A magnetic continuum in the cobalt-based honeycomb magnet BaCo2(AsO4)2.

Quantum spin liquids (QSLs) are topologically ordered states of matter that host fractionalized excitations. A particular route towards a QSL is via strongly bond-dependent interactions on the hexagonal lattice. A number of Ru- and Ir-based candidate Kitaev QSL materials have been pursued, but all have appreciable non-Kitaev interactions. Using time-domain terahertz spectroscopy, we observed a broad magnetic continuum over a wide range of temperatures and fields in the honeycomb cobalt-based magnet BaCo2(AsO4)2, which has been proposed to be a more ideal version of a Kitaev QSL. Applying an in-plane magnetic field of ~0.5 T suppresses the magnetic order, and at higher fields, applying the field gives rise to a spin-polarized state. Under a 4 T magnetic field that was oriented principally out of plane, a broad magnetic continuum was observed that may be consistent with a field-induced QSL. Our results indicate BaCo2(AsO4)2 is a promising QSL candidate.

ROI-Finder: machine learning to guide region-of-interest scanning for X-ray fluorescence microscopy.

The microscopy research at the Bionanoprobe (currently at beamline 9-ID and later 2-ID after APS-U) of Argonne National Laboratory focuses on applying synchrotron X-ray fluorescence (XRF) techniques to obtain trace elemental mappings of cryogenic biological samples to gain insights about their role in critical biological activities. The elemental mappings and the morphological aspects of the biological samples, in this instance, the bacterium Escherichia coli (E. Coli), also serve as label-free biological fingerprints to identify E. coli cells that have been treated differently. The key limitations of achieving good identification performance are the extraction of cells from raw XRF measurements via binary conversion, definition of features, noise floor and proportion of cells treated differently in the measurement. Automating cell extraction from raw XRF measurements across different types of chemical treatment and the implementation of machine-learning models to distinguish cells from the background and their differing treatments are described. Principal components are calculated from domain knowledge specific features and clustered to distinguish healthy and poisoned cells from the background without manual annotation. The cells are ranked via fuzzy clustering to recommend regions of interest for automated experimentation. The effects of dwell time and the amount of data required on the usability of the software are also discussed.

Membrane adhesion and the formation of heterogeneities: biology, biophysics, and biotechnology.

Membrane adhesion is essential to many vital biological processes. Sites of membrane adhesion are often associated with heterogeneities in the lipid and protein composition of the membrane. These heterogeneities are thought to play functional roles by facilitating interactions between proteins. However, the causal links between membrane adhesion and membrane heterogeneities are not known. Here we survey the state of the field and indicate what we think are understudied areas ripe for development.

Maternally-derived zinc transporters ZIP6 and ZIP10 drive the mammalian oocyte-to-egg transition.

Rapid cellular zinc influx regulates early mammalian development during the oocyte-to-egg transition through modulation of the meiotic cell cycle. Despite the physiological necessity of this zinc influx, the molecular mechanisms that govern such accumulation are unknown. Here we show that the fully grown mammalian oocyte does not employ a transcriptionally based mechanism of zinc regulation involving metal response element-binding transcription factor-1 (MTF-1), as demonstrated by a lack of MTF-1 responsiveness to environmental zinc manipulation. Instead, the mammalian oocyte controls zinc uptake through two maternally derived and cortically distributed zinc transporters, ZIP6 and ZIP10. Targeted disruption of these transporters using several approaches during meiotic maturation perturbs the intracellular zinc quota and results in a cell cycle arrest at a telophase I-like state. This arrest phenocopies established models of zinc insufficiency during the oocyte-to-egg transition, indicating the essential function of these maternally expressed transporters. Labile zinc localizes to punctate cytoplasmic structures in the human oocyte, and ZIP6 and ZIP10 are enriched in the cortex. Altogether, we demonstrate a mechanism of metal regulation required for female gamete development that may be evolutionarily conserved.

Monopolar and dipolar relaxation in spin ice Ho2Ti2O7.

