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To form a proper mitotic spindle, all kinetochores must capture at least one microtubule plus end. A new computational modeling study shows that a search-and-capture mechanism based on spatially unbiased microtubule dynamic instability is too slow to account for the experimentally observed rate of chromosome capture.
BACKGROUND: The Medtronic EnPulse pacemaker incorporates the new atrial capture management (ACM) algorithm to automatically measure atrial capture thresholds and subsequently manage atrial pacing outputs. OBJECTIVES: The purpose of this study was to evaluate the clinical performance of ACM. METHODS: Two hundred patients with an indication for a dual-chamber pacemaker underwent implantation. ACM thresholds and manually measured atrial pacing thresholds were assessed at follow-up visits. Clinical equivalence was defined as the ACM-measured threshold being within -0.25 V to +0.5 V of the manually measured threshold. The clinician analyzed all ACM measurements performed in-office for evidence of proarrhythmia. RESULTS: All 200 implanted patients had a 1-month visit, and validated manual and in-office ACM threshold data were available for 123 patients. The ACM threshold was 0.595 +/- 0.252 V, and the manual threshold was 0.584 +/- 0.233 V. The mean difference was 0.010 V with a 95% confidence interval of (-0.001, 0.021). The mean difference over all visits was 0.011 V. For all patients, the individual threshold differences were within the range of clinical equivalence at all visits. No atrial arrhythmias were observed during 892 ACM tests in 193 patients. CONCLUSION: This study demonstrated that the ACM algorithm is safe, accurate, and reliable over time. ACM was demonstrated to be clinically equivalent to the manual atrial threshold test in all patients at 1 month and over the entire follow-up period of up to 6 months. ACM ensures atrial capture, may save time during follow-up, and can be used to manage atrial pacing outputs.
The possible use of divalent alkaline-earth metal ions, including Mg2+, Ca2+, Sr2+, and Ba2+, as charge carrier for electron capture dissociation of peptides was investigated. Model peptides of RGGGVGGGR and NGGGWGGGN were used to simplify the interpretation of spectral information. It was demonstrated that useful electron capture dissociation (ECD) tandem mass spectra of these metalated peptides could be generated. Interestingly, peptides metalated with different alkaline-earth metal ions generated very similar ECD tandem mass spectra. Metalated c-ions and z-ions were the predominant fragment ions. Only Mg2+-metalated peptides gave somewhat different results. Some nonmetalated c-ions were observed from ECD of [RGGGVGGGR + Mg]2+ but not from [NGGGWGGGN + Mg]2+. Together with some ab initio calculations, it was established that the bound metal ions might activate the acidity of the amide hydrogen. With the presence of high proton affinity moiety, such as N-terminal amino group and/or side chain of the arginine residues, the metalated peptide ions could exist predominantly in their zwitterion forms, in which one or two backbone amide group(s) was deprotonated and the high proton affinity functional group(s) was protonated. It was believed that electron capture leads primarily to the reduction of the mobile proton rather than the metal ions. With this zwitterion model, the formation of nonmetalated c-fragments and the generation of similar ECD spectra for peptides metalated with various alkaline-earth metal ions could readily to be explained. Another interesting observation in the ECD mass spectra of metalated peptides is related to the enhanced formation of the minor ECD products, i.e., (c - 1)(+*) and (z + 1)+ ions. Together with ab initio calculations using a truncated peptide model, various possible reaction mechanisms for the formation of these minor ECD products were evaluated. It was concluded that hydrogen transfer between the initiated formed c and z(.) species plays an important role in the formation (c - 1)(+*) and (z + 1)+ ions. Although peptides metalated with these metal ions do not have better ECD efficiency compared to the multiply-protonated peptides, it provides practical accessibility of ECD methods to analyze small peptides with no basic amino acid residues.
Electron capture dissociation (ECD) of the peptide Substance P (SubP) complexed with divalent metals has been investigated. ECD of [SubP + H + M]3+ (M2+ = Mg2+ -Ba2+ and Mn2+ -Zn2+) allowed observation of a larger number of product ions than previous investigations of doubly charged metal-containing peptides. ECD of Mg-Ba, Mn, Fe, and Zn-containing complexes resulted in product ions with and without the metal from cleavage of backbone amine bonds (c' and z* -type ions). By contrast, ECD of Co and Ni-containing complexes yielded major bond cleavages within the C-terminal methionine residue (likely to be the metal ion binding site). Cu-containing complexes displayed yet another behavior: amide bond cleavage (b and y'-type ions). We believe some results can be rationalized both within the hot hydrogen atom mechanism and mechanisms involving electron capture into excited states, such as the recently proposed amide superbase mechanism. However, some behavior, including formation of (cn 'M - H)+ ions for Ca-Ba, is best explained within the latter mechanisms with initial electron capture at the metal. In addition, the ECD behavior appears to correlate with the metal second ionization energy (IE2). Co and Ni (displaying sequestered fragmentation) have IE2s of 17.1 and 18.2 eV, respectively, whereas IE2s for Mg-Ba, Mn, and Fe (yielding random cleavage) are 10.0 to 16.2 eV. This behavior is difficult to explain within the hot hydrogen atom mechanism because hydrogen transfer should not be influenced by IE2s. However, the drastically different fragmentation patterns for Co, Ni, and Cu compared to the other metals can also be explained by their higher propensity for nitrogen (as opposed to oxygen) binding. Nevertheless, these results imply that directed fragmentation can be accomplished via careful selection of the cationizing agent.
