Direct Structural Analysis Research Articles

Capillary electrophoresis of urinary prostate glycoproteins assists in the diagnosis of prostate cancer

Prostate marker assays are widely used for detection of prostate cancer (PCa) but are associated with considerable sensitivity and specificity problems. Therefore, we investigated prostatic protein glycosylation profiles as a potential biomarker. We determined the urinary asparagine-linked glycan (N-glycan) profile of prostatic proteins of healthy volunteers (n = 25), patients with benign prostate hyperplasia (BPH; n = 62) and newly diagnosed PCa patients (n = 42) using DNA-sequencer-assisted fluorophore-assisted carbohydrate electrophoresis. Through squeezing of the prostate, a sufficient amount of prostatic proteins was obtained for direct structural analyses of N-glycan structures. N-glycans of PCa compared to BPH were characterized by a significant decrease in triantennary structures (p = 0.047) and overall fucosylation (p = 0.026). Prostate-specific antigen (PSA) and the urinary glycoprofile marker showed comparable overall receiver operating characteristic curve analysis as well as in the diagnostic gray zone with serum PSA values between 4 and 10 μg/L. However, when combining PSA and the urinary glycoprofile marker, the latter gave an additive diagnostic value to serum PSA (p ≤ 0.001). In conclusion, N-glycosylation profiling demonstrated differences between BPH and PCa. These changes could lead to the discovery of a new biomarker for PCa.

The better model to predict and improve pediatric health care quality

The perpetual search for ways to improve pediatric health care quality has resulted in a multitude of assessments and strategies; however, there is little research evidence as to their conditions for maximum effectiveness. A major reason for the lack of evaluation research and successful quality improvement initiatives is the methodological challenge of measuring quality from the parent perspective. Comparison of performance-only and importance-performance models was done to determine the better predictor of pediatric health care quality and more successful method for improving the quality of care provided to children. Fourteen pediatric health care centers serving approximately 250,000 patients in 70,000 households in three West Central Florida counties were studied. A cross-sectional design was used to determine the importance and performance of 50 pediatric health care attributes and four global assessments of pediatric health care quality. Exploratory factor analysis revealed five dimensions of care (physician care, access, customer service, timeliness of services, and health care facility). Hierarchical multiple regression compared the performance-only and the importance-performance models. In-depth interviews, participant observations, and a direct cognitive structural analysis identified 50 health care attributes included in a mailed survey to parents(n = 1,030). The tailored design method guided survey development and data collection. The importance-performance multiplicative additive model was a better predictor of pediatric health care quality. Attribute importance moderates performance and quality, making the importance-performance model superior for measuring and providing a deeper understanding of pediatric health care quality and a better method for improving the quality of care provided to children. Regardless of attribute performance, if the level of attribute importance is not taken into consideration, health care organizations may spend valuable resources targeting the wrong areas for improvement. Consequently, this finding aids in health care quality research and policy decisions on organizational improvement strategies.

Characterization of mycobacterial triacylglycerols and monomeromycolyl diacylglycerols from Mycobacterium smegmatis biofilm by electrospray ionization multiple-stage and high-resolution mass spectrometry

The storage of triacylglycerols (TAGs) is essential for non-replicating persistence relevant to survival and the re-growth of mycobacteria during their exit from non-replicating state stress conditions. However, the detailed structures of this lipid family in mycobacteria largely remain unexplored. In this contribution, we describe a multiple-stage linear ion-trap mass spectrometric approach with high resolution mass spectrometry toward direct structural analysis of the TAGs, including a novel lipid subclass previously defined as monomeromycolyl diacylglycerol (MMDAG) isolated from biofilm of Mycobacterium smegmatis, a rapidly growing, non-pathogenic mycobacterium that has been used as a tool for molecular analysis of mycobacteria. Our results demonstrate that the major isomer in each of the molecular species of TAGs and MMDAGs consists of the common structure in which Δ(9)18:1- and 16:0-fatty acyl substituents are exclusively located at sn-1 and sn-2, respectively. Several isomers were found for most of the molecular species, and thus hundreds of structures are present in this lipid family. More importantly, this study revealed the structures of MMDAG, a novel subclass of TAG that has not been previously reported by direct mass spectrometric approaches.

