The quest for indicators of paroxysmal atrial fibrillation in sinus rhythm - the DETECT AF trial.

Background: Atrial fibrillation (AF) is related to an increased stroke risk. At present, differentiation between patients with paroxysmal AF (pAF) and without is only possible during AF episodes and not during sinus rhythm. If AF could be diagnosed more quickly and reliably, anticoagulation therapy may be administered and prevent from cardioembolic strokes. The DETECT AF trial evaluated the hypothesis that propagation of electric activities in patients with pAF differs from propagation in healthy atria and that this can be detected with a three-dimensional electrocardiogram in patients during sinus rhythm. Methods: We conducted a case-control study including patients with a history of pAF and a control group with no history of AF. Vectorcardiographic beat-to-beat variability of atrial activation in sinus rhythm was tested and compared between the two groups. Results: One hundred and eight patients with a history of pAF in sinus rhythm and 121 controls in sinus rhythm were included. With a combination of specific vectorcardiographic beat-to-beat variability parameters discrimination between the two groups was possible with a specificity of 82% and a sensitivity of 71% (p≤.01). Using heart rate independent parameters, both specificity and sensitivity were reduced to 70%. Conclusions: Analysis of atrial vectorcardiographic beat-to-beat variability indicates that atrial conduction variability in patients with pAF differs from patients without AF and may be used as an indicator to estimate the risk for pAF in patients during sinus rhythm. Further studies to investigate the potential of this parameter are needed. Clinical trial registration number: NCT02270112.

LaAlO3 stoichiometry is key to electron liquid formation at LaAlO3/SrTiO3 interfaces.

Emergent phenomena, including superconductivity and magnetism, found in the two-dimensional electron liquid (2-DEL) at the interface between the insulators lanthanum aluminate (LaAlO3) and strontium titanate (SrTiO3) distinguish this rich system from conventional 2D electron gases at compound semiconductor interfaces. The origin of this 2-DEL, however, is highly debated, with focus on the role of defects in the SrTiO3, while the LaAlO3 has been assumed perfect. Here we demonstrate, through experiments and first-principle calculations, that the cation stoichiometry of the nominal LaAlO3 layer is key to 2-DEL formation: only Al-rich LaAlO3 results in a 2-DEL. Although extrinsic defects, including oxygen deficiency, are known to render LaAlO3/SrTiO3 samples conducting, our results show that in the absence of such extrinsic defects an interface 2-DEL can form. Its origin is consistent with an intrinsic electronic reconstruction occurring to counteract a polarization catastrophe. This work provides insight for identifying other interfaces where emergent behaviours await discovery.

Enhanced ordering temperatures in antiferromagnetic manganite superlattices.

The disorder inherent to doping by cation substitution in the complex oxides can have profound effects on collective-ordered states. Here, we demonstrate that cation-site ordering achieved through digital-synthesis techniques can dramatically enhance the antiferromagnetic ordering temperatures of manganite films. Cation-ordered (LaMnO3)m/(SrMnO3)2m superlattices show Néel temperatures (TN) that are the highest of any La(1-x)Sr(x)MnO3 compound, approximately 70 K greater than compositionally equivalent randomly doped La(1/3)Sr(2/3)MnO3. The antiferromagnetic order is A-type, consisting of in-plane double-exchange-mediated ferromagnetic sheets coupled antiferromagnetically along the out-of-plane direction. Through synchrotron X-ray scattering, we have discovered an in-plane structural modulation that reduces the charge itinerancy and hence the ordering temperature within the ferromagnetic sheets, thereby limiting TN. This modulation is mitigated and driven to long wavelengths by cation ordering, enabling the higher TN values of the superlattices. These results provide insight into how cation-site ordering can enhance cooperative behaviour in oxides through subtle structural phenomena.

Metal-insulator transition and its relation to magnetic structure in (LaMnO3)2n/(SrMnO3)n superlattices.