Ferromagnetically interacting Ising spins on the pyrochlore lattice of corner-sharing tetrahedra form a highly degenerate manifold of low-energy states. A spin flip relative to this "spin-ice" manifold can fractionalize into two oppositely charged magnetic monopoles with effective Coulomb interactions. To understand this process, we have probed the low-temperature magnetic response of spin ice to time-varying magnetic fields through stroboscopic neutron scattering and SQUID magnetometry on a new class of ultrapure Ho2Ti2O7 crystals. Covering almost 10 decades of time scales with atomic-scale spatial resolution, the experiments resolve apparent discrepancies between prior measurements on more disordered crystals and reveal a thermal crossover between distinct relaxation processes. Magnetic relaxation at low temperatures is associated with monopole motion through the spin-ice vacuum, while at elevated temperatures, relaxation occurs through reorientation of increasingly spin-like monopolar bound states. Spin fractionalization is thus directly manifest in the relaxation dynamics of spin ice.

Talin at myotendinous junctions.

Junctions formed by skeletal muscles where they adhere to tendons, called myotendinous junctions, are sites of tight adhesion and where forces generated by the cell are placed on the substratum. In this regard, myotendinous junctions and focal contacts of fibroblasts in vitro are analogues. Talin is a protein located at focal contacts that may be involved in force transmission from actin filaments to the plasma membrane. This study investigates whether talin is also found at myotendinous junctions. Protein separations on SDS polyacrylamide gels and immunolabeling procedures show that talin is present in skeletal muscle. Immunofluorescence microscopy using anti-talin indicates that talin is found concentrated at myotendinous junctions and in lesser amounts in periodic bands over nonjunctional regions. Electron microscopic immunolabeling shows talin is a component of the digitlike processes of muscle cells that extend into tendons at myotendinous junctions. These findings indicate that there may be similarities in the molecular composition of focal contacts and myotendinous junctions in addition to functional analogies.

Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles, and development in Dictyostelium.

To investigate the intracellular role of the clathrin heavy chain in living cells, we have used "antisense" RNA to engineer mutant Dictyostelium discoideum cells that are severely deficient in clathrin heavy chain expression. Immunoblots stained with an anti-clathrin heavy chain antiserum revealed that mutant cells contained undetectable amounts of clathrin heavy chain protein. Similarly, Northern blots showed an absence of clathrin heavy chain mRNA. Clathrin heavy chain-deficient Dictyostelium cells were viable, but exhibited growth rates twofold slower than parental cells. Whereas many morphological features of the mutant cells were normal, mutant cells lacked coated pits and coated vesicles. Clathrin-deficient cells were also missing large translucent vacuoles that serve as endosomes and contractile vacuoles. In the absence of clathrin heavy chain, mutant cells displayed three distinct functional defects: (a) impairment in endocytosis of fluid phase markers, but competence in another endocytic pathway, the phagocytosis of solid particles; (b) defects in osmoregulation; and (c) inability to complete the starvation-induced development cycle.

Expression of Dictyostelium myosin tail segments in Escherichia coli: domains required for assembly and phosphorylation.

The assembly of myosins into filaments is a property common to all conventional myosins. The ability of myosins to form filaments is conferred by the tail of the large asymmetric molecule. We are studying cloned portions of the Dictyostelium myosin gene expressed in Escherichia coli to investigate functional properties of defined segments of the myosin tail. We have focused on five segments derived from the 68-kD carboxyl-terminus of the myosin tail. These have been expressed and purified to homogeneity from E. coli, and thus the boundaries of each segment within the myosin gene and protein sequence are known. We identified an internal 34-kD segment of the tail, N-LMM-34, which is required and sufficient for assembly. This 287-amino acid domain represents the smallest tail segment purified from any myosin that is capable of forming highly ordered paracrystals characteristic of myosin. Because the assembly of Dictyostelium myosin can be regulated by phosphorylation of the heavy chain, we have studied the in vitro phosphorylation of the expressed tail segments. We have determined which segments are phosphorylated to a high level by a Dictyostelium myosin heavy chain kinase purified from developed cells. While LMM-68, the 68-kD carboxyl terminus of Dictyostelium myosin, or LMM-58, which lacks the 10-kD carboxyl terminus of LMM-68, are phosphorylated to the same extent as purified myosin, subdomains of these segments do not serve as efficient substrates for the kinase. Thus LMM-58 is one minimal substrate for efficient phosphorylation by the myosin heavy chain kinase purified from developed cells. Taken together these results identify two functional domains in Dictyostelium myosin: a 34-kD assembly domain bounded by amino acids 1533-1819 within the myosin sequence and a larger 58-kD phosphorylation domain bounded by amino acids 1533-2034 within the myosin sequence.

Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.

Metalloregulatory DNA-binding protein encoded by the merR gene: isolation and characterization.

The MerR protein mediates the induction of the mercury resistance phenotype in bacteria; it has been isolated in order to study the effects of metal-ion induced changes in the metabolism of prokaryotic cells at the molecular level. After DNA sequences responsible for negative autoregulation were removed, the 16-kilodalton protein was overproduced and purified to more than 90 percent homogeneity by a salt extraction procedure that yields about 5 milligrams of protein per gram of cells. Complementation data, amino terminal analysis, gel filtration, and deoxyribonuclease I protection studies demonstrate that the purified merR gene product is a dimer under nondenaturing conditions and that it binds specifically to DNA, in the presence and absence of mercury, at a palindromic site which is directly between the -10 and -35 regions of the structural genes and adjacent to its own promoter. These initial results indicate that MerR is a DNA-binding metalloregulatory protein that plays a central role in this heavy metal responsive system and they delineate an operator site in the mer operon.

Transition metals in control of gene expression.

Metalloproteins play structural and catalytic roles in gene expression. The metalloregulatory proteins are a subclass that exerts metal-responsive control of genes involved in respiration, metabolism, and metal-specific homeostasis or stress-response systems, such as iron uptake and storage, copper efflux, and mercury detoxification. Two allosteric mechanisms for control of gene expression were first discovered in metalloregulatory systems: an iron-responsive translational control mechanism for ferritin production and a mercury-responsive DNA-distortion mechanism for transcriptional control of detoxification genes. These otherwise unrelated mechanisms give rise to a rapid physiological response when metal ion concentrations exceed a dangerous threshold. Molecular recognition in these allosteric metal ion receptors is achieved through atypical coordination geometries, cluster formation, or complexes with prosthetic groups, such as sulfide and heme. Thus, many of the inorganic assemblies that otherwise buttress the structure of biopolymers or catalyze substrate transformation in active sites of enzymes have also been adapted to serve sensor functions in the metalloregulatory proteins. Mechanistic studies of these metal-sensor protein interactions are providing new insights into fundamental aspects of inorganic chemistry, molecular biology, and cellular physiology.

Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis.

Intracellular zinc is thought to be available in a cytosolic pool of free or loosely bound Zn(II) ions in the micromolar to picomolar range. To test this, we determined the mechanism of zinc sensors that control metal uptake or export in Escherichia coli and calibrated their response against the thermodynamically defined free zinc concentration. Whereas the cellular zinc quota is millimolar, free Zn(II) concentrations that trigger transcription of zinc uptake or efflux machinery are femtomolar, or six orders of magnitude less than one atom per cell. This is not consistent with a cytosolic pool of free Zn(II) and suggests an extraordinary intracellular zinc-binding capacity. Thus, cells exert tight control over cytosolic metal concentrations, even for relatively low-toxicity metals such as zinc.

Mercury-199 NMR of the metal receptor site in MerR and its protein-DNA complex.

Structural insights have been provided by mercury-199 nuclear magnetic resonance (NMR) into the metal receptor site of the MerR metalloregulatory protein alone and in a complex with the regulatory target, DNA. The one- and two-dimensional NMR data are consistent with a trigonal planar Hg-thiolate coordination environment consisting only of Cys side chains and resolve structural aspects of both metal ion recognition and the allosteric mechanism. These studies establish 199Hg NMR techniques as useful probes of the metal coordination environment of regulatory proteins, copper enzymes, and zinc transcription factor complexes as large as 50 kilodaltons.

Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase.

The copper chaperone for the superoxide dismutase (CCS) gene is necessary for expression of an active, copper-bound form of superoxide dismutase (SOD1) in vivo in spite of the high affinity of SOD1 for copper (dissociation constant = 6 fM) and the high intracellular concentrations of both SOD1 (10 microM in yeast) and copper (70 microM in yeast). In vitro studies demonstrated that purified Cu(I)-yCCS protein is sufficient for direct copper activation of apo-ySOD1 but is necessary only when the concentration of free copper ions ([Cu]free) is strictly limited. Moreover, the physiological requirement for yCCS in vivo was readily bypassed by elevated copper concentrations and abrogation of intracellular copper-scavenging systems such as the metallothioneins. This metallochaperone protein activates the target enzyme through direct insertion of the copper cofactor and apparently functions to protect the metal ion from binding to intracellular copper scavengers. These results indicate that intracellular [Cu]free is limited to less than one free copper ion per cell and suggest that a pool of free copper ions is not used in physiological activation of metalloenzymes.

Metal ion chaperone function of the soluble Cu(I) receptor Atx1.

Reactive and potentially toxic cofactors such as copper ions are imported into eukaryotic cells and incorporated into target proteins by unknown mechanisms. Atx1, a prototypical copper chaperone protein from yeast, has now been shown to act as a soluble cytoplasmic copper(I) receptor that can adopt either a two- or three-coordinate metal center in the active site. Atx1 also associated directly with the Atx1-like cytosolic domains of Ccc2, a vesicular protein defined in genetic studies as a member of the copper-trafficking pathway. The unusual structure and dynamics of Atx1 suggest a copper exchange function for this protein and related domains in the Menkes and Wilson disease proteins.

Accuracy of electrocardiographic criteria for atrial enlargement: validation with cardiovascular magnetic resonance.

BACKGROUND: Anatomic atrial enlargement is associated with significant morbidity and mortality. However, atrial enlargement may not correlate with clinical measures such as electrocardiographic (ECG) criteria. Past studies correlating ECG criteria with anatomic measures mainly used inferior M-mode or two-dimensional echocardiographic data. We sought to determine the accuracy of the ECG to predict anatomic atrial enlargement as determined by volumetric cardiovascular magnetic resonance (CMR). METHODS: ECG criteria for left (LAE) and right atrial enlargement (RAE) were compared to CMR atrial volume index measurements for 275 consecutive subjects referred for CMR (67% males, 51 +/- 14 years). ECG criteria for LAE and RAE were assessed by an expert observer blinded to CMR data. Atrial volume index was computed using the biplane area-length method. RESULTS: The prevalence of CMR LAE and RAE was 28% and 11%, respectively, and by any ECG criteria was 82% and 5%, respectively. Though nonspecific, the presence of at least one ECG criteria for LAE was 90% sensitive for CMR LAE. The individual criteria P mitrale, P wave axis < 30 degrees , and negative P terminal force in V1 (NPTF-V1) > 0.04s.mm were 88-99% specific although not sensitive for CMR LAE. ECG was insensitive but 96-100% specific for CMR RAE. CONCLUSION: The presence of at least one ECG criteria for LAE is sensitive but not specific for anatomic LAE. Individual criteria for LAE, including P mitrale, P wave axis < 30 degrees , or NPTF-V1 > 0.04s.mm are highly specific, though not sensitive. ECG is highly specific but insensitive for RAE. Individual ECG P wave changes do not reliably both detect and predict anatomic atrial enlargement.

Spatially regulated recruitment of clathrin to the plasma membrane during capping and cell translocation.

