities, fractional anisotropy and streamline count. AD patients showed lower fractional anisotropy (P=0.01) and streamline count (P=0.029), and higher radial diffusivity (P=0.014) than controls in the cingulum. After correction for white matter atrophy, only fractional anisotropy and radial diffusivity remained significantly lower in AD compared to controls (P=0.003 and P=0.05). In the parahippocampal bundle, AD patients had lower mean and radial diffusivities (P=0.048 and P=0.013) compared to controls, from which only radial diffusivity survived for white matter adjustment (P=0.05). Regression models revealed that cognitive performance is also accounted for by white matter microstructural values. Structural connectivity within the DMN is important to the execution of high-complexity tasks, probably due to its relevant role in the integration of the network.

Electronic And Structural Study Of Pt-modified Au Vicinal Surfaces: A Model System For Pt-au Catalysts.

Two single crystalline surfaces of Au vicinal to the (111) plane were modified with Pt and studied using scanning tunneling microscopy (STM) and X-ray photoemission spectroscopy (XPS) in ultra-high vacuum environment. The vicinal surfaces studied are Au(332) and Au(887) and different Pt coverage (θPt) were deposited on each surface. From STM images we determine that Pt deposits on both surfaces as nanoislands with heights ranging from 1 ML to 3 ML depending on θPt. On both surfaces the early growth of Pt ad-islands occurs at the lower part of the step edge, with Pt ad-atoms being incorporated into the steps in some cases. XPS results indicate that partial alloying of Pt occurs at the interface at room temperature and at all coverage, as suggested by the negative chemical shift of Pt 4f core line, indicating an upward shift of the d-band center of the alloyed Pt. Also, the existence of a segregated Pt phase especially at higher coverage is detected by XPS. Sample annealing indicates that the temperature rise promotes a further incorporation of Pt atoms into the Au substrate as supported by STM and XPS results. Additionally, the catalytic activity of different PtAu systems reported in the literature for some electrochemical reactions is discussed considering our findings.

A New Platinum Complex With Tryptophan: Synthesis, Structural Characterization, Dft Studies And Biological Assays In Vitro Over Human Tumorigenic Cells.

A new platinum(II) complex with the amino acid L-tryptophan (trp), named Pt-trp, was synthesized and characterized. Elemental, thermogravimetric and ESI-QTOF mass spectrometric analyses led to the composition [Pt(C11H11N2O2)2]⋅6H2O. Infrared spectroscopic data indicate the coordination of trp to Pt(II) through the oxygen of the carboxylate group and also through the nitrogen atom of the amino group. The (13)C CP/MAS NMR spectroscopic data confirm coordination through the oxygen atom of the carboxylate group, while the (15)N CP/MAS NMR data confirm coordination of the nitrogen of the NH2 group to the metal. Density functional theory (DFT) studies were applied to evaluate the cis and trans coordination modes of trp to platinum(II). The trans isomer was shown to be energetically more stable than the cis one. The Pt-trp complex was evaluated as a cytotoxic agent against SK-Mel 103 (human melanoma) and Panc-1 (human pancreatic carcinoma) cell lines. The complex was shown to be cytotoxic over the considered cells.

Structural And Biomechanical Changes In The Achilles Tendon After Chronic Treatment With Statins.

Cases of tendinopathy and tendon ruptures have been reported as side effects associated with statin therapy. This work assessed possible changes in the structural and biomechanical properties of the tendons after chronic treatment with statins. Wistar rats were divided into the following groups: treated with atorvastatin (A-20 and A-80), simvastatin (S-20 and S-80) and the group that received no treatment (C). The doses of statins were calculated using allometric scaling, based on the doses of 80 mg/day and 20 mg/day recommended for humans. The morphological aspect of the tendons in A-20, S-20 and S-80 presented signals consistent with degeneration. Both the groups A-80 and S-80 showed a less pronounced metachromasia in the compression region of the tendons. Measurements of birefringence showed that A-20, A-80 and S-80 groups had a lower degree of organization of the collagen fibers. In all of the groups treated with statins, the thickness of the epitenon was thinner when compared to the C group. In the biomechanical tests the tendons of the groups A-20, A-80 and S-20 were less resistant to rupture. Therefore, statins affected the organization of the collagen fibers and decreased the biomechanical strength of the tendons, making them more predisposed to ruptures.