Robust structural analysis of native biological macromolecules from multi-crystal anomalous diffraction data.

Structure determinations for biological macromolecules that have no known structural antecedents typically involve the incorporation of heavier atoms than those found natively in biological molecules. Currently, selenomethionyl proteins analyzed using single- or multi-wavelength anomalous diffraction (SAD or MAD) data predominate for such de novo analyses. Naturally occurring metal ions such as zinc or iron often suffice in MAD or SAD experiments, and sulfur SAD has been an option since it was first demonstrated using crambin 30 years ago; however, SAD analyses of structures containing only light atoms (Zmax ≤ 20) have not been common. Here, robust procedures for enhancing the signal to noise in measurements of anomalous diffraction by combining data collected from several crystals at a lower than usual X-ray energy are described. This multi-crystal native SAD method was applied in five structure determinations, using between five and 13 crystals to determine substructures of between four and 52 anomalous scatterers (Z ≤ 20) and then the full structures ranging from 127 to 1200 ordered residues per asymmetric unit at resolutions from 2.3 to 2.8 Å. Tests were devised to assure that all of the crystals used were statistically equivalent. Elemental identities for Ca, Cl, S, P and Mg were proven by f'' scattering-factor refinements. The procedures are robust, indicating that truly routine structure determination of typical native macromolecules is realised. Synchrotron beamlines that are optimized for low-energy X-ray diffraction measurements will facilitate such direct structural analysis.

Direct Probe of Interplay between Local Structure and Superconductivity in FeTe0.55Se0.45

The relationship between atomically defined structures and physical properties in functional materials remains a subject of constant interest. We explore the interplay between local crystallographic structure, composition, and local superconductive properties in iron chalcogenide superconductors. Direct structural analysis of scanning tunneling microscopy data allows local lattice distortions and structural defects across an FeTe0.55Se0.45 surface to be explored on a single unit-cell level. Concurrent superconducting gap (SG) mapping reveals suppression of the SG at well-defined structural defects, identified as a local structural distortion. The strong structural distortion causes the vanishing of the superconducting state. This study provides insight into the origins of superconductivity in iron chalcogenides by providing an example of atomic-level studies of the structure-property relationship.

Development of liquid microjunction extraction strategy for improving protein identification from tissue sections

MALDI Mass Spectrometry Imaging has shown important potential for molecular classification and pathology marker discovery. Protein markers identification is therefore of prime importance. Direct structural analysis from tissue sections has shown limitations for protein identification because of the high degree of complexity of tissues. Only proteins of major abundance are identified this way. On the contrary, conventional proteomics approaches clearly allow for reliable identification of complex protein extracts but do not provide fine correlation with protein location in their original context. Here is presented an approach to obtain identification of proteins of various abundances while keeping their localization within the section. On-tissue trypsin digestion followed by micro-extraction using a liquid micro-junction interface is an efficient strategy to extract tryptic peptides and further identify the associated proteins off tissues. It was shown that conventional Reverse Phase Liquid Chromatography separation on the extracted material followed by MS/MS analysis on a HR FTMS instrument enabled the identification of 1500 proteins on average with high confidence from an area of about 650μm in diameter, which corresponds to an estimated number of 1900 cells in average. The approach can be easily integrated in the MALDI MSI workflow and should provide interesting insights for clinical applications.

Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration

Leukotriene (LT)A₄ and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA₄ is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide configuration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities sufficient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog 14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, confirming the proposed structure of eoxin A₄. Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA₄ and converts 5R-HPETE to the cis epoxide 5-epi-LTA₄, establishing substrate chirality as a determinant of the cis or trans epoxide configuration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations.

Direct structure analysis of advanced nanomaterials by high-resolution electron microscopy

AbstractHigh-resolution electron microscopy (HREM) analysis has contributed to the direct structure analysis of advanced nanostructured materials, of which the properties of these materials are strongly dependent on the atomic arrangements. In the present article, the direct structure analysis of nanostructured materials such as boride and oxide materials was described and the high-resolution imaging methods were applied to boron nitride nanomaterials such as nanotubes and nanoparticles. An aberration correction technique is also expected as an advanced nanostructure analysis with higher resolution. The HREM image of TlBa2Ca3Cu4O11 was taken with the incident beam parallel to the a axis together with a structure model after image processing.