Clathrin-coated vesicles bud from selected cellular membranes to traffic-specific intracellular proteins. To study the dynamic properties of clathrin-coated membranes, we expressed clathrin heavy chain tagged with green fluorescent protein (GFP) in Dictyostelium cells. GFP-clathrin was functional and retained the native properties of clathrin: the chimeric protein formed classic clathrin lattices on cellular membranes and also rescued phenotypic defects of clathrin null cells. GFP-clathrin distributed into punctate loci found throughout the cytoplasm, on the plasma membrane, and concentrated to a perinuclear location. These clathrin-coated structures were remarkably motile and capable of rapid and bidirectional transport across the cell. We identified two local domains of the plasma membrane as sites for clathrin recruitment in motile cells. First, as cells translocated or changed shape and retracted their tails, clathrin was transiently concentrated on the membrane at the back of the cell tail. Second, as cells capped their cell surface receptors, clathrin was recruited locally to the membrane under the tight cap of cross-linked receptors. This suggests that local sites for clathrin polymerization on specific domains of the plasma membrane undergo rapid and dynamic regulation in motile cells.

Crystal structure of the Atx1 metallochaperone protein at 1.02 A resolution.

BACKGROUND: Metallochaperone proteins function in the trafficking and delivery of essential, yet potentially toxic, metal ions to distinct locations and particular proteins in eukaryotic cells. The Atx1 protein shuttles copper to the transport ATPase Ccc2 in yeast cells. Molecular mechanisms for copper delivery by Atx1 and similar human chaperones have been proposed, but detailed structural characterization is necessary to elucidate how Atx1 binds metal ions and how it might interact with Ccc2 to facilitate metal ion transfer. RESULTS: The 1.02 A resolution X-ray structure of the Hg(II) form of Atx1 (HgAtx1) reveals the overall secondary structure, the location of the metal-binding site, the detailed coordination geometry for Hg(II), and specific amino acid residues that may be important in interactions with Ccc2. Metal ion transfer experiments establish that HgAtx1 is a functional model for the Cu(I) form of Atx1 (CuAtx1). The metal-binding loop is flexible, changing conformation to form a disulfide bond in the oxidized apo form, the structure of which has been solved to 1.20 A resolution. CONCLUSIONS: The Atx1 structure represents the first structure of a metallochaperone protein, and is one of the largest unknown structures solved by direct methods. The structural features of the metal-binding site support the proposed Atx1 mechanism in which facile metal ion transfer occurs between metal-binding sites of the diffusible copper-donor and membrane-tethered copper-acceptor proteins. The Atx1 structural motif represents a prototypical metal ion trafficking unit that is likely to be employed in a variety of organisms for different metal ions.

Imaging skills for transthoracic echocardiography in cardiology fellows: The value of motion metrics.

BACKGROUND: Proficiency in transthoracic echocardiography (TTE) requires an integration of cognitive knowledge and psychomotor skills. Whereas cognitive knowledge can be quantified, psychomotor skills are implied after repetitive task performance. We applied motion analyses to evaluate psychomotor skill acquisition during simulator-based TTE training. METHODS AND RESULTS: During the first month of their fellowship training, 16 cardiology fellows underwent a multimodal TTE training program for 4 weeks (8 sessions). The program consisted of online and live didactics as well as simulator training. Kinematic metrics (path length, time, probe accelerations) were obtained at the start and end of the course for 8 standard TTE views using a simulator. At the end of the course TTE image acquisition skills were tested on human models. After completion of the training program the trainees reported improved self-perceived comfort with TTE imaging. There was also an increase of 8.7% in post-test knowledge scores. There was a reduction in the number of probe accelerations [median decrease 49.5, 95% CI = 29-73, adjusted P < 0.01], total time [median decrease 10.6 s, 95% CI = 6.6-15.5, adjusted P < 0.01] and path length [median decrease 8.8 cm, 95% CI = 2.2-17.7, adjusted P < 0.01] from the start to the end of the course. During evaluation on human models, the trainees were able to obtain all the required TTE views without instructor assistance. CONCLUSION: Simulator-derived motion analyses can be used to objectively quantify acquisition of psychomotor skills during TTE training. Such an approach could be used to assess readiness for clinical practice of TTE.