Serotype-Specific Sugars Impact Structure but Not Functions of the Trimeric Autotransporter Adhesin EmaA of Aggregatibacter actinomycetemcomitans.

The human oral pathobiont Aggregatibacter actinomycetemcomitans expresses multiple virulence factors, including the trimeric, extracellular matrix protein adhesin A (EmaA). The posttranslational modification of EmaA is proposed to be dependent on the sugars and enzymes associated with O-polysaccharide (O-PS) synthesis of the lipopolysaccharide (LPS). This modification is important for the structure and function of this adhesin. To determine if the composition of the sugars alters structure and/or function, the prototypic 202-kDa protein was expressed in a non-serotype b, emaA mutant strain. The transformed strain displayed EmaA adhesins similar in appearance to the prototypic adhesin as observed by two-dimensional (2D) electron microscopy of whole-mount negatively stained bacterial preparations. Biochemical analysis indicated that the protein monomers were posttranslationally modified. 3D electron tomographic reconstruction and structure analyses of the functional domain revealed three well-defined subdomains (SI, SII, and SIII) with a linker region between SII and SIII. Structural changes were observed in all three subdomains and the linker region of the adhesins synthesized compared with the known structure. These changes, however, did not affect the ability of the strain to bind collagen or form biofilms. The data suggest that changes in the composition of the glycan moiety alter the 3D structure of the molecule without negatively affecting the function(s) associated with this adhesin. IMPORTANCE The human oral pathogen A. actinomycetemcomitans is a causative agent of periodontal and several systemic diseases. EmaA is a trimeric autotransporter protein adhesin important for colonization by this pathobiont in vivo. This adhesin is modified with sugars associated with the O-polysaccharide (O-PS), and the modification is mediated using the enzymes involved in lipopolysaccharide (LPS) biosynthesis. The interaction with collagen is not mediated by the specific binding between the glycans and collagen but is attributed to changes in the final quaternary structure necessary to maintain an active adhesin. In this study, we have determined that the composition of the sugars utilized in the posttranslational modification of this adhesin is exchangeable without compromising functional activities.

Interactions between the Trimeric Autotransporter Adhesin EmaA and Collagen Revealed by Three-Dimensional Electron Tomography.

Bacterial adhesion to host tissues is considered the first and critical step of microbial infection. The extracellular matrix protein adhesin A (EmaA) is a collagen-binding adhesin of the periodontal pathogen Aggregatibacter actinomycetemcomitans Three 202-kDa EmaA monomers form antenna-like structures on the bacterial surface with the functional domain located at the apical end. The structure of the 30-nm functional domain has been determined by three-dimensional (3D) electron tomography and subvolume averaging. The region exhibits a complex architecture composed of three subdomains (SI to SIII) and a linker between subdomains SII and SIII. However, the molecular interaction between the adhesin receptor complexes has yet to be revealed. This study provides the first detailed 3D structure of reconstituted EmaA/collagen complexes obtained using 3D electron tomography and image processing techniques. The observed interactions of EmaA with collagen were not to whole, intact fibrils, but rather to individual collagen triple helices dissociated from the fibrils. The majority of the contacts with the EmaA functional domain encompassed subdomains SII and SIII and in some cases the tip of the apical domain, involving SI. These data suggest a multipronged mechanism for the interaction of Gram-negative bacteria with collagen.IMPORTANCE Bacterial adhesion is a crucial step for bacterial colonization and infection. In recent years, the number of antibiotic-resistant strains has dramatically increased; therefore, there is a need to search for novel antimicrobial agents. Thus, great efforts are being devoted to develop a clear understanding of the bacterial adhesion mechanism for preventing infections. In host/pathogen interactions, once repulsive forces are overcome, adhesins recognize and tightly bind to specific receptors on the host cell or tissue components. Here, we present the first 3D structure of the interaction between the collagen-binding adhesin EmaA and collagen, which is critical for the development of endocarditis in humans.

The LYR protein subunit NB4M/NDUFA6 of mitochondrial complex I anchors an acyl carrier protein and is essential for catalytic activity.