Bacterial endotoxins (BEs) are integrated part of Escherichia coli, a microorganism widely used for the production of recombinant proteins. BEs should be eliminated in the course of down stream processing of target protein produced by these bacteria. Supermacroporous monolith (continuous bed) columns, so called cryogel columns, with immobilized polyethyleneimine (PEI), polymyxin B (PMB) and lysozyme were employed for BEs capture. Due to the large interconnected pores it was possible to use cryogel columns at flow rates as high as 10 ml/min. The columns packed with Sepharose CL-4B with immobilized PEI, PMB and lysozyme were impossible to use at these high flow rates due to the collapse of the bed. The dynamic capacities of the cryogel columns were nearly independent of the flow rate. In the presence of EDTA, BEs were quantitatively captured from mixtures with a model protein, bovine serum albumin (BSA) at pH 7.2 with practically no protein losses. At pH 3.6 BEs were captured directly from non-clarified E. coli cell lysate resulting in more than 10(4) times BEs clearance.
We study experimentally the dynamics of drops impacting horizontal fibers and characterize the ability of these objects to capture the drops. We first show that a drop larger than a critical radius cannot be trapped by a fiber whatever its velocity. We determine this critical size as a function of the fiber radius. Then we show that for smaller drops, different situations can occur: at a low impact velocity, the drop is entirely captured by the fiber, whereas some liquid is ejected when arriving faster. We quantify the threshold velocity of capture.
OBJECTIVES: Publication bias is a problem in systematic reviews of randomized controlled trials. The aim of this study was to assess the influence of publication bias in a systematic review of the effectiveness of Progressive Resistance Training (PRT) in older people. STUDY DESIGN AND SETTING: The relevant studies were ascertained from three sources: electronic databases, experts, and handsearching. Capture-recapture, visual inspection of funnel plots, two statistical tests, and two methods that make adjustments for publication bias, were employed to check the robustness of the conclusions of the systematic review. RESULTS: The methods employed gave broadly consistent results. Capture-recapture estimated that 3 (95% CI ) relevant studies were missed, while Trim and Fill suggested 16 studies had been missed. Both Egger's test for bias and a funnel plot regression approach suggested that publication bias was present. A selection model approach suggested that the funnel plot asymmetry observed may not be entirely due to publication bias. CONCLUSION: Capture-recapture is a potentially useful method for assessing publication bias. Further research in the form of simulation studies is required, using a variety of scenarios to investigate the extent to which each method approximates the truth.
The SfiI endonuclease is a prototype for DNA looping. It binds two copies of its recognition sequence and, if Mg(2+) is present, cuts both concertedly. Looping was examined here on supercoiled and relaxed forms of a 5.5 kb plasmid with three SfiI sites: sites 1 and 2 were separated by 0.4 kb, and sites 2 and 3 by 2.0 kb. SfiI converted this plasmid directly to the products cut at all three sites, though DNA species cleaved at one or two sites were formed transiently during a burst phase. The burst revealed three sets of doubly cut products, corresponding to the three possible pairings of sites. The equilibrium distribution between the different loops was evaluated from the burst phases of reactions initiated by adding MgCl(2) to SfiI bound to the plasmid. The short loop was favored over the longer loops, particularly on supercoiled DNA. The relative rates for loop capture were assessed after adding SfiI to solutions containing the plasmid and MgCl(2). On both supercoiled and relaxed DNA, the rate of loop capture across 0.4 kb was only marginally faster than over 2.0 kb or 2.4 kb. The relative strengths and rates of looping were compared to computer simulations of conformational fluctuations in DNA. The simulations concurred broadly with the experimental data, though they predicted that increasing site separations should cause a shallower decline in the equilibrium constants than was observed but a slightly steeper decline in the rates for loop capture. Possible reasons for these discrepancies are discussed.