Superlattices of (LaMnO3){2n}/(SrMnO3){n} (1or=3. Measurements of transport, magnetization, and polarized neutron reflectivity reveal that the ferromagnetism is relatively uniform in the metallic state, and is strongly modulated in the insulating state, being high in LaMnO3 and suppressed in SrMnO3. The modulation is consistent with a Mott transition driven by the proximity between the (LaMnO3)/(SrMnO3) interfaces. The insulating state for n>or=3 obeys variable range hopping at low temperatures. We suggest that this is due to states at the Fermi level that emerge at the (LaMnO3)/(SrMnO3) interfaces and are localized by disorder.

Genetic analyses involving interactions between the ergosterol biosynthetic enzymes, lanosterol synthase (Erg7p) and 3-ketoreductase (Erg27p), in the yeast Saccharomyces cerevisiae.

Protein-protein interaction studies in the Saccharomyces cerevisiae ergosterol biosynthetic pathway suggest that enzymes in this pathway may act as an integrated multienzyme complex. The yeast sterol 3-ketoreductase (Erg27p) required for C-4 demethylation of sterols has previously been shown to also be required for the function of the upstream oxidosqualene cyclase/lanosterol synthase (Erg7p); thus, erg27 mutants accumulate oxidosqualenes as precursors rather than 3-ketosterones. In the present study, we have created various mutations in the ERG27 gene. These mutations include 5 C-terminal truncations, 6 internal deletions, and 32 point mutants of which 14 were obtained by site-directed mutagenesis and 18 by random mutagenesis. We have characterized these ERG27 mutations by determining the following: Erg27 and Erg7 enzyme activities, presence of Erg27p as determined by western immunoblots, ability to grow on various sterol substrates and GC sterol profiles. Mutations of the predicted catalytic residues, Y202F and K206A, resulted in the endogenous accumulation of 3-ketosterones rather than oxidosqualenes suggesting retention of Erg7 enzyme activity. This novel phenotype demonstrated that the catalytic function of Erg27p can be separated from its Erg7p chaperone ability. Other erg27 mutations resulted in proteins that were present, as determined by western immunoblotting, but unable to interact with the Erg7 protein. We also classify Erg27p as belonging to the SDR (short-chain dehydrogenase/reductase) family of enzymes and demonstrate the possibility of homo- or heterodimerization of the protein. This study provides new insights into the role of Erg27p in sterol biosynthesis.

Viscous spin exchange torque on precessional magnetization in (LaMnO3)2n/(SrMnO3)n superlattices.

Photoinduced magnetization dynamics is investigated in chemically ordered (LaMnO3)2n/(SrMnO3)n superlattices using the time-resolved magneto-optic Kerr effect. A monotonic frequency-field dependence is observed for the n=1 superlattice, indicating a single spin population consistent with a homogeneous hole distribution. In contrast, for n> or =2 superlattices, a large precession frequency is observed at low fields indicating the presence of an exchange torque in the dynamic regime. We attribute the emergence of exchange torque to the coupling between two spin populations-viscous and fast spins.

Collagen content contributes to arterial narrowing in human atherosclerotic plaques.

Collagen is a known component in human atherosclerotic plaques. However, little investigation into the use of collagen deposition as a treatment target has been performed. In this study, collagen deposition in the left anterior descending branch of the left coronary artery (LAD) and the posterior intraventricular branch of the right coronary artery (PIV) appears to be a uniform event. Using arterial samples from human cadavers, the mean thickness of collagen deposition in arterial samples was 671.9 microm for the LAD and 670.2 microm for the PIV. This study also shows that collagen deposition in the PIV is slightly greater in females than for males. However, data on collagen contributions in males or females in other arteries was not obtainable at this time. This data suggests that collagen deposition may function as an early target to treat the atherosclerotic state and not just later complications.

Suppressed magnetization at the surfaces and interfaces of ferromagnetic metallic manganites.