Portal-Large Terminase Interactions of the Bacteriophage T4 DNA Packaging Machine Implicate a Molecular Lever Mechanism for Coupling ATPase to DNA Translocation

DNA packaging by double-stranded DNA bacteriophages and herpesviruses is driven by a powerful molecular machine assembled at the portal vertex of the empty prohead. The phage T4 packaging machine consists of three components: dodecameric portal (gp20), pentameric large terminase motor (gp17), and 11- or 12-meric small terminase (gp16). These components dynamically interact and orchestrate a complex series of reactions to produce a DNA-filled head containing one viral genome per head. Here, we analyzed the interactions between the portal and motor proteins using a direct binding assay, mutagenesis, and structural analyses. Our results show that a portal binding site is located in the ATP hydrolysis-controlling subdomain II of gp17. Mutations at key residues of this site lead to temperature-sensitive or null phenotypes. A conserved helix-turn-helix (HLH) that is part of this site interacts with the portal. A recombinant HLH peptide competes with gp17 for portal binding and blocks DNA translocation. The helices apparently provide specificity to capture the cognate prohead, whereas the loop residues communicate the portal interaction to the ATPase center. These observations lead to a hypothesis in which a unique HLH-portal interaction in the symmetrically mismatched complex acts as a lever to position the arginine finger and trigger ATP hydrolysis. Transiently connecting the critical parts of the motor; subdomain I (ATP binding), subdomain II (controlling ATP hydrolysis), and C-domain (DNA movement), the portal-motor interactions might ensure tight coupling between ATP hydrolysis and DNA translocation.

Observation of conducting filament growth in nanoscale resistive memories

Nanoscale resistive switching devices, sometimes termed memristors, have recently generated significant interest for memory, logic and neuromorphic applications. Resistive switching effects in dielectric-based devices are normally assumed to be caused by conducting filament formation across the electrodes, but the nature of the filaments and their growth dynamics remain controversial. Here we report direct transmission electron microscopy imaging, and structural and compositional analysis of the nanoscale conducting filaments. Through systematic ex-situ and in-situ transmission electron microscopy studies on devices under different programming conditions, we found that the filament growth can be dominated by cation transport in the dielectric film. Unexpectedly, two different growth modes were observed for the first time in materials with different microstructures. Regardless of the growth direction, the narrowest region of the filament was found to be near the dielectric/inert-electrode interface in these devices, suggesting that this region deserves particular attention for continued device optimization.

Single-Molecule Analysis of the Functional Structure of Telomerase RNP

Telomerase is a cellular ribonucleoprotein (RNP) complex comprised of telomerase RNA, telomerase reverse transcriptase (TERT) and other protein cofactors. The activity of the telomerase enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report the development and application of a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from individual enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures. In particular, we have established that the phylogenetically conserved pseudoknot motif within telomerase RNA is properly folded in catalytically active enzymes. Interestingly, the pseudoknot motif is misfolded in the absence of proteins, and the protein subunits of the telomerase holoenzyme counteract RNA misfolding and allow a significant fraction of the RNPs to form the pseudoknot structure. Only those RNP complexes containing a properly folded pseudoknot are catalytically active. These results not only demonstrate the functional importance of various telomerase RNA regions such as the pseudoknot but also establish a direct method for elucidating how the structure of telomerase enables its function.

Allosteric Communication in Cysteinyl tRNA Synthetase: A NETWORK OF DIRECT AND INDIRECT READOUT

Protein structure networks are constructed for the identification of long-range signaling pathways in cysteinyl tRNA synthetase (CysRS). Molecular dynamics simulation trajectory of CysRS-ligand complexes were used to determine conformational ensembles in order to gain insight into the allosteric signaling paths. Communication paths between the anticodon binding region and the aminoacylation region have been identified. Extensive interaction between the helix bundle domain and the anticodon binding domain, resulting in structural rigidity in the presence of tRNA, has been detected. Based on the predicted model, six residues along the communication paths have been examined by mutations (single and double) and shown to mediate a coordinated coupling between anticodon recognition and activation of amino acid at the active site. This study on CysRS clearly shows that specific key residues, which are involved in communication between distal sites in allosteric proteins but may be elusive in direct structure analysis, can be identified from dynamics of protein structure networks.