Mitochondrial complex I is the largest and most complicated enzyme of the oxidative phosphorylation system. It comprises a number of so-called accessory subunits of largely unknown structure and function. Here we studied subunit NB4M [NDUFA6, LYR motif containing protein 6 (LYRM6)], a member of the LYRM family of proteins. Chromosomal deletion of the corresponding gene in the yeast Yarrowia lipolytica caused concomitant loss of the mitochondrial acyl carrier protein subunit ACPM1 from the enzyme complex and paralyzed ubiquinone reductase activity. Exchanging the LYR motif and an associated conserved phenylalanine by alanines in subunit NB4M also abolished the activity and binding of subunit ACPM1. We show, by single-particle electron microscopy and structural modeling, that subunits NB4M and ACPM1 form a subdomain that protrudes from the peripheral arm in the vicinity of central subunit domains known to be involved in controlling the catalytic activity of complex I.

Pulmonary pulse transit time: a novel echocardiographic indicator of hemodynamic and vascular alterations in pulmonary hypertension and pulmonary fibrosis.

INTRODUCTION: Pulse transit time (PTT) is generally assumed to be a surrogate marker for blood pressure changes and arterial stiffness. The aim was to evaluate whether pulmonary PTT (pPTT) may be noninvasively measured by Doppler echocardiography and whether it might be valuable for detecting pulmonary hemodynamic and vascular alterations. METHODS: We defined pPTT as the interval between R-wave in the ECG and the corresponding peak late systolic pulmonary vein flow velocity measured by pw-Doppler in the pulmonary vein. Twelve consecutive patients with pulmonary hypertension (PH) and 12 subjects without any cardiovascular or respiratory disease were included in the study. All patients underwent a standard echocardiography including pPTT measurement. RESULTS: In the PH group, 5 patients had idiopathic pulmonary arterial hypertension (WHO 1), 1 patient PH associated with connective tissue disease (CTD, WHO 1) without pulmonary fibrosis (PF), and 6 patients PH associated with PF either due to CTD (WHO 1) or other etiology (WHO 3). Mean pPTT was significantly shorter in the PH group (138.0 ± 16.78 msec; P < 0.0001) than in the control group (383.5 ± 23.84 msec). Within the PH group, the subgroup of patients with PF showed significantly shorter mean pPTT (93.50 ± 15.47 msec; P = 0.004) than the subgroup of patients with PH without PF (182.6 ± 14.35 msec). CONCLUSIONS: The results of this study suggest that pPTT might be an interesting surrogate marker of pulmonary hemodynamic and vascular alterations in PH and PF. Further studies are warranted to evaluate the possible influence of other variables on pPTT.

Functional dissection of the proton pumping modules of mitochondrial complex I.

Mitochondrial complex I, the largest and most complicated proton pump of the respiratory chain, links the electron transfer from NADH to ubiquinone to the pumping of four protons from the matrix into the intermembrane space. In humans, defects in complex I are involved in a wide range of degenerative disorders. Recent progress in the X-ray structural analysis of prokaryotic and eukaryotic complex I confirmed that the redox reactions are confined entirely to the hydrophilic peripheral arm of the L-shaped molecule and take place at a remarkable distance from the membrane domain. While this clearly implies that the proton pumping within the membrane arm of complex I is driven indirectly via long-range conformational coupling, the molecular mechanism and the number, identity, and localization of the pump-sites remains unclear. Here, we report that upon deletion of the gene for a small accessory subunit of the Yarrowia complex I, a stable subcomplex (nb8mΔ) is formed that lacks the distal part of the membrane domain as revealed by single particle analysis. The analysis of the subunit composition of holo and subcomplex by three complementary proteomic approaches revealed that two (ND4 and ND5) of the three subunits with homology to bacterial Mrp-type Na(+)/H(+) antiporters that have been discussed as prime candidates for harbouring the proton pumps were missing in nb8mΔ. Nevertheless, nb8mΔ still pumps protons at half the stoichiometry of the complete enzyme. Our results provide evidence that the membrane arm of complex I harbours two functionally distinct pump modules that are connected in series by the long helical transmission element recently identified by X-ray structural analysis.

