The Smc5/6 Core Complex Is a Structure-Specific DNA Binding and Compacting Machine.

The structural organization of chromosomes is a crucial feature that defines the functional state of genes and genomes. The extent of structural changes experienced by genomes of eukaryotic cells can be dramatic and spans several orders of magnitude. At the core of these changes lies a unique group of ATPases-the SMC proteins-that act as major effectors of chromosome behavior in cells. The Smc5/6 proteins play essential roles in the maintenance of genome stability, yet their mode of action is not fully understood. Here we show that the human Smc5/6 complex recognizes unusual DNA configurations and uses the energy of ATP hydrolysis to promote their compaction. Structural analyses reveal subunit interfaces responsible for the functionality of the Smc5/6 complex and how mutations in these regions may lead to chromosome breakage syndromes in humans. Collectively, our results suggest that the Smc5/6 complex promotes genome stability as a DNA micro-compaction machine.

Bacillus subtilis RecA with DprA-SsbA antagonizes RecX function during natural transformation.

Bacillus subtilis DprA and RecX proteins, which interact with RecA, are crucial for efficient chromosomal and plasmid transformation. We showed that RecA, in the rATP·Mg2+ bound form (RecA·ATP), could not compete with RecX, SsbA or SsbB for assembly onto single-stranded (ss)DNA, but RecA·dATP partially displaced these proteins from ssDNA. RecX promoted reversible depolymerization of preformed RecA·ATP filaments. The two-component DprA-SsbA mediator reversed the RecX negative effect on RecA filament extension, but not DprA or DprA and SsbB. In the presence of DprA-SsbA, RecX added prior to RecA·ATP inhibited DNA strand exchange, but this inhibition was reversed when RecX was added after RecA. We propose that RecA nucleation is more sensitive to RecX action than is RecA filament growth. DprA-SsbA facilitates formation of an active RecA filament that directly antagonizes the inhibitory effects of RecX. RecX and DprA enable chromosomal transformation by altering RecA filament dynamics. DprA-SsbA and RecX proteins constitute a new regulatory network of RecA function. DprA-SsbA contributes to the formation of an active RecA filament and directly antagonizes the inhibitory effects of RecX during natural transformation.

Dependence of the structure and mechanics of metaphase chromosomes on oxidized cysteines.

We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues may play a structural role in mitotic chromosome organization, in accord with classical studies by Dounce et al. (J Theor Biol 42:275-285, 1973) and Sumner (J Cell Sci 70:177-188, 1984a). Human chromosomes isolated into buffer unfold when exposed to dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In micromanipulation experiments which allow us to examine the mechanics of individual metaphase chromosomes, we have found that the gel-like elastic stiffness of native metaphase chromosomes is dramatically suppressed by DTT and TCEP, even before the chromosomes become appreciably unfolded. We also report protein labeling experiments on human metaphase chromosomes which allow us to tag oxidized and reduction-sensitive cysteine residues. PAGE analysis using fluorescent labels shows a small number of labeled bands. Mass spectrometry analysis of similarly labeled proteins provides a list of candidates for proteins with oxidized cysteines involved in chromosome organization, notably including components of condensin I, cohesin, the nucleosome-interacting proteins RCC1 and RCC2, as well as the RNA/DNA-binding protein NONO/p54NRB.

Age-associated alterations in the micromechanical properties of chromosomes in the mammalian egg.

PURPOSE: The incidence of aneuploidy in eggs from women of advanced reproductive age can exceed 60%, making the mammalian egg a unique model system to study the mechanisms of chromosome segregation errors. METHODS: Here we applied a novel biophysical chromosome stretching approach to quantify mechanical stiffness of meiotic chromosomes in the mammalian egg and then documented how these properties changed in a mouse model of physiologic reproductive aging. RESULTS: We found significant differences in chromosome micromechanics, and thus in higher order chromosome structure, coincident with advanced reproductive age, a time that is also unequivocally associated with an increase in egg aneuploidy. CONCLUSIONS: These findings have important implications for both reproductive and cancer biology where aneuploidy plays a central role in aging related disease states.

Three-dimensional speckle tracking echocardiography.

