Gap-like feature observed in the non-magnetic topological insulators.

Non-magnetic gap at the Dirac point of topological insulators remains an open question in the field. Here, we present angle-resolved photoemission spectroscopy experiments performed on Cr-doped Bi2Se3 and showed that the Dirac point is progressively buried by the bulk bands and a low spectral weight region in the vicinity of the Dirac point appears. These two mechanisms lead to spectral weight suppression region being mistakenly identified as an energy gap in earlier studies. We further calculated the band structure and found that the original Dirac point splits into two nodes due to the impurity resonant states and the energy separation between the nodes is the low density of state region which appears to be like an energy gap in potoemission experiments. We supported our arguments by presenting photoemission experiments carried out with on- and off- resonant photon energies. Our observation resolves the widely debated questions of apparent energy gap opening at the Dirac point without long range ferromagnetic order in topological insulators.

Nematicity and Charge Order in Superoxygenated La_{2-x}Sr_{x}CuO_{4+y}.

In this Letter, we report a resonant x-ray scattering measurement of stripelike charge order in the 1/8th doped component of electronically phase-separated, orthorhombic La_{2}CuO_{4+y}. This observation is coupled to the absence of any resonant (001) peak, which at different resonant energies has been identified with the presence of low-temperature-tetragonal-like structural tilt patterns or nematicity in the CuO planes. Thus, we provide evidence that structural pinning is not necessary for the formation of static charge stripes and that the relationship between charge nematicity and stripes may not be simple.

Structural phase diagram for ultra-thin epitaxial Fe3O4 / MgO(0 0 1) films: thickness and oxygen pressure dependence.

A systematic investigation of the thickness and oxygen pressure dependence for the structural properties of ultra-thin epitaxial magnetite (Fe3O4) films has been carried out; for such films, the structural properties generally differ from those for the bulk when the thickness ⩽10 nm. Iron oxide ultra-thin films with thicknesses varying from 3 nm to 20 nm were grown on MgO (0 0 1) substrates using molecular beam epitaxy under different oxygen pressures ranging from 1 × 10(-7) torr to 1 × 10(-5) torr. The crystallographic and electronic structures of the films were characterized using low energy electron diffraction (LEED) and x-ray photoemission spectroscopy (XPS), respectively. The quality of the epitaxial Fe3O4 ultra-thin films was judged by magnetic measurements of the Verwey transition, along with complementary XPS spectra. It was observed that under the same growth conditions the stoichiometry of ultra-thin films under 10 nm transforms from the Fe3O4 phase to the FeO phase. In this work, a phase diagram based on thickness and oxygen pressure has been constructed to explain the structural phase transformation. It was found that high-quality magnetite films with thicknesses ⩽20 nm formed within a narrow range of oxygen pressure. An optimal and controlled growth process is a crucial requirement for the accurate study of the magnetic and electronic properties for ultra-thin Fe3O4 films. Furthermore, these results are significant because they may indicate a general trend in the growth of other oxide films, which has not been previously observed or considered.

An appraisal of methods for the analysis of longitudinal categorical data with MAR drop-outs.

A number of methods for analysing longitudinal ordinal categorical data with missing-at-random drop-outs are considered. Two are maximum-likelihood methods (MAXLIK) which employ marginal global odds ratios to model associations. The remainder use weighted or unweighted generalized estimating equations (GEE). Two of the GEE use Cholesky-decomposed standardized residuals to model the association structure, while another three extend methods developed for longitudinal binary data in which the association structures are modelled using either Gaussian estimation, multivariate normal estimating equations or conditional residuals. Simulated data sets were used to discover differences among the methods in terms of biases, variances and convergence rates when the association structure is misspecified. The methods were also applied to a real medical data set. Two of the GEE methods, referred to as Cond and ML-norm in this paper and by their originators, were found to have relatively good convergence rates and mean squared errors for all sample sizes (80, 120, 300) considered, and one more, referred to as MGEE in this paper and by its originators, worked fairly well for all but the smallest sample size, 80.