CMR reveals myocardial damage from cardiotoxic oncologic therapies in breast cancer patients.

BACKGROUND: Breast cancer is a common and increasingly treatable disease. However, survivors have a significantly elevated risk of cardiac events afterwards. This study aimed to characterise cardiac changes during cardiotoxic cancer therapy using cardiovascular magnetic resonance (CMR) imaging. METHODS: This study involved 34 patients with histologically proven breast cancer and planned cardiotoxic therapy. All patients underwent CMR before starting therapy, and 6 and 12 months thereafter. The CMR protocol included volumetric and functional analyses, parametric mapping, and deformation analysis using feature tracking. As the control group, 10 healthy female volunteers were scanned using the same protocol. RESULTS: With therapy, there was a significant reduction of left ventricular and right ventricular ejection fractions (both p < 0.05) without reaching pathologic values. Left ventricular radial (p = 0.008), circumferential (p = 0.010), and longitudinal strain (p = 0.036) were also reduced at follow-up. In the parametric mapping, there was a significant increase in native T1 time (start: 1037 ± 41 ms vs. 6 months: 1068 ± 51 ms vs. 12 months: 1017 ± 57 ms, p < 0.001) and T2 time (start: 55 ± 4 ms vs. 6 months: 59 ± 3 ms vs. 12 months: 57 ± 3 ms, p = 0.001), with unchanged extracellular volume and relative late gadolinium enhancement. Twelve months after cancer diagnosis, the breast cancer patients exhibited significant impairments in left ventricular global radial (p = 0.001), circumferential (p = 0.001), and longitudinal strain (p = 0.002) and T2 time (p = 0.008) compared to the healthy controls. DISCUSSION: Breast cancer patients receiving cardiotoxic chemotherapy show persistent deterioration in left ventricular strain values. This is accompanied by inflammatory changes in non-invasive tissue characterisation. Larger studies with longer follow-up periods are needed to identify patients at risk and establish preventive and therapeutic approaches.

Ultrastructural analysis of the rugose cell envelope of a member of the Pasteurellaceae family.

Bacterial membranes serve as selective environmental barriers and contain determinants required for bacterial colonization and survival. Cell envelopes of Gram-negative bacteria consist of an outer and an inner membrane separated by a periplasmic space. Most Gram-negative bacteria display a smooth outer surface (e.g., Enterobacteriaceae), whereas members of the Pasteurellaceae and Moraxellaceae families show convoluted surfaces. Aggregatibacter actinomycetemcomitans, an oral pathogen representative of the Pasteurellaceae family, displays a convoluted membrane morphology. This phenotype is associated with the presence of morphogenesis protein C (MorC). Inactivation of the morC gene results in a smooth membrane appearance when visualized by two-dimensional (2D) electron microscopy. In this study, 3D electron microscopy and atomic force microscopy of whole-mount bacterial preparations as well as 3D electron microscopy of ultrathin sections of high-pressure frozen and freeze-substituted specimens were used to characterize the membranes of both wild-type and morC mutant strains of A. actinomycetemcomitans. Our results show that the mutant strain contains fewer convolutions than the wild-type bacterium, which exhibits a higher curvature of the outer membrane and a periplasmic space with 2-fold larger volume/area ratio than the mutant bacterium. The inner membrane of both strains has a smooth appearance and shows connections with the outer membrane, as revealed by visualization and segmentation of 3D tomograms. The present studies and the availability of genetically modified organisms with altered outer membrane morphology make A. actinomycetemcomitans a model organism for examining membrane remodeling and its implications in antibiotic resistance and virulence in the Pasteurellaceae and Moraxellaceae bacterial families.

Projection-based volume alignment.