Speckle tracking echocardiography (STE) was popularized in the first decade of this century. Analysis of cardiac mechanics has been the focus of ultrasonics, and the breakthrough came with STE. Beyond analysis solely of left ventricular ejection fraction, STE allows the assessment of various pathophysiologies, including myocardial layer-specific myocardial function, twist and rotation, and dyssynchrony. Recent developments in the technology have resulted in commercially available 3-dimensional (D)-STE systems. Through experimental studies and clinical investigations, the reliability and feasibility of 3D-STE-derived data have been validated, and the advantages of 3D-STE over 2D-STE have been revealed. In addition, because of the 3D nature of the technology, 3D-STE provides novel deformation parameters (ie, 3D-strain and area change ratio) that have the potential for more accurate assessment of overall and regional myocardial function. Recently, various preliminary studies using 3D-STE have reported on myocardial characteristics, novel mechanics in the left ventricle, prediction of therapeutic effects, observations of cardiac function through interventions, and challenges for left atrial and right ventricular functions. In this review, we focus on the features of the methodology, validation, and clinical application of 3D-ST.

Left ventricular activation imaging by 3-dimensional speckle-tracking echocardiography. Comparison with electrical activation mapping.

BACKGROUND: Activation imaging with 3-dimensional speckle-tracking echocardiography (3D-STE) aims to visualize the time required for the onset of regional contraction from QRS onset. We hypothesized that the optimal setting of activation imaging was associated with electrical activation. This study was designed to determine an optimal setting of activation imaging with 3D-STE in comparison with that of a voltage mapping system and to assess the feasibility of this imaging method. METHODS AND RESULTS: We enrolled 7 patients who underwent electrical voltage mapping. Regional deformation was measured by area change ratio (ACR) with 3D-STE. Activation imaging data were obtained at 10%, 25%, 50%, and 100% of maximal ACR values as the threshold for onset of regional contraction. Duration of LV electrical intraventricular activation time (IVAT(electrical)) by voltage mapping and mechanical IVAT (IVAT(mechanical)) by activation imaging was defined as the time difference between the first and latest endocardial activation sites. We obtained 21 data sets under various conduction patterns and pacing configurations. The strongest correlation between IVAT(mechanical) and IVAT(electrical) was observed at 25% of maximal ACR values (IVAT(electrical)=0.47 * IVAT(mechanical)+20, R=0.80, P<0.001). Concordance of the first and latest activated segments between activation imaging and voltage mapping was 90.5% at this setting (19 studies). CONCLUSIONS: Activation imaging with 3D-STE may be a feasible noninvasive method of dyssynchrony imaging based on electromechanical coupling.

Application of 3-dimensional speckle tracking imaging to the assessment of right ventricular regional deformation.

BACKGROUND: The aim of this study was to carry out 3-dimensional speckle tracking imaging (3DSTI) of the right ventricle (RV) and evaluate RV regional wall deformation. METHODS AND RESULTS: 3DSTI of the RV was performed in 35 normal subjects, 8 patients with arrhythmogenic right ventricular cardiomyopathy, and 8 patients with pulmonary arterial hypertension. Peak systolic area change ratio and regional contraction timing relative to global systolic time (time to peak strain/time to end-systole×100) were measured in each segment. Good-quality images were acquired of the inflow segment in 87%, apex in 87%, outflow in 57%, and septum in 94% of the 35 normal subjects. In normal subjects, peak systolic area change ratio of the inflow anterior wall was -41±14%; inflow inferior wall, -35±9%; apical anterior wall, -41±10%; apical inferior wall, -31±11%; outflow, -31±9%; and septum wall, -36±11%. Contraction timing of the apical anterior wall and septum wall were earlier than those of other segments. In patients with RV dysfunction, 3DSTI indicated low peak systolic area change ratio in the damaged area. CONCLUSIONS: RV 3DSTI indicated segmental heterogeneity in magnitude and timing of RV contraction. 3DSTI may be a promising modality for providing precise quantitative information on complex RV wall motion.

Significant improvement of left atrial and left atrial appendage function after catheter ablation for persistent atrial fibrillation.

