Review and classification of variability analysis techniques with clinical applications

Background

Representation of independent biophysical sources using Fourier analysis can be inefficient because the basis is sinusoidal and general. When complex fractionated atrial electrograms (CFAE) are acquired during atrial fibrillation (AF), the electrogram morphology depends on the mix of distinct nonsinusoidal generators. Identification of these generators using efficient methods of representation and comparison would be useful for targeting catheter ablation sites to prevent arrhythmia reinduction.

Method

A data-driven basis and transform is described which utilizes the ensemble average of signal segments to identify and distinguish CFAE morphologic components and frequencies. Calculation of the dominant frequency (DF) of actual CFAE, and identification of simulated independent generator frequencies and morphologies embedded in CFAE, is done using a total of 216 recordings from 10 paroxysmal and 10 persistent AF patients. The transform is tested versus Fourier analysis to detect spectral components in the presence of phase noise and interference. Correspondence is shown between ensemble basis vectors of highest power and corresponding synthetic drivers embedded in CFAE.

Results

The ensemble basis is orthogonal, and efficient for representation of CFAE components as compared with Fourier analysis (p ≤ 0.002). When three synthetic drivers with additive phase noise and interference were decomposed, the top three peaks in the ensemble power spectrum corresponded to the driver frequencies more closely as compared with top Fourier power spectrum peaks (p ≤ 0.005). The synthesized drivers with phase noise and interference were extractable from their corresponding ensemble basis with a mean error of less than 10%.

Conclusions

The new transform is able to efficiently identify CFAE features using DF calculation and by discerning morphologic differences. Unlike the Fourier transform method, it does not distort CFAE signals prior to analysis, and is relatively robust to jitter in periodic events. Thus the ensemble method can provide a useful alternative for quantitative characterization of CFAE during clinical study.     

The timing and nature of neovascularization of jejunal free flaps: an experimental study in a large animal model.

The present study was designed (1) to determine whether a free jejunal transfer in a large animal model can develop collateral circulation that is adequate to maintain viability after division of the pedicle and (2) to determine the earliest time pedicle ligation is safe after transplantation. A 15-cm jejunal segment was transferred to the necks of 18 dogs weighing 25 to 35 kg. The bowel segment was inset longitudinally under the skin on one side of the neck, partially covered by the neck muscles, and the mesenteric vessels were anastomosed to recipient vessels in the neck. The proximal and distal bowel stomas were exteriorized through skin openings 12 cm apart and matured. The dogs were subjected to ligation of the vascular pedicle at different intervals: postoperative day 7 (group I, n = 3), day 14 (group II, n = 5), day 21 (group III, n = 5), and day 28 (group IV, n = 5). Blood perfusion was measured in the proximal and distal bowel stomas before pedicle division (control) and 24 hours later using hydrogen gas clearance and fluorescein dye. Bowel necrosis was analyzed using planimetry. The bowel was also stained with hematoxylin and eosin and factor VIII, and new blood vessels were counted. Mean values (+/- standard deviation) were compared with control values for each test and with normal values in the intact bowel using analysis of variance with Neumann-Keuls post-hoc test for multiple comparisons. No jejunal free flaps survived when the vascular pedicle was divided 1 week postoperatively. Bowel survival was 60 percent at 2 weeks, 83 percent at 3 weeks, and 100 percent at 4 weeks. Hydrogen gas clearance values (ml/min/100 g) were 49.6 +/- 8.7 in the mucosa of the intraabdominal jejunum and 37.9 +/- 9.4 in the jejunum that was transferred to the neck before division of the pedicle. Twenty-four hours after pedicle division, hydrogen gas clearance values were 2.8 +/- 6.4 in group I (p < 0.05), 22.4 +/- 12.4 in group II, 23.9 +/- 9.3 in group III, and 34.2 +/- 7.5 in group IV. FluoroScan readings in the transferred jejunum were 201 +/- 7.2 in the control group, 9.3 +/- 2.8 in group I (p < 0.05), 79.1 +/- 10.6 in group II, 66.2 +/- 7.3 in group III, and 164 +/- 11.9 in group IV. New vessel formation as identified by factor VIII staining correlated with increasing bowel perfusion and flap survival rate. Bowel neovascularization, perfusion, and survival increased progressively 1 week after transfer. Significant portions of the transferred bowel will neovascularize and survive as early as 2 weeks postoperatively. However, a minimum of 4 weeks before ligation of the pedicle is necessary to maximize flap perfusion and guarantee survival.     

BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2.     

Recovery of time on limits of stability from functional fatigue in Division II collegiate athletes

Health and fitness professionals working with athletes could establish effective and safe practice and training programs if recovery time on dynamic balance from exertion was available. Research investigating the time needed to recover dynamic limits of stability (LOS) from exertion has not been reported. The purpose of this study was to determine the recovery timeline on LOS from functional fatigue in collegiate athletes. Eighteen athletes (11 men, 7 women) from Division II collegiate soccer team who passed prescreening tests to identify their fitness levels were randomly tested on 2 different days by condition (fatigue or nonfatigue). Functional fatigue was determined by using the Borg 15-point rating of perceived exertion (RPE) scale. Subjects were tested on LOS on the Biodex Balance System pre, post, 10, 15, and 20 minutes for each condition. The main effect for condition was not significant (F() = 0.004, p = 0.948), whereas the main effect for time was significant (F(4,64) = 6.167, p < 0.001). The RPE scoring revealed the significant main effect in FATIGUE (F(2.69, 45.73) = 234.8, p < 0.001). In conclusion, 20 minutes of functional activity will likely have a negative influence on dynamic balance, with balance recovery occurring within 10 minutes after the cessation of exercise in Division II collegiate soccer athletes. Moreover, the level of exertion measured by RPE would correspond to athletes' ability to control their center of mass.     

Evolutionary dynamics of the kinetochore network in eukaryotes as revealed by comparative genomics

During eukaryotic cell division, the sister chromatids of duplicated chromosomes are pulled apart by microtubules, which connect via kinetochores. The kinetochore is a multiprotein structure that links centromeres to microtubules, and that emits molecular signals in order to safeguard the equal distribution of duplicated chromosomes over daughter cells. Although microtubule‐mediated chromosome segregation is evolutionary conserved, kinetochore compositions seem to have diverged. To systematically inventory kinetochore diversity and to reconstruct its evolution, we determined orthologs of 70 kinetochore proteins in 90 phylogenetically diverse eukaryotes. The resulting ortholog sets imply that the last eukaryotic common ancestor (LECA) possessed a complex kinetochore and highlight that current‐day kinetochores differ substantially. These kinetochores diverged through gene loss, duplication, and, less frequently, invention and displacement. Various kinetochore components co‐evolved with one another, albeit in different manners. These co‐evolutionary patterns improve our understanding of kinetochore function and evolution, which we illustrated with the RZZ complex, TRIP13, the MCC, and some nuclear pore proteins. The extensive diversity of kinetochore compositions in eukaryotes poses numerous questions regarding evolutionary flexibility of essential cellular functions.     

Use of genomic DNA control features and predicted operon structure in microarray data analysis: ArrayLeaRNA – a Bayesian approach

Background  

Microarrays are widely used for the study of gene expression; however deciding on whether observed differences in expression are significant remains a challenge.     

The Effect of Coumarin on Growth, Production of Dry Matter, Protein and Nucleic Acids in Roots of Maize and Wheat and the Interaction of Coumarin with Metabolic Inhibitors