What happens to ferromagnetism at the surfaces and interfaces of manganites? With the competition between charge, spin, and orbital degrees of freedom, it is not surprising that the surface behaviour may be profoundly different to that of the bulk. Using a powerful combination of two surface probes, tunnelling and polarized x-ray interactions, this paper reviews our work on the nature of the electronic and magnetic states at manganite surfaces and interfaces. The general observation is that ferromagnetism is not the lowest energy state at the surface or interface, which results in a suppression or even loss of ferromagnetic order at the surface. Two cases will be discussed ranging from the surface of the quasi-2D bilayer manganite (La(2-2x)Sr(1+2x)Mn(2)O(7)) to the 3D perovskite (La(2/3)Sr(1/3)MnO(3))/SrTiO(3) interface. For the bilayer manganite, which is ferromagnetic and conducting in the bulk, these probes present clear evidence for an intrinsic insulating non-ferromagnetic surface layer atop adjacent subsurface layers that display the full bulk magnetization. This abrupt intrinsic magnetic interface is attributed to the weak inter-bilayer coupling native to these quasi-two-dimensional materials. This is in marked contrast to the situation for the non-layered manganite system (La(2/3)Sr(1/3)MnO(3)/SrTiO(3)), whose magnetization near the interface is less than half the bulk value at low temperatures and decreases with increasing temperature at a faster rate than that for the bulk.

Doping controlled superconductor-insulator transition in Bi2Sr2-xLaxCaCu2O8+delta.

We show that the doping-controlled superconductor-insulator transition (SIT) in a high critical temperature cuprate system (Bi(2)Sr(2-x)La(x)CaCu(2)O(8+delta)) exhibits a fundamentally different behavior than is expected from conventional SIT. At the critical doping, the sheet resistance seems to diverge in the zero-temperature limit. Above the critical doping, the transport is universally scaled by a two-component conductance model. Below, it continuously evolves from weakly to strongly insulating behavior. The two-component conductance model suggests that a collective electronic phase-separation mechanism may be responsible for this unconventional SIT behavior.

Data-driven analysis approach for biomarker discovery using molecular-profiling technologies.

High-throughput molecular-profiling technologies provide rapid, efficient and systematic approaches to search for biomarkers. Supervised learning algorithms are naturally suited to analyse a large amount of data generated using these technologies in biomarker discovery efforts. The study demonstrates with two examples a data-driven analysis approach to analysis of large complicated datasets collected in high-throughput technologies in the context of biomarker discovery. The approach consists of two analytic steps: an initial unsupervised analysis to obtain accurate knowledge about sample clustering, followed by a second supervised analysis to identify a small set of putative biomarkers for further experimental characterization. By comparing the most widely applied clustering algorithms using a leukaemia DNA microarray dataset, it was established that principal component analysis-assisted projections of samples from a high-dimensional molecular feature space into a few low dimensional subspaces provides a more effective and accurate way to explore visually and identify data structures that confirm intended experimental effects based on expected group membership. A supervised analysis method, shrunken centroid algorithm, was chosen to take knowledge of sample clustering gained or confirmed by the first step of the analysis to identify a small set of molecules as candidate biomarkers for further experimentation. The approach was applied to two molecular-profiling studies. In the first study, PCA-assisted analysis of DNA microarray data revealed that discrete data structures exist in rat liver gene expression and correlated with blood clinical chemistry and liver pathological damage in response to a chemical toxicant diethylhexylphthalate, a peroxisome-proliferator-activator receptor agonist. Sixteen genes were then identified by shrunken centroid algorithm as the best candidate biomarkers for liver damage. Functional annotations of these genes revealed roles in acute phase response, lipid and fatty acid metabolism and they are functionally relevant to the observed toxicities. In the second study, 26 urine ions identified from a GC/MS spectrum, two of which were glucose fragment ions included as positive controls, showed robust changes with the development of diabetes in Zucker diabetic fatty rats. Further experiments are needed to define their chemical identities and establish functional relevancy to disease development.