Recombinant integrin alpha v beta6 was evaluated as a capture ligand in a sandwich ELISA for the detection and serotyping of foot-and-mouth disease (FMD) virus. Our routinely applied method employs seven serotype-specific rabbit polyclonal antibodies as capture ligands and seven serotype-specific guinea pig polyclonal antibodies as detecting reagents. The recombinant integrin bound FMD virus of all seven serotypes but not that of another vesicular disease, swine vesicular disease (SVD). Considerable heterotypic cross-reactions were evident when using the integrin capture ligand in combination with guinea pig detecting antibodies but totally type-specific reactions resulted when serotype-specific monoclonal antibodies (mabs) were used instead of the guinea pig reagents. The specificity of reaction of the integrin capture/mab detector combination was superior to that of our routinely employed rabbit/guinea pig ELISA and offers an improvement for test interpretation. As a universal trapping reagent for all FMD virus serotypes the alpha v beta6 recombinant protein also has the potential for application in other test procedures for viral identification (e.g. pen-side chromatographic strip-tests, biosensors, immunocapture RT-PCR, antigenic characterization procedures and monoclonal antibody profiling of emerging field virus strains) and in antibody detection assays employed for the diagnosis of FMD.
This report describes the production of several MAbs against N195 protein, a major immunodomain of SARS CoV nucleocapsid protein [He, Q., Chong, K.H., Chang, H.H., Leung, B., Ling, A.E., Wei, T., Chan, S.W., Ooi, E.E., Kwang, J., 2004. Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin. Diagn. Lab. Immunol. 11 (2) 417-422.]. One representative IgG1 monoclonal antibody (MAb), S-A5D5, was selected and characterized. S-A5D5 reacted specifically react with both recombinant and native nucleocapsid protein of SARS CoV. The reactivity of S-A5D5 with purified N195 protein and utilization of the MAb as a detector antibody to develop an antigen capture ELISA was assessed. As little as 37.5 pg of purified N protein and 50 TCID(50) of SARS CoV could be detected by the antigen capture ELISA. Specific binding of the MAb S-A5D5 to both purified N195 and SARS CoV nucleocapsid antigen was effectively inhibited by human SARS positive serum and guinea pig anti-N195 serum. The N protein in N195-spike recombinant baculovirus-infected Sf-9 cells could also be identified. N protein was detected in 18 IFA IgM-positive serum samples collected from SARS confirmed patients, but not in nine samples collected from SARS recovery patient. No false positive results were given when 60 samples from healthy individuals were tested, and no cross-reaction occurred when infectious bronchitis virus (IBV), chicken coronavirus, was tested. This monoclonal antibody-based antigen capture ELISA is thus a powerful tool for early diagnosis of SARS CoV infection.
OBJECTIVES: Neurologic complications during carotid artery stenting (CAS) are most clearly associated with embolization of visible debris. Distal filter devices may provide cerebral protection by capturing clinically significant debris. However, they increase procedural time and expense and have their own set of complications. The current study was undertaken to identify the clinical factors predictive for the presence or absence of visible debris captured by distal filter devices during CAS. METHODS: Patients undergoing CAS with use of a distal filter device (n = 279) were prospectively entered into an investigational carotid registry. Recorded variables were classified as patient-, lesion-, or procedure-related. The filter was assessed for visible debris in each case. The odds ratio (OR) and 95% confidence interval (CI) were determined for each variable to predict visible debris. The ability of each variable to predict the absence of visible debris was assessed by calculating the individual negative predictive value (NPV). RESULTS: Visible debris was present in 169 filters (60.3%). There was an increased risk of visible debris found with several variables (OR, 95% CI): hypertension (2.9, 1.7 to 5.2), hypercholesterolemia (2.3, 1.4 to 3.9), stent diameter >9 mm (16.6, 9.0 to 30.0), and any neurologic event (4.2, 1.5 to 9.9). The NPV failed to exceed 0.80 (80%) for any variable. The NPV of the variables with a significantly elevated OR was as follows: hypertension (0.60), hypercholesterolemia (0.52), stent diameter >9 mm (0.75), and any neurologic event (0.38). CONCLUSIONS: Several clinical variables are associated with the presence of visible debris captured by distal filter devices. The current study failed to identify any variables capable of consistently predicting the absence of visible debris. These findings support the routine rather than the selective use of cerebral protection during CAS.