Direct structural analysis of modified RNA by fluorescent in-line probing

Chemical probing is a common method for the structural characterization of RNA. Typically, RNA is radioactively end-labelled, subjected to probing conditions, and the cleavage fragment pattern is analysed by gel electrophoresis. In recent years, many chemical modifications, like fluorophores, were introduced into RNA, but methods are lacking that detect the influence of the modification on the RNA structure with single-nucleotide resolution. Here, we first demonstrate that a 5′-terminal 32P label can be replaced by a dye label for in-line probing of riboswitch RNAs. Next, we show that small, highly structured FRET-labelled Diels–Alderase ribozymes can be directly probed, using the internal or terminal FRET dyes as reporters. The probing patterns indeed reveal whether or not the attachment of the dyes influences the structure. The existence of two dye labels in typical FRET constructs is found to be beneficial, as ‘duplexing’ allows observation of the complete RNA on a single gel. Structural information can be derived from the probing gels by deconvolution of the superimposed band patterns. Finally, we use fluorescent in-line probing to experimentally validate the structural consequences of photocaging, unambiguously demonstrating the intentional destruction of selected elements of secondary or tertiary structure.

Role of Key Aromatic Residues in the Ligand-binding Domain of α7 Nicotinic Receptors in the Agonist Action of β-Amyloid

Soluble β-amyloid (Aβ) resides in certain regions of the brain at or near picomolar concentration, rising in level during the prodromic stage of Alzheimer disease. Recently, we identified the homomeric α7 nicotinic acetylcholine receptor (α7-nAChR) as one possible functional target for picomolar Aβ. This study was aimed at addressing which residues in α7-nAChRs potentially interact with Aβ to regulate the presynaptic function of this receptor. Site-directed mutagenesis was carried out to study the key aromatic residues in the mouse α7-nAChR agonist-binding pocket. Mutations of tyrosine188 resulted in a decrease in activation of presynaptic α7-nAChRs by ACh and Aβ but with no change in response to nicotine, indicating the critical role of Tyr-188 in presynaptic regulation by Aβ. Coimmunoprecipitation additionally revealed direct binding of Aβ to α7-nAChRs and to the Tyr-188 mutant receptor. In contrast, mutations of Tyr-195 in α7-nAChR led to decreased activation by nicotine without apparent effects on ACh- or Aβ-induced responses. Agonist-induced responses of Tyr-93 mutant α7-nAChRs indicated possible interactions of nicotine and Aβ with its hydroxyl group, but there was no change in presynaptic responses after mutation of Trp-149. All of the mutants were shown to be expressed on the plasma membrane using cell surface labeling. Together, these results directly demonstrate an essential role for the aromatic residue Tyr-188 as a key component in the agonist binding domain for the activation of α7-nAChRs by Aβ.

Use of Aberration-Corrected STEM for Direct Structure and Chemistry Analysis of Catalytic Metal Particles

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Packing of the Extracellular Domain Hydrophobic Core Has Evolved to Facilitate Pentameric Ligand-gated Ion Channel Function

Protein function depends on conformational flexibility and folding stability. Loose packing of hydrophobic cores is not infrequent in proteins, as the enhanced flexibility likely contributes to their biological function. Here, using experimental and computational approaches, we show that eukaryotic pentameric ligand-gated ion channels are characterized by loose packing of their extracellular domain β-sandwich cores, and that loose packing contributes to their ability to rapidly switch from closed to open channel states in the presence of ligand. Functional analyses of GABA(A) receptors show that increasing the β-core packing disrupted GABA-mediated currents, with impaired GABA efficacy and slowed GABA current activation and desensitization. We propose that loose packing of the hydrophobic β-core developed as an evolutionary strategy aimed to facilitate the allosteric mechanisms of eukaryotic pentameric ligand-gated ion channels.