Isolation, characterization, and regulation of the Candida albicans ERG27 gene encoding the sterol 3-keto reductase.

The Candida albicans ERG27 gene which encodes the 3-keto reductase enzyme required for sterol C-4 demethylation was isolated and found to encode a 349 amino acid protein that is 60% identical at the amino acid level to the Saccharomyces cerevisiae Erg27p. A C. albicans erg27 null was created in a strain containing an integrated ERG27 rescue cassette under the control of the pMAL2 inducible promoter. The C. albicans erg27 strain was able to grow only in the presence of maltose indicating that the ERG27 gene is essential. The C. albicans erg27 null showed complete loss of both 3-keto reductase and oxidosqualene cyclase (Erg7p) activities compromising all sterol synthesis. These results suggest that Erg27p inhibitors might be effective antifungals. To explore ERG27 regulation, an erg11 null strain was generated. C. albicans erg6 and erg24 mutants were also employed along with the inhibitors, itraconazole and zaragozic acid A, to characterize ERG27 expression using Northern analysis. Expression was increased two- to fourfold in erg11, erg6 and erg24 backgrounds. However, itraconazole which targets Erg11p (lanosterol demethylase) increased ERG27 expression 10-fold and zaragozic acid A which targets the Erg9p (squalene synthase) increased ERG27 expression fivefold. The azole and erg11 results support other observations that azoles may affect non-sterol targets.

Ergosterol gene expression in wild-type and ergosterol-deficient mutants of Candida albicans.

The ergosterol pathway is the major target of the azole antifungals. We have developed a panel of five viable ergosterol biosynthetic mutants (erg2, erg3, erg6, erg11 and erg24) and have performed Northern analyses to study transcriptional regulation using probes to four ergosterol biosynthetic genes (ERG2, ERG7, ERG11 and ERG25), as well as probes to two additional genes encoding ergosterol cytochrome coenzymes (CYB5 and NCP1). ERG11, which encodes the sterol 14-demethylase, the direct target of the azole antifungals, was the most up-regulated gene followed by ERG7 and ERG25. Transcription of the four ergosterol genes was most up-regulated in erg24 and erg6 mutant backgrounds, deficient in C-14 reductase and the C-24 sterol transmethylase, respectively. Unexpectedly, we also found that the two cytochrome genes, CYB5 encoding cytochrome b5 and NCP1 encoding the cytochrome P450 reductase, were not regulated markedly different from wild-type in the erg2, erg3, erg6, erg11 and erg24 strains of Candida albicans.

Disruption of the Candida albicans CYB5 gene results in increased azole sensitivity.

Sterol synthesis in fungi is an aerobic process requiring molecular oxygen and, for several cytochrome-mediated reactions, aerobically synthesized heme. Cytochrome b(5) is required for sterol C5-6 desaturation and the encoding gene, CYB5, is nonessential in Saccharomyces cerevisiae. Cyb5p and Ncp1p (cytochrome P-450 reductase) appear to have overlapping functions in this organism, with disruptions of each alone being viable. The cytochrome P-450 reductase phenotype has also been shown to demonstrate increased sensitivity to azole antifungals. Based on this phenotype, the CYB5 gene in the human pathogen Candida albicans was investigated to determine whether the cyb5 genotype was viable and would also demonstrate azole sensitivity. Sequential disruption of the CYB5 alleles by direct transformation resulted in viability, presumably conferred by the presence of a third copy of the CYB5 gene. Subsequent disruption procedures with a pMAL2-CYB5 rescue cassette and a CYB5-URA3 blaster cassette resulted in viable cyb5 strains with no third copy. The C. albicans CYB5 gene is concluded to be nonessential. Thus, the essentiality of this gene and whether we observed two or three alleles was dependent upon the gene disruption protocol. The C. albicans cyb5 strains produced a sterol profile containing low ergosterol levels and sterol intermediates similar to that reported for the S. cerevisiae cyb5. The C. albicans cyb5 shows increased sensitivity to azoles and terbinafine, an inhibitor of squalene epoxidase, and, unexpectedly, increased resistance to morpholines, which inhibit the ERG2 and ERG24 gene products. These results indicate that an inhibitor of Cyb5p would not be lethal but would make the cell significantly more sensitive to azole treatment.