When heterogeneous samples of macromolecular assemblies are being examined by 3D electron microscopy (3DEM), often multiple reconstructions are obtained. For example, subtomograms of individual particles can be acquired from tomography, or volumes of multiple 2D classes can be obtained by random conical tilt reconstruction. Of these, similar volumes can be averaged to achieve higher resolution. Volume alignment is an essential step before 3D classification and averaging. Here we present a projection-based volume alignment (PBVA) algorithm. We select a set of projections to represent the reference volume and align them to a second volume. Projection alignment is achieved by maximizing the cross-correlation function with respect to rotation and translation parameters. If data are missing, the cross-correlation functions are normalized accordingly. Accurate alignments are obtained by averaging and quadratic interpolation of the cross-correlation maximum. Comparisons of the computation time between PBVA and traditional 3D cross-correlation methods demonstrate that PBVA outperforms the traditional methods. Performance tests were carried out with different signal-to-noise ratios using modeled noise and with different percentages of missing data using a cryo-EM dataset. All tests show that the algorithm is robust and highly accurate. PBVA was applied to align the reconstructions of a subcomplex of the NADH: ubiquinone oxidoreductase (Complex I) from the yeast Yarrowia lipolytica, followed by classification and averaging.

Correlation of the amino-acid sequence and the 3D structure of the functional domain of EmaA from Aggregatibacter actinomycetemcomitans.

Adhesion to collagen is an important virulence determinant for the periodontal pathogen Aggregatibacter actinomycetemcomitans. Binding to collagen is mediated by the extracellular-matrix protein adhesin-A (EmaA). EmaA is a homotrimeric autotransporter protein that forms flexible antenna-like appendages on the bacterium surface. An ellipsoidal structure at the distal end of the appendage, composed of three subdomains, contains the functional domain of the molecule. A correlation between amino-acid sequence and subdomain structure (SI and SII) was proposed based on an analysis of the volume/molecular weight ratio. EmaA from three mutant strains (deletions of amino-acids 70-206 and 70-386 and a substitution mutation G162S) has been studied by electron microscopy to test this hypothesis. 3D structures were analyzed using single-axis tilt tomography of negatively stained preparations of bacteria combined with subvolume averaging. Additionally, a large number of 2D images of the apical domain of the adhesins from the mutants were extracted from micrographs of the bacterial surface, aligned and classified. The combined data showed that amino-acids 70-206 localize to subdomain SI and 70-386 comprise subdomains SI and SII. Moreover, we showed that the substitution mutation G162S, which abolishes collagen binding activity, does not affect the overall structural integrity of the functional domain. However, the structure of subdomain SI in this mutant is slightly altered with respect to the wild-type strain. These data also have allowed us to interpret the architectural features of each subdomain of EmaA in more detail and to correlate the 3D structure of the functional domain of EmaA with the amino-acid sequence.

A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I.

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast Yarrowia lipolytica comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination, the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits, including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence-based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I.

Probabilistic principal component analysis with expectation maximization (PPCA-EM) facilitates volume classification and estimates the missing data.

We have developed a new method for classifying 3D reconstructions with missing data obtained by electron microscopy techniques. The method is based on principal component analysis (PCA) combined with expectation maximization. The missing data, together with the principal components, are treated as hidden variables that are estimated by maximizing a likelihood function. PCA in 3D is similar to PCA for 2D image analysis. A lower dimensional subspace of significant features is selected, into which the data are projected, and if desired, subsequently classified. In addition, our new algorithm estimates the missing data for each individual volume within the lower dimensional subspace. Application to both a large model data set and cryo-electron microscopy experimental data demonstrates the good performance of the algorithm and illustrates its potential for studying macromolecular assemblies with continuous conformational variations.

Architecture of complex I and its implications for electron transfer and proton pumping.

