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.

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.

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.

Non-contrast-enhanced magnetic resonance angiography is equal to contrast-enhanced multislice computed tomography for correct aortic sizing before transcatheter aortic valve implantation.

BACKGROUND: Correct sizing of the aortic annulus in aortic valve stenosis is crucial for successful transcatheter aortic valve implantation (TAVI). Multislice computed tomography (MSCT) seems to be most promising imaging modality for this pre-interventional diagnostic work-up, but has the disadvantage of exposing mostly co-morbid patients to iodine and nephrotoxic contrast agents. To establish a useful sizing method for TAVI without the use of contrast media, we compared measurements of a non-contrast magnetic resonance imaging (MRI) technique with MSCT serving as the reference standard. METHODS: Fifty-two patients who underwent TAVI were previously examined with MSCT and MRI, respectively. MRI examination included a 3D steady-state free-precession sequence covering the entire ascending aorta. Perimeter and area of the aortic root were analyzed by two blinded readers in consensus using a dedicated software. Decisions for Sapien 3 valve size of both imaging modalities were compared using the mean derived annulus diameter. RESULTS: Mean age of the study cohort was 82.2 ± 4.9 years, log EuroScore was 25.2 ± 4.8 %. Mean aortic annulus perimeter as measured by MSCT was 76.7 ± 6.9 mm. MRI yielded a mean perimeter of 76.5 ± 6.7 mm with a good correlation coefficient (r = 0.93, p < 0.0001). Decision for valve size showed good correlation between both imaging modalities (r = 0.94, p < 0.0001). CONCLUSION: In conclusion, non-contrast MRI shows good correlation to MSCT in the assessment of the aortic annulus and valve sizing. This non-contrast technique might be a reasonable alternative for aortic root sizing before TAVI without the use of nephrotoxic contrast agents, especially in patients with severely reduced kidney function.

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.

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.

MCJ/DnaJC15, an endogenous mitochondrial repressor of the respiratory chain that controls metabolic alterations.

Mitochondria are the main engine that generates ATP through oxidative phosphorylation within the respiratory chain. Mitochondrial respiration is regulated according to the metabolic needs of cells and can be modulated in response to metabolic changes. Little is known about the mechanisms that regulate this process. Here, we identify MCJ/DnaJC15 as a distinct cochaperone that localizes at the mitochondrial inner membrane, where it interacts preferentially with complex I of the electron transfer chain. We show that MCJ impairs the formation of supercomplexes and functions as a negative regulator of the respiratory chain. The loss of MCJ leads to increased complex I activity, mitochondrial membrane potential, and ATP production. Although MCJ is dispensable for mitochondrial function under normal physiological conditions, MCJ deficiency affects the pathophysiology resulting from metabolic alterations. Thus, enhanced mitochondrial respiration in the absence of MCJ prevents the pathological accumulation of lipids in the liver in response to both fasting and a high-cholesterol diet. Impaired expression or loss of MCJ expression may therefore result in a "rapid" metabolism that mitigates the consequences of metabolic disorders.

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.

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.

The effects of combined acamprosate and integrative behaviour therapy in the outpatient treatment of alcohol dependence: a randomized controlled trial.

AIMS: The aim of this randomized, controlled, multisite trial was to evaluate the efficacy of combined treatment with integrative behaviour therapy (IBT) and acamprosate on drinking behaviour in detoxified alcohol-dependent patients. METHODS: A total of 371 patients were randomized to one of the three treatment conditions: IBT plus acamprosate, IBT plus placebo, or supportive counselling ('treatment as usual', TAU) plus acamprosate. The main outcome was success rate, i.e., rate of abstinence plus improvement according to the criteria of Feuerlein and Küfner (1989), at the end of the six-month treatment phase and at the subsequent six-month follow-up. Drinking status was validated by blood parameters (CDT, GGT, and MCV). Data were analyzed by an intent-to-treat model and missing data were classified as relapse. RESULTS: The success rates at the end of treatment under both TAU plus acamprosate (37.7%) and IBT plus placebo (48%) almost reached the levels derived from the literature. However, adding acamprosate to IBT did not result in the expected increase in success rate (IBT plus acamprosate: 47.6%), and success rates did not differ significantly between groups. Similarly, there was no significant difference between treatment success rates at follow-up. CONCLUSION: The results suggest that the combination of acamprosate and IBT is not more effective than treatment with either IBT or acamprosate alone. However, the two acamprosate conditions differed in success rate by about 10%, which might constitute a clinically relevant though statistically non-significant effect.

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.

The crystal structures of eukaryotic phosphofructokinases from baker's yeast and rabbit skeletal muscle.

Phosphofructokinase 1 (PFK) is a multisubunit allosteric enzyme that catalyzes the principal regulatory step in glycolysis-the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by ATP. The activity of eukaryotic PFK is modulated by a number of effectors in response to the cell's needs for energy and building blocks for biosynthesis. The crystal structures of eukaryotic PFKs-from Saccharomyces cerevisiae and rabbit skeletal muscle-demonstrate how successive gene duplications and fusion are reflected in the protein structure and how they allowed the evolution of new functionalities. The basic framework inherited from prokaryotes is conserved, and additional levels of structural and functional complexity have evolved around it. Analysis of protein-ligand complexes has shown how PFK is activated by fructose 2,6-bisphosphate (a powerful PFK effector found only in eukaryotes) and reveals a novel nucleotide binding site. Crystallographic results have been used as the basis for structure-based effector design.

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.