Artificial dielectric superlattices with broken inversion symmetry.

A variety of three-constituent superlattice patterns were made in atomic layer-by-layer films, with patterns breaking inversion symmetry giving effective permanent bias fields ranging up to about 200 kV/cm. Dielectric constants at room temperature were nearly 10(3), with loss tangents under 0.01. Most of the response came from discrete dipoles comprising multiple unit cells, but without any ferroelectric phase transition.

Atomic scale imaging and spectroscopy of a CuO(2) plane at the surface of Bi(2)Sr(2)CaCu(2)O(8+delta).

We have used a scanning tunneling microscope to demonstrate that a single CuO2 plane can form a stable and atomically ordered layer at the surface of Bi(2)Sr(2)CaCu(2)O(8+delta). In contrast to previous studies on high-T(c) surfaces, the CuO2-terminated surface exhibits a strongly suppressed tunneling conductance at low voltages. We consider a number of different explanations for this phenomena and propose that it may be caused by how the orbital symmetry of the CuO2 plane's electronic states affects the tunneling process.

Candida albicans sterol C-14 reductase, encoded by the ERG24 gene, as a potential antifungal target site.

The incidence of fungal infections has increased dramatically, which has necessitated additional and prolonged use of the available antifungal agents. Increased resistance to the commonly used antifungal agents, primarily the azoles, has been reported, thus necessitating the discovery and development of compounds that would be effective against the major human fungal pathogens. The sterol biosynthetic pathway has proved to be a fertile area for antifungal development, and steps which might provide good targets for novel antifungal development remain. The sterol C-14 reductase, encoded by the ERG24 gene, could be an effective target for drug development since the morpholine antifungals, inhibitors of Erg24p, have been successful in agricultural applications. The ERG24 gene of Candida albicans has been isolated by complementation of a Saccharomyces cerevisiae erg24 mutant. Both copies of the C. albicans ERG24 gene have been disrupted by using short homologous regions of the ERG24 gene flanking a selectable marker. Unlike S. cerevisiae, the C. albicans ERG24 gene was not required for growth, but erg24 mutants showed several altered phenotypes. They were demonstrated to be slowly growing, with doubling times at least twice that of the wild type. They were also shown to be significantly more sensitive to an allylamine antifungal and to selected cellular inhibitors including cycloheximide, cerulenin, fluphenazine, and brefeldin A. The erg24 mutants were also slightly resistant to the azoles. Most importantly, erg24 mutants were shown to be significantly less pathogenic in a mouse model system and failed to produce germ tubes upon incubation in human serum. On the basis of these characteristics, inhibitors of Erg24p would be effective against C. albicans.

Identification of the human cytochromes p450 responsible for in vitro formation of R- and S-norfluoxetine.

The formation of R- and S-norfluoxetine was analyzed in vitro in human liver microsomes. Low apparent K(m) values for R-norfluoxetine formation of < or =8 microM and S-norfluoxetine of <0.2 microM were determined. R-Norfluoxetine formation rates in a characterized microsomal bank correlated with the catalytic activities for cytochrome P450 (CYP) 2D6, CYP2C9, and CYP2C8. Expressed CYP2C9, CYP2C19, and CYP2D6 formed R-norfluoxetine following incubation with 1 microM R-fluoxetine and exhibited apparent K(m) values of 9.7, 8.5, and 1.8 microM, respectively. Multivariate correlation analysis identified CYP2C9 and CYP2D6 as significant regressors with R-norfluoxetine formation. Antibodies to the CYP2C subfamily and CYP2D6 each exhibited moderate inhibition of R-norfluoxetine formation. Therefore, CYP2D6 and CYP2C9 contribute to this biotransformation. At pharmacological concentrations of S-fluoxetine, S-norfluoxetine formation rates in the bank of microsomes were found to correlate only with CYP2D6 catalytic activity and only expressed CYP2D6 was found to be capable of forming S-norfluoxetine. Thus, it would appear that both CYP2D6 and CYP2C9 contribute to the formation of R-norfluoxetine, whereas only CYP2D6 is responsible for the conversion to S-norfluoxetine. Since the enantiomers of fluoxetine and norfluoxetine are inhibitors of CYP2D6, upon chronic dosing, the CYP2D6-mediated metabolism of the fluoxetine enantiomers would likely be inhibited, resulting in R-norfluoxetine formation being mediated by CYP2C9 and S-norfluoxetine formation being mediated by multiple high K(m) enzymes.