The evolution of strategy variation: will an ESS evolve?

Evolutionarily stable strategy (ESS) models are widely viewed as predicting the strategy of an individual that when monomorphic or nearly so prevents a mutant with any other strategy from entering the population. In fact, the prediction of some of these models is ambiguous when the predicted strategy is "mixed", as in the case of a sex ratio, which may be regarded as a mixture of the subtraits "produce a daughter" and "produce a son." Some models predict only that such a mixture be manifested by the population as a whole, that is, as an "evolutionarily stable state"; consequently, strategy monomorphism or polymorphism is consistent with the prediction. The hawk-dove game and the sex-ratio game in a panmictic population are models that make such a "degenerate" prediction. We show here that the incorporation of population finiteness into degenerate models has effects for and against the evolution of a monomorphism (an ESS) that are of equal order in the population size, so that no one effect can be said to predominate. Therefore, we used Monte Carlo simulations to determine the probability that a finite population evolves to an ESS as opposed to a polymorphism. We show that the probability that an ESS will evolve is generally much less than has been reported and that this probability depends on the population size, the type of competition among individuals, and the number of and distribution of strategies in the initial population. We also demonstrate how the strength of natural selection on strategies can increase as population size decreases. This inverse dependency underscores the incorrectness of Fisher's and Wright's assumption that there is just one qualitative relationship between population size and the intensity of natural selection.

A new statistical test of fitness set data from reciprocal transplant experiments involving intermediate phenotypes.

Experimental biologists use reciprocal transplant experiments (RTEs) involving divergent forms to test hypotheses about fitness trade-offs across, and local adaptation to, native environments. Additional evolutionary hypotheses about diversifying selection, the evolution of specialization, and the coexistence of specialists and generalists are only testable when the RTE also includes intermediate (or alternatively generalist) forms. Environmental variation makes such RTEs challenging, and so strategies that increase their effectiveness are useful. Here, we focus on improvements to the efficiency of RTEs involving intermediate forms with respect to the experimental design and the analysis of the resulting data. We provide a likelihood ratio-based test that offers increased statistical power and robustness relative to another test involving nonlinear regression, when used both for simulated data sets and for data from a study of two divergent fish species and their hybrids transplanted between two lake habitats. The test can be used with unequal numbers of observations, unequal variances, and binomial-type survival data and other nonnormal data. Simulations suggest that having equal numbers of experimental units in each phenotype-environment combination is reasonable. The intentional pairing of observations between environmental conditions (by using clones, full sibs, or half-sibs) is beneficial when paired observations have fitnesses that are negatively related between conditions but is detrimental with positive relatedness. Our methods can be extended to study more than two divergent forms.

Increased power with modified forms of the Levene (Med) test for heterogeneity of variance.

While the conventional Levene (Med) test is a widely used and robust test for detecting heterogeneity of variance, it does not take notice of either the linear dependencies among the residuals involved or the possibility of a mean (or median)-variance relationship. This paper explores the substantial improvements in power possible by investigating the benefits both of removing such linear dependencies (structural zeros) and of modeling (even roughly) such relationships.

Cell density and contraction regulate p38 MAP kinase-dependent responses in neonatal rat cardiac myocytes.