Effects of coumarin on fresh weight, dry matter, protein and nucleic acid content per cell in attached roots of maize and wheat and in whole excised elongation zones of maize were determined. The inhibition in cell length exerted by coumarin did not correspond to an inhibition of the net synthetic capacity. Coumarin treatment increased the cell surface, the production of dry matter and the protein content per cell. The dry matter and the protein content per unit surface was slightly increased or unaffected. The effect of coumann on cell shape seemed to be independent of that on dry matter production and net protein synthesis. The same was found in excised elongation zones. —The net DNA-synthesis per cell was slightly increased in attached roots by coumann treatment, but this effect was probably not correlated with the morphogenetic changes. Inhibition of DNA-synthesis with hydroxyurea did not alter the coumarin induced changes in cell shape. —The net RNA-synthesis per cell was slightly decreased after coumarin treatment, but the net RNA-synthesis per cell and the morphogenetic effects exerted by coumarin were not related with each other. Inhibition of m-RNA-synthesis with actinomycin D did not prevent the effects of coumarin on cell division, cell expansion, dry matter production and net protein synthesis. The same was true for inhibitors of protein synthesis, puromycin and p-fluorophenyl-alanine. The findings are in support of the view that coumarin affects already existing structures or enzymes. —Comparisons between coumarin and the uncouplers, DNP and dicoumarol, showed that the effects of coumarin were not, solely, due to uncoupling. SH-protecting agents, BAL, DTE and glutathione, did, with few exceptions, not reduce the morphogenetic effects of coumarin.     

High resolution crystal structure of Clostridium propionicum β‐alanyl‐CoA:ammonia lyase,a new member of the “hot dog fold” protein superfamily

Clostridium propionicum is the only organism known to ferment β‐alanine, a constituent of coenzyme A (CoA) and the phosphopantetheinyl prosthetic group of holo‐acyl carrier protein. The first step in the fermentation is a CoA‐transfer to β‐alanine. Subsequently, the resulting β‐alanyl‐CoA is deaminated by the enzyme β‐alanyl‐CoA:ammonia lyase (Acl) to reversibly form ammonia and acrylyl‐CoA. We have determined the crystal structure of Acl in its apo‐form at a resolution of 0.97 Å as well as in complex with CoA at a resolution of 1.59 Å. The structures reveal that the enyzme belongs to a superfamily of proteins exhibiting a so called “hot dog fold” which is characterized by a five‐stranded antiparallel β‐sheet with a long α‐helix packed against it. The functional unit of all “hot dog fold” proteins is a homodimer containing two equivalent substrate binding sites which are established by the dimer interface. In the case of Acl, three functional dimers combine to a homohexamer strongly resembling the homohexamer formed by YciA‐like acyl‐CoA thioesterases. Here, we propose an enzymatic mechanism based on the crystal structure of the Acl·CoA complex and molecular docking. Proteins 2014; 82:2041–2053. © 2014 Wiley Periodicals, Inc.     

Herpesvirus saimiri antagonizes nuclear domain 10-instituted intrinsic immunity via an ORF3-mediated selective degradation of cellular protein Sp100

In recent studies, the nuclear domain 10 (ND10) components PML, Sp100, human Daxx (hDaxx), and ATRX were identified to be cellular restriction factors that are able to inhibit the replication of several herpesviruses. The antiviral function of ND10, however, is antagonized by viral effector proteins by a variety of strategies, including degradation of PML or relocalization of ND10 proteins. In this study, we analyzed the interplay between infection with herpesvirus saimiri (HVS), the prototypic rhadinovirus, and cellular defense by ND10. In contrast to other herpesviruses, we found that HVS specifically degraded the cellular ND10 component Sp100, whereas other factors like PML or hDaxx remained intact. We could further identify the ORF3 tegument protein of HVS, which shares homology with the cellular formylglycinamide ribotide amidotransferase (FGARAT) enzyme, to be the viral factor that induces the proteasomal degradation of Sp100. Interestingly, recent studies showed that the ORF3-homologous proteins ORF75c of murine gammaherpesvirus 68 and BNRF-1 of Epstein-Barr virus modulate the ND10 proteins PML and ATRX, respectively, suggesting that the ND10 targets of viral FGARAT-homologous proteins diversified during evolution. Furthermore, a virus with the ORF3 deletion was efficiently complemented in Sp100-depleted cells, indicating that Sp100 is able to inhibit HVS in the absence of antagonistic mechanisms. In contrast, we observed that PML, which was neither degraded nor redistributed after HVS infection, strongly restricted both wild-type HVS and virus with the ORF3 deletion. Thus, HVS may lack a factor that efficiently counteracts the repressive function of PML, which may foster latency as the outcome of infection.