Specificity of natural and artificial substrates for human Cdc25A.

Cdc25A is a dual-specific protein phosphatase involved in the regulation of the kinase activity of Cdk-cyclin complexes in the eukaryotic cell cycle. To understand the mechanism of this important regulator, we have generated highly purified biochemical reagents to determine the kinetic constants for human Cdc25A with respect to a set of peptidic, artificial, and natural substrates. Cdc25A and its catalytic domain (dN25A) demonstrate very similar kinetics toward the artificial substrates p-nitrophenyl phosphate (k(cat)/K(m) = 15-25 M(-1) s(-1)) and 3-O-methylfluorescein phosphate (k(cat)/K(m) = 1.1-1.3 x 10(4) M(-1) s(-1)). Phospho-peptide substrates exhibit extremely low second-order rate constants and a flat specificity profile toward Cdc25A and dN25A (k(cat)/K(m) = 1 to 10 M(-1) s(-1)). In contrast to peptidic substrates, Cdc25A and dN25A are highly active phosphatases toward the natural substrate, T14- and Y15-bis-phosphorylated Cdk2/CycA complex (Cdk2-pTpY/CycA) with k(cat)/K(m) values of 1.0-1.1 x 10(6) M(-1) s(-1). In the context of the Cdk2-pTpY/CycA complex, phospho-threonine is preferred over phospho-tyrosine by more than 10-fold. The highly homologous catalytic domain of Cdc25c is essentially inactive toward Cdk2-pTpY/CycA. Taken together these data indicate that a significant degree of the specificity of Cdc25 toward its Cdk substrate resides within the catalytic domain itself and yet is in a region(s) that is outside the phosphate binding site of the enzyme.

A novel gene conserved from yeast to humans is involved in sterol biosynthesis.

The ERG28 gene was originally identified by microarray expression profiling as possibly involved in the Saccharomyces cerevisiae sterol pathway. Microarray analyses suggested that the transcription pattern of ERG28 closely followed that of genes involved in sterol synthesis. ERG28 was also found in Schizosaccharomyces pombe and Arabidopsis as well as humans, and in the latter was shown to be highly expressed in adult testis tissue. All four proteins contain potential transmembrane domain(s). Gas chromatography-mass spectrometry analysis of an ERG28-deleted S. cerevisiae strain (which is slow growing but not auxotrophic for ergosterol) indicates a lesion in sterol C-4 demethylation. Sterol profiles indicate accumulation of 3-keto and carboxylic acid sterol intermediates, which are involved in removing the two C-4 methyl groups from the sterol A ring. Similar intermediates have previously been demonstrated to accumulate in erg26 (sterol dehydrogenase/decarboxylase) and erg27 (3-ketoreductase) mutants in yeast. We speculate that the role of the Erg28 protein (Erg28p) may be either to tether Erg26p and Erg27p to the endoplasmic reticulum or to facilitate interaction between these proteins.-Gachotte, D., J. Eckstein, R. Barbuch, T. Hughes, C. Roberts, and M. Bard. A novel gene conserved from yeast to humans is involved in sterol biosynthesis. J. Lipid Res. 2001. 42: 150;-154.