In vitro cardiac myocyte hypertrophy is characterized by increased cell size, sarcomere organization, and induction of several genes including atrial natriuretic factor (ANF). The hypertrophic growth program has been associated with activation of various mitogen-activated protein kinase (MAP) kinase family members, one of which is a stress kinase, p38. In this study, we found that the p38-specific inhibitor SB-203580 failed to inhibit phenylephrine-induced ANF-driven gene expression in low-density myocyte cultures but did inhibit gene expression in higher density cultures. Dense myocyte cultures also had a higher metabolic activity and contraction rate than cells plated at low density. We found that mimicking this effect by rapid electrical pacing activated ANF-driven gene expression and that this expression was inhibited by inactivation of p38. However, addition of SB-203580 at time points ranging between 1 and 72 h suggests that the effect of p38 on the ANF promoter may be both direct and indirect. Electrical pacing induced a small, but consistent, increase in p38 phosphorylation (phospho-p38) at time points ranging from 30 min to 4 h, but at later times phospho-p38 levels were reduced. When myocytes were treated with phenylephrine or electrically paced in the presence of the p38 inhibitor, there was little discernible change in morphology or rates of protein synthesis from DMSO-treated cells at 48 or 72 h. These data indicate that cell density and myocyte contraction may modulate p38-dependent pathways for ANF gene expression, but these pathways may not be direct and have limited effects on hypertrophic morphology.

A low-affinity serum response element allows other transcription factors to activate inducible gene expression in cardiac myocytes.

Hypertrophic growth of cardiac muscle cells is induced by a variety of physiological and pathological stimuli and is associated with a number of changes, including activation of genes such as atrial natriuretic factor. We found that two serum response element (SRE)-like DNA elements, one of which does not meet the consensus sequence and binds serum response factor (SRF) with low affinity, regulate the activity of this promoter. Surprisingly, the ability to induce the promoter by two different physiologic stimuli, as well as various activated transcription factors, including SRF-VP16, was primarily dependent upon the nonconsensus rather than the consensus SRE. This SRE controls the induction of gene expression via an unusual mechanism in that it is required to allow some, but not all, active transcription factors at unrelated sites on the promoter to stimulate gene expression. Thus, in addition to regulation of SRF activity by growth stimuli, regulation of a low-affinity SRE element controls inducible gene expression by modulating the ability of other transcription factors to stimulate the transcription machinery.

Identification of yeast proteins from two-dimensional gels: working out spot cross-contamination.

With the complete sequence of the yeast genome now available, efforts by many laboratories are underway to identify each of the spots on two-dimensional (2-D) gels corresponding to the most abundant yeast proteins. The high mass accuracy now attainable using matrix assisted laser desorption/ionization (MALDI)-mass spectrometry equipped with delayed extraction simplifies the process of identification, such that many spots can be unambiguously identified in a short period of time merely by using peptide mass fingerprinting and generally available database matching programs. Although it is not always possible to match spots between gels run by different laboratories, proteins generally yield the same abundant proteolytic fragments when tryptic digestions are performed. Databases containing these signature peptides not only simplify the task of reidentifying proteins from different gels, but also make it possible to identify small amounts of cross-contaminating proteins from different spots, as well as common extraneous contaminants such as human keratins. In this paper, we present data on the identification of > 20 previously unreported yeast proteins from 2-D gels. Some novel proteins were identified from randomly analyzed spots. Focusing on 14 spots in a narrow-pH-range gel, we demonstrate how organizing peak-table data and peptide match-list data into databases enables the identification of a larger percentage of the peaks.

Ras and rho are required for galphaq-induced hypertrophic gene expression in neonatal rat cardiac myocytes.

The hypertrophic response is characterized by increased myofibril/sarcomere organization, induction of the cardiac specific atrial natriuretic factor (ANF) and myosin light chain-2 (MLC-2v) genes, and an increase in total cell volume. The alpha1-adrenergic agonist phenylephrine induces both the morphological and biochemical markers of hypertrophy in cultured neonatal rat ventricular cardiomyocytes. Previous studies have suggested a functional requirement for the heterotrimeric G-protein, Galphaq, for a subset of the hypertrophic phenotypes. The small GTPases Ras and Rho have also been implicated in phenylephrine-induced hypertrophy. To further delineate the role of Galphaq in hypertrophy, a constitutively active mutant of Galphaq was transiently transfected in primary rat ventricular cardiomyocytes. This molecule was sufficient to induce ANF-, AP1-, and MLC-2-driven gene expression. Co-transfection of Galphaq and dominant negative Ras or dominant negative Raf resulted in dose-dependent inhibition of ANF-driven expression. Both dominant negative Rho, and the Rho inhibitor C3-transferase, also attenuated Galphaq- and Ras-induced ANF-driven gene expression. Cells transfected with active Galphaq did not show a detectable increase in activation of the mitogen activated protein kinases ERK or SAPK. However, activity of the MAP-kinases appears to be important for Galphaq-induced gene expression since the MAP-kinase phosphatase Clone 100 and catalytically inactive SAPK strongly inhibited Galphaq-induced ANF expression. Thus, our studies indicate Galphaq-induced hypertrophic gene expression requires the small G-proteins Ras and Rho. The data also indicates that Galphaq mediated gene expression is dependent on functional MAP-kinases and that multiple signaling pathways contribute to Galphaq-mediated cardiac cell hypertrophy.