Structural design optimization of racing motor boat based on nonlinear finite element analysis

Since 1980's, optimum design techniques for ship structural design have been developed to the preliminary design which aims at minimum weight or minimum cost design of mid-ship section based on analytic structural analysis. But the optimum structural design researches about the application for the detail design of local structure based on FEA have been still insufficient. This paper presents optimization technique for the detail design of a racing motor boat. To improve the performance and reduce the damage of a real existing racing boat, direct structural analyses; static and non-linear transient dynamic analyses, were carried out to check the constraints of minimum weight design. As a result, it is shown that the optimum structural design of a racing boat has to be focused on reducing impulse response from pitching motion than static response because the dynamic effect is more dominant. Optimum design algorithm based on nonlinear finite element analysis for a racing motor boat was developed and coded to ANSYS, and its applicability for actual structural design was verifed.

Structure and Dynamics of Cholesterol-Containing Polyunsaturated Lipid Membranes Studied by Neutron Diffraction and NMR

A direct and quantitative analysis of the internal structure and dynamics of a polyunsaturated lipid bilayer composed of 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6n3-PC) containing 29 mol% cholesterol was carried out by neutron diffraction, 2H-NMR and 13C-MAS NMR. Scattering length distribution functions of cholesterol segments as well as of the sn-1 and sn-2 hydrocarbon chains of 18:0-22:6n3-PC were obtained by conducting experiments with specifically deuterated cholesterol and lipids. Cholesterol orients parallel to the phospholipids, with the A-ring near the lipid glycerol and the terminal methyl groups 3 Å away from the bilayer center. Previously, we reported that the density of polyunsaturated docosahexaenoic acid (DHA, 22:6n3) chains was higher near the lipid–water interface. Addition of cholesterol partially redistributes DHA density from near the lipid–water interface to the center of the hydrocarbon region. Cholesterol raises chain-order parameters of both stearic acid and DHA chains. The fractional order increase for stearic acid methylene carbons C8–C18 is larger, reflecting the redistribution of DHA chain density toward the bilayer center. The correlation times of DHA chain isomerization are short and mostly unperturbed by the presence of cholesterol. The uneven distribution of saturated and polyunsaturated chain densities and the cholesterol-induced balancing of chain distributions may have important implications for the function and integrity of membrane receptors, such as rhodopsin.

Structural design optimization of racing motor boat based on nonlinear finite element analysis

Since 1980's, optimum design techniques for ship structural design have been developed to the preliminary design which aims at minimum weight or minimum cost design of mid-ship section based on analytic structural analysis. But the optimum structural design researches about the application for the detail design of local structure based on FEA have been still insufficient. This paper presents optimization technique for the detail design of a racing motor boat. To improve the performance and reduce the damage of a real existing racing boat, direct structural analyses; static and non-linear transient dynamic analyses, were carried out to check the constraints of minimum weight design. As a result, it is shown that the optimum structural design of a racing boat has to be focused on reducing impulse response from pitching motion than static response because the dynamic effect is more dominant. Optimum design algorithm based on nonlinear finite element analysis for a racing motor boat was developed and coded to ANSYS, and its applicability for actual structural design was verifed.

Recurrent Inversion Events at 17q21.31 Microdeletion Locus Are Linked to the MAPT H2 Haplotype

The chromosomal band 17q21.31, containing the microtubule-associated protein tau (MAPT) gene, is a hotspot for chromosomal rearrangements. It is known to contain a common inversion polymorphism of approximately 900 kb in populations with European ancestry. The inverted configuration is linked to a distinct MAPT haplotype, H2, which is relatively common in Europeans but nearly absent in Asian and African populations. Recent studies have demonstrated that the H2 haplotype is ancestral in hominoids, and under positive selection in Europeans. This haplotype is also linked to events leading to the 17q21.31 microdeletion syndrome, one of the most common causes of ‘idiopathic’ mental retardation in people of European descent. We performed direct analysis of the chromosome structure by fluorescence in situ hybridization and observed heterozygosity of the inversion status for the H2 chromosomes, but not for the H1 haplotype. Inversion heterozygosity was also observed in a mother homozygous for the H2 haplotype, who transmitted the chromosome with the deletion to a proband with 17q21.31 microdeletion syndrome. Our results highlight an allele-specific sensitivity to chromosome rearrangements and suggest that it is the heterozygosity of inversion status that predisposes to the 17q21.31 microdeletion syndrome.