BACKGROUND: The long-term effects of catheter ablation (CA) on the left atrium and left atrial appendage (LAA) are unknown in persistent atrial fibrillation (AF). This study investigated left atrial (LA) reverse remodeling and evolution of LA/LAA function after successful CA for persistent AF and identified predictors for maintenance of sinus rhythm (SR) and LA reverse remodeling. METHODS AND RESULTS: CA was performed in 123 patients with persistent AF. LA volumes, LA strain and LAA wall velocity were assessed both at baseline and at 12 months after ablation. Patients who maintained SR were divided into 2 groups according to whether LA volume decreased by ≥15% at follow-up (responders) or not (non-responders). During a follow-up period of 18±2 months, AF recurred in 45 patients (37%). Of the remaining 78 patients (63%) without recurrent AF, 62 patients (79%) were classified as responders. LA/LAA function significantly improved and the prevalence of spontaneous echo contrast decreased only in responders at follow-up. LA systolic strain and LAA wall velocity were independent predictors of both maintenance of SR (odds ratio [OR], 2.57; P=0.003; OR, 3.02; P=0.002, respectively) and LA reverse remodeling (OR, 4.44; P=0.007; OR, 3.52; P=0.01, respectively). CONCLUSIONS: Successful CA is associated with LA reverse remodeling and LA/LAA functional recovery in patients with persistent AF. LA systolic strain and LAA wall velocity at baseline predicted both maintenance of SR and LA reverse remodeling.

Dislodged and mechanically distorted stent stuck within a previously implanted drug-eluting stent.

Coronary stent loss during percutaneous coronary intervention is rare and is often associated with significant morbidity. Several retrieval techniques, overlying stent deployment and crushing, and surgical removal can be used to deal with a stent lost in the coronary system. We successfully treated a dislodged and mechanically distorted coil stent stuck within a previously implanted drug-eluting stent (DES) by stent-crush technique. This case might provide insight into the mechanisms responsible for the longitudinal fragility of cobalt alloy and coil-structure stents and stent fracture of DES. In the DES era, careful attention should be paid to such complications when attempting to deliver a stent to a distal vessel through a pre-existing DES.

Nanomedicine for cancer: lipid-based nanostructures for drug delivery and monitoring.

Recent advances in nanotechnology, materials science, and biotechnology have led to innovations in the field of nanomedicine. Improvements in the diagnosis and treatment of cancer are urgently needed, and it may now be possible to achieve marked improvements in both of these areas using nanomedicine. Lipid-coated nanoparticles containing diagnostic or therapeutic agents have been developed and studied for biomedical applications and provide a nanomedicine strategy with great potential. Lipid nanoparticles have cationic headgroups on their surfaces that bind anionic nucleic acids and contain hydrophobic drugs at the lipid membrane and hydrophilic drugs inside the hollow space in the interior. Moreover, researchers can design nanoparticles to work in combination with external stimuli such as magnetic field, light, and ionizing radiation, which adds further utility in biomedical applications. In this Account, we review several examples of lipid-based nanoparticles and describe their potential for cancer treatment and diagnosis. (1) The development of a lipid-based nanoparticle that included a promoter-enhancer and transcriptional activator greatly improved gene therapy. (2) The addition of a radiosensitive promoter to lipid nanoparticles was sufficient to confer radioisotope-activated expression of the genes delivered by the nanoparticles. (3) We successfully tailored lipid nanoparticle composition to increase gene transduction in scirrhous gastric cancer cells. (4) When lipophilic photosensitizing molecules were incorporated into lipid nanoparticles, those particles showed an increased photodynamic cytotoxic effect on the target cancer. (5) Coating an Fe(3)O(4) nanocrystal with lipids proved to be an efficient strategy for magnetically guided gene-silencing in tumor tissues. (6) An Fe(16)N(2)/lipid nanocomposite displayed effective magnetism and gene delivery in cancer cells. (7) Lipid-coated magnetic hollow capsules carried aqueous anticancer drugs and delivered them in response to a magnetic field. (8) Fluorescent lipid-coated and antibody-conjugated magnetic nanoparticles detected cancer-associated antigen in a microfluidic channel. We believe that the continuing development of lipid-based nanomedicine will lead to the sensitive minimally invasive treatment of cancer. Moreover, the fusion of different scientific fields is accelerating these developments, and we expect these interdisciplinary efforts to have considerable ripple effects on various fields of research.

Tissue Doppler imaging dyssynchrony parameter derived from the myocardial active wall motion improves prediction of responders for cardiac resynchronization therapy.