Multilocus evolutionarily stable strategy models: additive effects.

A common reservation about Evolutionarily Stable Strategy analyses is that they fail to account explicitly for Mendelian genetics, and therefore may produce biologically unfeasible predictions. This note shows that Evolutionarily Stable Strategy and multilocus genetic analyses agree for one simple model of frequency-dependent selection under the assumption that the effects of alleles and loci are completely additive. A numeric example illustrated the concepts and results involved.

Accurate mass measurements using MALDI-TOF with delayed extraction.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is now an essential tool in biopolymer analysis. Sensitivity and mass range are unsurpassed, but mass measurement accuracy and resolution have been limited. With delayed extraction and a reflecting analyzer, mass measurements using MALDI-TOF can be made with an accuracy of a few parts per million (ppm). It is possible to distinguish Lys from Gln in peptides, and to determine the elemental composition of smaller molecules (mass 100-500). In database searching strategies, a smaller mass window, resulting from an increase in mass accuracy, greatly decreases the number of possible candidates. Mass measurement accuracy with errors less than 5 ppm is demonstrated on a mixture of 12 peptides ranging in mass from ca. 900 to 3700 Da. Mass measurements on 13 peaks in an unseparated tryptic digest of myoglobin gave results with an overall average error less than 3.5 ppm, with a maximum error of 7 ppm.

Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: implications for proteome mapping.

Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of alpha- and beta-chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa alpha-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.

Primary structures for a mammalian cellular and serum copper amine oxidase.

The 6-hydroxydopa quinone-containing active site peptide from bovine serum amine oxidase has been found to be highly homologous to a segment of a cloned human kidney amiloride-binding protein (Barbry, P., Champe, M., Chassande, O., Munemitsu, S., Champigny, G., Lingueglia, E., Maes, P., Frelin, C., Tartar, A., Ullrich, A., and Lazdunski, M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7347-7351). Additionally, a second 38-residue tryptic peptide (peptide XI) isolated from bovine serum amine oxidase shows 82% identity with a portion near the carboxyl terminus of the human kidney amiloride-binding protein. When an extended active site peptide was isolated from porcine kidney diamine oxidase (Janes, S. M., Palcic, M. M., Scaman, C. H., Smith, A. J., Brown, D. E., Dooley, D. M., Mure, M., and Klinman, J. P. (1992) Biochemistry 31, 12147-12154), it was found to be fully contained in the human kidney amiloride-binding protein. Examination of amiloride binding to bovine serum amine oxidase and porcine kidney diamine oxidase reveals dissociation constants of 196 and 9.1 microM, respectively. Taken together, these findings indicate that the cDNA isolated for human kidney amiloride-binding protein encodes a human kidney diamine oxidase. Two oligonucleotides, based on the tryptic peptide XI and active-site peptide of bovine serum amine oxidase, were used to amplify a portion of cDNA from a commercial bovine liver cDNA library through the use of the polymerase chain reaction. A full-length clone (2.7 kilobase pairs) for bovine serum amine oxidase was subsequently obtained through screening of the same cDNA library with the amplified 0.7-kilobase pair cDNA. These studies provide the first primary sequences for a mammalian cellular and serum copper amine oxidase. Computer alignment of amine oxidase cDNA-derived protein sequences reveals three conserved histidine residues, which are likely to be ligands to copper.