Proton pumping NADH:ubiquinone oxidoreductase (complex I) is the largest and remains by far the least understood enzyme complex of the respiratory chain. It consists of a peripheral arm harbouring all known redox active prosthetic groups and a membrane arm with a yet unknown number of proton translocation sites. The ubiquinone reduction site close to iron-sulfur cluster N2 at the interface of the 49-kDa and PSST subunits has been mapped by extensive site directed mutagenesis. Independent lines of evidence identified electron transfer events during reduction of ubiquinone to be associated with the potential drop that generates the full driving force for proton translocation with a 4H(+)/2e(-) stoichiometry. Electron microscopic analysis of immuno-labelled native enzyme and of a subcomplex lacking the electron input module indicated a distance of 35-60 A of cluster N2 to the membrane surface. Resolution of the membrane arm into subcomplexes showed that even the distal part harbours subunits that are prime candidates to participate in proton translocation because they are homologous to sodium/proton antiporters and contain conserved charged residues in predicted transmembrane helices. The mechanism of redox linked proton translocation by complex I is largely unknown but has to include steps where energy is transmitted over extremely long distances. In this review we compile the available structural information on complex I and discuss implications for complex I function.

Interferon beta-1b therapy in chronic viral dilated cardiomyopathy--is there a role for specific therapy?

BACKGROUND: Myocardial biopsy can be used for the detection of viral genome in dilated cardiomyopathy (DCM). Pilot studies have previously reported beneficial effects on clinical outcome and safety of an antiviral therapy using interferon beta-1b in chronic viral DCM. METHODS AND RESULTS: Myocardial biopsies were taken from patients with DCM. Using polymerase chain reaction and Southern Blot analysis, viral genome could be detected in 49% of patients. In 42 patients with viral infection, off-label use with interferon beta-1b was initiated. A further 68 patients formed the control group. The outcome was evaluated after follow-up with echocardiography, exercise electrocardiogram, and New York Heart Association class. A total of 81 men and 29 women with a median left ventricular ejection fraction of 34% were included. The follow-up period was 36 months. In 33 (79%) patients with interferon beta-1b treatment, minor adverse reactions occurred, but no major adverse events were reported. No significant benefit for interferon beta-1b treatment on clinical outcome could be detected during follow-up. CONCLUSIONS: Off-label use with interferon beta-1b in patients with viral DCM is feasible and safe under routine clinical practice. Concerning the herein evaluated clinical outcome parameters, promising results from pilot studies could not be confirmed. High prevalence of parvovirus B19 (92%) might influence the results.

3D structure of phosphofructokinase from Pichia pastoris: Localization of the novel gamma-subunits.

The largest and one of the most complex ATP-dependent allosteric phosphofructokinase (Pfk) has been found in the methylotrophic yeast, Pichia pastoris. The enzyme is a hetero-oligomer ( approximately 1MDa) composed of three distinct subunits (alpha, beta and gamma) with molecular masses of 109, 104 and 41kDa, respectively. While the alpha- and beta-subunits show sequence similarities to other phosphofructokinase subunits, the gamma-subunit does not show high homology to any known protein in the databases. We have determined the first quaternary structure of P. pastoris phosphofructokinase by 3D electron microscopy. Random conical techniques and tomography have been instrumental to ascertain the quality of the sample preparations for structural studies and to obtain a reliable 3D structure. The final reconstruction of P. pastoris Pfk resembles its yeast counterparts with four additional densities, assigned to four gamma-subunits, bridging the N-terminal domains of the four pairs of alpha- and beta-subunits. Our data has evidenced novel interactions between the gamma- and the alpha-subunits comparable in intensity to the interactions, shown by cross-linking and limited proteolytic degradation experiments, between the gamma- and beta-subunits. The structural data provides clear insights into the allosteric fine-tuned regulation of the enzyme by ATP and AMP observed in this yeast species.

On cross-correlations, averages and noise in electron microscopy.