BACKGROUND: The aim of this study was to propose modified tissue Doppler imaging (TDI) parameters derived from the first active wall motion and to assess them for the better prediction of cardiac resynchronization therapy (CRT) responders in comparison with to original TDI parameters. METHODS AND RESULTS: In 61 patients with CRT, time from QRS onset to peak velocities by TDI (Ts), which were derived from active wall motion identified by longitudinal strain rate (LSR) value, were assessed. Time from QRS onset to the negative peak of LSR (TLSR) was also assessed. Modified standard deviation of Ts in 12 left ventricular (LV) segments (Ts-SD), that of TLSR (TLSR-SD), differences of Ts between septum and lateral wall (Ts-SL), and that of TLSR (TLSR-SL) were calculated. Original Ts-SD and Ts-SL were calculated by previously described methods. Responders were defined as patients with LV end-systolic volume reduction (>15%) at 6 months after CRT: 35 patients (57%) were identified as CRT responders. Area under the receiver-operating characteristics curve (AUC) of modified Ts-SD (0.87) was significantly higher than that of Ts-SD (0.65), Ts-SL (0.62), and TLSR-SL (0.69). AUC of modified Ts-SL was significantly higher than those of Ts-SD, and Ts-SL. AUC of TLSR-SD (0.82) also was significantly higher than that of Ts-SD. CONCLUSIONS: Modified TDI dyssynchrony parameters derived from the first active wall motion improve the ability to predict responders to CRT.

Micromechanics of human mitotic chromosomes.

Eukaryote cells dramatically reorganize their long chromosomal DNAs to facilitate their physical segregation during mitosis. The internal organization of folded mitotic chromosomes remains a basic mystery of cell biology; its understanding would likely shed light on how chromosomes are separated from one another as well as into chromosome structure between cell divisions. We report biophysical experiments on single mitotic chromosomes from human cells, where we combine micromanipulation, nano-Newton-scale force measurement and biochemical treatments to study chromosome connectivity and topology. Results are in accord with previous experiments on amphibian chromosomes and support the 'chromatin network' model of mitotic chromosome structure. Prospects for studies of chromosome-organizing proteins using siRNA expression knockdowns, as well as for differential studies of chromosomes with and without mutations associated with genetic diseases, are also discussed.

Ferromagnetic FePt-nanoparticles/polycation hybrid capsules designed for a magnetically guided drug delivery system.

The present Article describes the synthesis of ferromagnetic capsules approximately 330 nm in diameter with a nanometer-thick shell to apply to magnetic carriers in a magnetically guided drug delivery system. The magnetic shell of 5 nm in thickness is a nanohybrid, composed of ordered alloy FePt nanoparticles of approximately 3-4 nm in size and a polymer layer of a cationic polyelectrolyte, poly(diaryldimethylammonium chloride) (PDDA). The magnetic capsules have an excellent capacity for carrying medical drugs and genes. Surface-modified silica particles with PDDA were used as a template for the capsules. FePt nanoparticles were deposited on the PDDA-modified silica particles through a polyol method followed by dissolving the silica particles with a NaOH solution, resulting in the formation of the magnetic capsules as the final product. A three-dimensional hollow structure is maintained by the nanohybrid shell. The FePt-nanoparticles/PDDA nanohybrid shell also exhibits a ferromagnetic feature at room temperature because the FePt nanoparticles of an ordered-alloy phase are formed with the aid of PDDA despite the small size (3-4 nm).

High-resolution, genome-wide mapping of positive supercoiling in chromosomes.

Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to Escherichia coli and Saccharomyces cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently oriented genes, consistent with the 'twin-domain' model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin-binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

Value of religious care for relief of psycho-existential suffering in Japanese terminally ill cancer patients: the perspective of bereaved family members.

OBJECTIVE: This study aimed to clarify the experience of bereaved family members of cancer patients regarding the usefulness of religious care (perceived usefulness). The value of this care to palliate psycho-existential suffering in future patients was also examined (predicted usefulness). METHODS: A questionnaire was sent to 592 bereaved family members of cancer patients who were admitted to certified palliative care units in Japan. Responses were obtained from 378 families, indicating whether the patient received religious care, the perceived usefulness of the care, and its predicted usefulness for palliation of psycho-existential suffering. RESULTS: About 25% (N=83) indicated that the patient had received religious care, whereas 75% (N=255) had not received it. Families of patients who had received religious care evaluated pastoral care workers (86%), religious services (82%), and religious music (80%) as 'very useful' or 'useful'. Families predicted usefulness of religious care for future patients: attending a religious service (very useful or useful, 56%; not useful or harmful, 44%), a religious atmosphere (48%, 52%), meeting with a pastoral care worker (50%, 50%), and religious care by physicians (26%, 74%), and nurses (27%, 73%). Families with a religion were significantly more likely to rate religious care as useful for future patients. CONCLUSIONS: Families of patients who received religious care generally evaluated this care to be very useful or useful. For future patients, some families felt that religious care would be useful, but some did not. In Japan, religious care is more likely to provide benefits to patients who have a religion.