Mass spectrometric characterization of a series of adenosylated peptides acting as bisubstrate analogs of protein kinases.

We are currently developing strategies to synthesize bisubstrate analogs as potential inhibitors of serine and tyrosine protein kinases; several such analogs have been synthesized. The initial target proteins were the cAMP dependent protein kinase (cAPK) and the Ca(+2)/calmodulin dependent protein kinase (CaM kiiase II). These bisubstrate analogs were based on either known peptide substrates such as kemptide, a seven amino acid peptide substrate of cAPK, or on inhibitory peptides such as a seventeen amino acid peptide encompassing the autoinhibitory domain of CaM kinase II. Peptides containing a single phosphoserine group were first synthesized and then adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), or adenosine 5'-triphosphate (ATP) was coupled through the serine phosphate with prior activation by 1,1-carbonyldiimidazole using either a solution or solid phase reaction scheme. In this current study, we report the characterization of the bisubstrate analogs by liquid secondary ionization mass spectrometry (LSIMS), matrix-assisted laser desorption mass spectrometry (MALDI), and tandem mass spectrometry (MS/MS).In the positive-ion mode, the LSIMS spectra of the bisubstrate analogs yielded a series of molecular ions containing mono-, di-, and trivalent cation adducts. Cation adducts were absent in the negative-ion mode where the dominant species were deprotonated molecular ions, [M - H](-), making this latter technique more useful for confirming product identity and assessing purity. Analysis of these compounds by MALDI in both the positive- and negative-ion modes yielded molecular ions which also contained metal ion adducts, although they were limited primarily to Fe(+2) adducts. Unlike LSIMS, the MALDI spectra showed no evidence for the elimination of the phosphoadenosine or other structural moieties. When these compounds were subjected to high energy collision-induced dissociation (CID), the dominant fragmentation pathways under positive-ion MS/MS conditions resulted from cleavage of the phosphate linkages to the adenosine moiety with charge retention on the peptide, although a major peak for 5'-deoxyadenosine was also seen at m/z 250. Charge retention in the negative-ion mode was most pronounced for ion fragments containing the highly acidic phosphate moieties and yielded phosphoadenosine related ions, for example, (AMP-H)(-), (AMP-H-H2O)(-), (ADP-H)(-), etc., as well as ions originating from the phosphate linker such as PO3 (-), H2PO4 (-), HP2O6 (-), H3P2O7 (-), and H2P3O9 (-). The largest phosphoadenosine ion in the negative-ion CID spectra for each bisubstrate analog, for example, m/z 426 (ADP-H)(-), m/z 506 (ATP-H)(-), or m/z 586 (AP4-H)(-), indicated that the desired covalent modification had been formed between the phosphoserine and APn moieties.

Low-mass ions produced from peptides by high-energy collision-induced dissociation in tandem mass spectrometry.

High-energy collision-induced dissociation (CID) mass spectrometry provides a rapid and sensitive means for determining the primary sequence of peptides. The low-mass region (below mass 300) of a large number of tandem CID spectra of peptides has been analyzed. This mass region contains several types of informative fragment ions, including dipeptide ions, immonium ions, and other related ions. Useful low-mass ions are also present in negative-ion CID spectra. Immonium ions (general structure [H2N=CH-R](+), where R is the amino acid side chain) and related ions characteristic of specific amino acid residues give information as to the presence or absence of these residues in the peptide being analyzed. Tables of observed immonium and reiated ions for the 20 standard amino acids and for a number of modified amino acids are presented. A database consisting of 228 high-energy CID spectra of peptides has been established, and the frequency of occurrence of various ions indicative of specific ammo acid residues has been determined. Two model computer-aided schemes for analysis of the ammo-acid content of unknown peptides have been developed and tested against the database.