Biological samples are radiation-sensitive and require imaging under low-dose conditions to minimize damage. As a result, images contain a high level of noise and exhibit signal-to-noise ratios that are typically significantly smaller than 1. Averaging techniques, either implicit or explicit, are used to overcome the limitations imposed by the high level of noise. Averaging of 2D images showing the same molecule in the same orientation results in highly significant projections. A high-resolution structure can be obtained by combining the information from many single-particle images to determine a 3D structure. Similarly, averaging of multiple copies of macromolecular assembly subvolumes extracted from tomographic reconstructions can lead to a virtually noise-free high-resolution structure. Cross-correlation methods are often used in the alignment and classification steps of averaging processes for both 2D images and 3D volumes. However, the high noise level can bias alignment and certain classification results. While other approaches may be implicitly affected, sensitivity to noise is most apparent in multireference alignments, 3D reference-based projection alignments and projection-based volume alignments. Here, the influence of the image signal-to-noise ratio on the value of the cross-correlation coefficient is analyzed and a method for compensating for this effect is provided.

Longitudinal strain assessed by cardiac magnetic resonance correlates to hemodynamic findings in patients with severe aortic stenosis and predicts positive remodeling after transcatheter aortic valve replacement.

AIMS: To assess left-ventricular strain parameters before and after transcatheter aortic valve replacement (TAVR) by feature tracking cardiac magnetic resonance imaging (FT CMR) and to correlate the findings to hemodynamic state and left-ventricular remodeling. METHODS AND RESULTS: Patients with symptomatic AS underwent FT CMR before and after TAVR. Patients were carefully evaluated by a comprehensive work-up including CMR, echocardiography and left and right heart catheterization. Thirty patients formed the study population. High-flow/high-gradient (HF/HG) aortic stenosis was diagnosed in 11 patients (36.7%), 6 patients (20.0%) exhibited low-flow/low-gradient AS (LF/LG) and 13 patients (43.3%) were classified to have so-called paradoxical low-flow/low-gradient (PLF/LG) AS. The HF/HG patients had a significantly reduced longitudinal strain which recovered after TAVR (-12.67 ± 4.60 to -15.46 ± 5.61%, p = 0.048). In the LF/LG group, an even more pronounced reduction of longitudinal strain and also an impairment of longitudinal velocity could be observed. Both parameters improved after therapy (strain: -5.06 ± 4.25 to -8.02 ± 3.28%, p = 0.045; velocity: 25.33 ± 9.63 to 37.13 ± 11.64 mm/s, p = 0.042). Patients with PLF/LG showed preserved longitudinal strain but a reduction of longitudinal velocity similar to the LF/LG group. These patients did not show a significant improvement of strain parameters after TAVR. Longitudinal velocity exhibited the highest predictive power for the identification of a low-flow state (sensitivity 75%, specificity 80%). CONCLUSION: Improvement of longitudinal strain parameters after TAVR is dependent on the initial hemodynamically defined AS subgroup.

Myocardial strain characteristics and outcomes after transcatheter aortic valve replacement.

BACKGROUND: Objective of this study was to make an assessment of standard functional and defor-mation parameters (strain) in patients after transcatheter aortic valve replacement (TAVR) by cardiac magnetic resonance imaging (CMR) and the evaluation of their prognostic impact. METHODS: Patients undergoing TAVR received CMR on a 1.5 T whole-body scanner at 3 months after the procedure. Deformation parameters (strain, strain rate, velocity, displacement) were assessed in lon-gitudinal, circumferential and radial orientation using a feature tracking approach. Primary outcome measure was defined according to Valve Academic Research Consortium-2 (VARC-2) criteria. RESULTS: Eighty-three patients formed the study population. Deformation parameters were significantly reduced in all three orientations for strain (longitudinal: -12.1 ± 5.4% vs. -15.9 ± 1.96%, p < 0.0001; radial: 34.4 ± 15.3% vs. 47.2 ± 11.4%, p < 0.0001; circumferential: -16.8 ± 4.3% vs. -21.1 ± 2.5%, p < 0.0001) and strain rate (longitudinal: -0.79 ± 0.33%/s vs. -0.91 ± 0.23%/s, p = 0.043; radial: 2.5 ± 1.2%/s vs. 2.9 ± 0.9%, p = 0.067; circumferential: -1.1 ± 0.6%/s vs. -1.3 ± 0.3%/s, p = 0.006) in comparison to a healthy control population. Median follow-up was 614 days. During this period, 13 endpoints occurred (cumulative event rate of 10.7%). Patients with event by trend exhibited poorer strain and strain rate in longitudinal and radial orientation without reaching statistical significance (longitudinal strain: -11.2 ± 5.4% vs. -12.3 ± 5.4%, p = 0.52; longitudinal strain rate: -0.73 ± ± 0.23%/s vs. 0.80 ± 0.35%/s, p = 0.53; radial strain: 29.5 ± 19.6% vs. 35.2 ± 14.5%, p = 0.24; radial strain rate: 2.2 ± 1.6%/s vs. 2.6 ± 1.2%/s, p = 0.31). CONCLUSIONS: Assessment of left ventricular deformation parameters by CMR revealed functional abnormalities in comparison to healthy controls. Prognostic significance remains to be further investi-gated.