Kinase Inhibitor Profile For Human Nek1, Nek6, And Nek7 And Analysis Of The Structural Basis For Inhibitor Specificity.

Human Neks are a conserved protein kinase family related to cell cycle progression and cell division and are considered potential drug targets for the treatment of cancer and other pathologies. We screened the activation loop mutant kinases hNek1 and hNek2, wild-type hNek7, and five hNek6 variants in different activation/phosphorylation statesand compared them against 85 compounds using thermal shift denaturation. We identified three compounds with significant Tm shifts: JNK Inhibitor II for hNek1(Δ262-1258)-(T162A), Isogranulatimide for hNek6(S206A), andGSK-3 Inhibitor XIII for hNek7wt. Each one of these compounds was also validated by reducing the kinases activity by at least 25%. The binding sites for these compounds were identified by in silico docking at the ATP-binding site of the respective hNeks. Potential inhibitors were first screened by thermal shift assays, had their efficiency tested by a kinase assay, and were finally analyzed by molecular docking. Our findings corroborate the idea of ATP-competitive inhibition for hNek1 and hNek6 and suggest a novel non-competitive inhibition for hNek7 in regard to GSK-3 Inhibitor XIII. Our results demonstrate that our approach is useful for finding promising general and specific hNekscandidate inhibitors, which may also function as scaffolds to design more potent and selective inhibitors.

Chemical And Structural Analyses Of Titanium Plates Retrieved From Patients.

The aim of this study was to evaluate the microscopic structure and chemical composition of titanium bone plates and screws retrieved from patients with a clinical indication and to relate the results to the clinical conditions associated with the removal of these devices. Osteosynthesis plates and screws retrieved from 30 patients between January 2010 and September 2013 were studied by metallographic, gas, and energy dispersive X-ray (EDX) analyses and the medical records of these patients were reviewed. Forty-eight plates and 238 screws were retrieved. The time elapsed between plate and screw insertion and removal ranged between 11 days and 10 years. Metallographic analysis revealed that all the plates were manufactured from commercially pure titanium (CP-Ti). The screw samples analyzed consisted of Ti-6Al-4V alloy, except four samples, which consisted of CP-Ti. Titanium plates studied by EDX analysis presented greater than 99.7% titanium by mass. On gas analysis of Ti-6Al-4V screws, three samples were outside the standard values. One CP-Ti screw sample and one plate sample also presented an oxygen analysis value above the standard. The results indicated that the physical properties and chemical compositions of the plates and screws did not correspond with the need to remove these devices or the time of retention.

Towards A Deeper Understanding Of Structural Biomass Recalcitrance Using Phase-contrast Tomography.

The development of technological routes to convert lignocellulosic biomass to liquid fuels requires an in-depth understanding of the cell wall architecture of substrates. Essential pretreatment processes are conducted to reduce biomass recalcitrance and usually increase the reactive surface area. Quantitative three-dimensional information about both bulk and surface structural features of substrates needs to be obtained to expand our knowledge of substrates. In this work, phase-contrast tomography (PCT) was used to gather information about the structure of a model lignocellulosic biomass (piassava fibers). The three-dimensional cellular organization of piassava fibers was characterized by PCT using synchrotron radiation. This technique enabled important physical features that describe the substrate piassava fibers to be visualized and quantified. The external surface area of a fiber and internal surface area of the pores in a fiber could be determined separately. More than 96% of the overall surface area available to enzymes was in the bulk substrate. The pore surface area and length exhibited a positive linear relationship, where the slope of this relationship depended on the plant tissue. We demonstrated that PCT is a powerful tool for the three-dimensional characterization of the cell wall features related to biomass recalcitrance. Original and relevant quantitative information about the structural features of the analyzed material were obtained. The data obtained by PCT can be used to improve processing routes to efficiently convert biomass feedstock into sugars.