Experimental validation of left ventricular transmural strain gradient with echocardiographic two-dimensional speckle tracking imaging.

AIMS: To validate intramural strain measured by a speckle tracking imaging (STI) system against that measured by sonomicrometry crystals. METHODS AND RESULTS: In 11 anaesthetized sheep, six sets of three sonomicrometry crystals were implanted in endo-, mid-wall, and epimyocardium on the anterior and lateral walls. Circumferential strain (CS) was calculated at three layers from endo-, mid-wall, and epicardial speckle. Radial strain (RS) was assessed for inner and outer halves of the myocardium. We compared ultrasound-derived strain measurements against those by sonomicrometry at baseline and during pharmacological stress and coronary occlusion. Intraclass correlation coefficients at baseline and during coronary occlusion were as follows: endocardial CS 0.80, 0.97; mid-wall CS 0.58, 0.89; epicardial CS 0.71, 0.81; endocardial RS 0.50, 0.78; epicardial RS 0.35, 0.83; and total RS 0.33, 0.71; respectively. At baseline, endocardial strains were higher than mid-wall and epicardial strains, resulting in an inner/outer wall RS gradient and inner/mid/outer wall CS gradients. Ischaemia caused significant reduction in all strains and disappearance of the strain gradient. CONCLUSION: A newly developed STI system can accurately assess the intramural heterogeneity of CS distribution in normal and ischaemic myocardial segments and has the potential to become a non-invasive bedside tool for characterizing myocardial strain gradient.

From bead to rod: Comparison of theories by measuring translational drag coefficients of micron-sized magnetic bead-chains in Stokes flow.

Frictional drag force on an object in Stokes flow follows a linear relationship with the velocity of translation and a translational drag coefficient. This drag coefficient is related to the size, shape, and orientation of the object. For rod-like objects, analytical solutions of the drag coefficients have been proposed based on three rough approximations of the rod geometry, namely the bead model, ellipsoid model, and cylinder model. These theories all agree that translational drag coefficients of rod-like objects are functions of the rod length and aspect ratio, but differ among one another on the correction factor terms in the equations. By tracking the displacement of the particles through stationary fluids of calibrated viscosity in magnetic tweezers setup, we experimentally measured the drag coefficients of micron-sized beads and their bead-chain formations with chain length of 2 to 27. We verified our methodology with analytical solutions of dimers of two touching beads, and compared our measured drag coefficient values of rod-like objects with theoretical calculations. Our comparison reveals several analytical solutions that used more appropriate approximation and derived formulae that agree with our measurement better.

mDia1 senses both force and torque during F-actin filament polymerization.

Formins, an important family of force-bearing actin-polymerizing factors, function as homodimers that bind with the barbed end of actin filaments through a ring-like structure assembled from dimerized FH2 domains. It has been hypothesized that force applied to formin may facilitate transition of the FH2 ring from an inhibitory closed conformation to a permissive open conformation, speeding up actin polymerization. We confirm this hypothesis for mDia1 dependent actin polymerization by stretching a single-actin filament in the absence of profilin using magnetic tweezers, and observe that increasing force from 0.5 to 10 pN can drastically speed up the actin polymerization rate. Further, we find that this force-promoted actin polymerization requires torsionally unconstrained actin filament, suggesting that mDia1 also senses torque. As actin filaments are subject to complex mechanical constraints in living cells, these results provide important insights into how formin senses these mechanical constraints and regulates actin organization accordingly.

Statistics of loop formation along double helix DNAs.

We compute relative position distributions of distant sites along discretized semiflexible polymers, focusing on encounter statistics for pairs of sites along a double-stranded DNA molecule (dsDNA), using a transfer-matrix approach. We generalize the usual semiflexible polymer, considering nonlinear elasticity effects arising from inhomogeneities which either appear at any position via thermal fluctuation, or which occur at specific "quenched" locations. We apply our theory to two problems associated with dsDNA looping. First, we discuss how local flexible defects in double-helix structure facilitate cyclization of short dsDNA molecules. Flexible defects greatly enhance cyclization rate, and strongly modify its dependence on the closure orientational boundary condition. This effect is relevant to free-solution cyclization experiments, and to loop formation in vivo. Second, we present calculations of force dependence of the probability of formation of loops along single dsDNAs which show how the probability of loop formation is suppressed by tension.