Cardiac magnetic resonance imaging derived quantification of myocardial ischemia and scar improves risk stratification and patient management in stable coronary artery disease.

BACKGROUND: Quantification of myocardial ischemia and necrosis might ameliorate prognostic models and lead to improved patient management. However, no standardized consensus on how to assess and quantify these parameters has been established. The aim of this study was to quantify these variables by cardiac magnetic resonance imaging (CMR) and to establish possible incremental implications in cardiovascular risk prediction. METHODS: This study is a retrospective analysis of patients with known or suspected coronary artery disease (CAD) referred for adenosine perfusion CMR was performed. Myocardial ischemia and necrosis were assessed and quantified using an algorithm based on standard first-pass perfusion imaging and late gadolinium enhancement (LGE). The combined primary endpoint was defined as cardiac death, non-fatal myocardial infarction, and stroke. RESULTS: 845 consecutive patients were enrolled into the study. During the median follow-up of 3.64 [1.03; 10.46] years, 61 primary endpoints occurred. Patients with primary endpoint showed larger extent of ischemia (10.7 ± 12.25% vs. 3.73 ± 8.29%, p < 0.0001) and LGE (21.09 ± 15.11% vs. 17.73 ± 10.72%, p < 0.0001). A risk prediction model containing the extent of ischemia and LGE proved to be superior in comparison to all other models (χ² increase: from 39.678 to 56.676, integrated discrimination index: 0.3851, p = 0.0033, net reclassification index: 0.11516, p = 0.0071). The ben-eficial effect of revascularization tended to be higher in patients with greater extents of ischemia, though statistical significance was not reached. CONCLUSIONS: Quantification of myocardial ischemia and LGE was shown to significantly improve existing risk prediction models and might thus lead to an improvement in patient management.

Biochemical and electron microscopic characterization of the F1F0 ATP synthase from the hyperthermophilic eubacterium Aquifex aeolicus.

The F(1)F(0) ATP synthase has been purified from the hyperthermophilic eubacterium Aquifex aeolicus and characterized. Its subunits have been identified by MALDI-mass spectrometry through peptide mass fingerprinting and MS/MS. It contains the canonical subunits alpha, beta, gamma, delta and epsilon of F(1) and subunits a and c of F(0). Two versions of the b subunit were found, which show a low sequence homology to each other. Most likely they form a heterodimer. An electron microscopic single particle analysis revealed clear structural details, including two stalks connecting F(1) and F(0). In several orientations the central stalk appears to be tilted and/or kinked. It is unclear whether there is a direct connection between the peripheral stalk and the delta subunit.

Three-dimensional reconstruction of single particles in electron microscopy image processing.

Three-dimensional electron microscopy of single macromolecular assemblies has made large strides forward over the last decade. A large number of image processing techniques have been developed and many have found general distribution. For the proper usage of the wide range of available techniques, a clear concept of all processing steps is essential. This chapter provides step-by-step instruction for the three-dimensional reconstruction of an unknown macromolecule. Where possible, the limitations of the techniques are explained. The chapter attempts to be sufficiently general such so as not to adhere to a single image processing system. Described are alignment techniques for two and three dimensions, classification procedures, and the usage of three-dimensional reconstruction algorithms.