BACKGROUND: Many laboratories use the DNA Hybrid Capture 2 HPV-high risk assay (Digene) to detect and type oncogenic HPV. The aim of this work was to compare this assay with a new HPV genotyping assay: HPV Consensus kit (Argène). Actually, this assay is not commercially available. MATERIALS AND METHODS: Ninety-four cervical samples were tested with both the routine assay Hybrid Capture 2 and the HPV Consensus kit. Discordant results were analysed by amplification with a nested PCR and sequencing of amplified products. RESULTS: Only 81 results could be analysed concerning the oncogenic risk. The overall concordance was 92,6%. But we find 13 "generic" results with the HPV Consensus kit, the generic probe including high risk and low risk genotypes. CONCLUSION: HPV Consensus kit results showed a better detection sensitivity for this assay than Hybrid Capture 2 assay. Nevertheless, "generic" results give no information about the oncogenic risk of the HPV detected in a sample.
A diverse group of microtubule-binding proteins has been linked through live-cell imaging of green fluorescent protein (GFP) fusion proteins. These proteins share the ability to associate with the plus ends of elongating microtubules and track with these tips as the microtubules grow, in a process known as "tip tracking". Several models have been proposed to explain the significance of this activity, including roles in delivering proteins to the cell periphery and in modulating microtubule dynamics. However, the recent observation that some of the tip trackers colocalize on structures undergoing search-capture suggests that tip tracking could be a fundamental aspect of the search-capture process. Focusing on the shared ability of these proteins to undergo tip tracking, this article is intended to place the search-capture model in the context of other proposed functions and to stimulate discussion in this area.
Basal cells play an undefined role in signaling the growth and differentiation of normal secretory epithelial cells in the human prostate. Because basal cells disappear during malignant transformation, we hypothesize that loss of basal cell function may have a permissive role in progression of prostate intraepithelial neoplasia into invasive carcinoma. We describe an immuno-laser capture microdissection approach to selectively capture basal cells. Using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, we identified several protein candidates selectively expressed in microdissected basal cells. We also demonstrate that the RNA derived form this technique is an excellent source for gene-array studies. Thus, we provide evidence that proteomic and microgenomic techniques can be successfully applied to investigate the expression profiles of basal and secretory cells after immuno-capture.
The 16S rRNA genes from eight isolates of Renibacterium salmoninarum with different origins and dates of isolation were sequenced to evaluate the possibility to construct a diagnostic PCR system with target sites within this gene. The sequences were found to be identical but for one single position in one of the isolates, and two regions with an adequate number of nucleotide differences as compared to closely related species were identified. Species-specific fluorescent PCR primers complementary to these regions were constructed as well as oligonucleotides for DNA preparation by sequence capture. A mimic molecule was constructed to be used as an internal control. The PCR was specific and allowed the detection of DNA equivalent to 1-10 R. salmoninarum genomes per reaction. The DNA preparation with sequence capture and analysis by PCR with a mimic was found to be a reliable method for analysis of kidneys from fish with BKD. The amount of PCR inhibiting substances present in the tissue was reduced, and the relevant DNA was concentrated in the capture step. Furthermore, the use of the mimic molecule in the system assured that false negative results could be identified.
This study was concerned with what stimulus information humans with amblyopia use to judge the shape of simple objects. We used a string of four Gabor patches to define a contour. A fifth, center patch served as the test pattern. The observers' task was to judge the location of the test pattern relative to the contour. The contour was either a straight line, or an arc with positive or negative curvature. We asked whether phase shifts in the inner or outer pairs of patches distributed along the contour influence the perceived shape. That is, we measured the phase shift influence function. Our results, consistent with previous studies, show that amblyopes are imprecise in shape discrimination, showing elevated thresholds for both lines and curves. We found that amblyopes often make much larger perceptual errors (biases) than do normal observers in the absence of phase shifts. These errors tend to be largest for curved shapes and at large separations. In normal observers, shifting the phase of inner patches of the string by 0.25 cycle results in almost complete phase capture (attraction) at the smallest separation (2 lambda), and the capture effect falls off rapidly with separation. A 0.25 cycle shift of the outer pair of patches has a much smaller effect, in the opposite direction (repulsion). While several amblyopic observers showed reduced capture by the phase of the inner patches, to our surprise, several of the amblyopes were sensitive to the phase of the outer patches. We used linear multiple regression to determine the weights of all cues to the task: the carrier phase of the inner patches, carrier phase of the outer patches and the envelope of the outer patches. Compared to normal observers, some amblyopes show a weaker influence of the phase of the inner patches, and a stronger influence of both the phase and envelope of the outer patches. We speculate that this may be a consequence of abnormal "crowding" of the inner patches by the outer ones.
Many nocturnal and crepuscular fish use vision to feed and function under low light levels. However, little is known about their ability to accommodate or their visual acuity under these light levels. We used Infrared Photoretinoscopy to track the refractive state of the eye during prey capture under low light in Apogon annularis, a nocturnal reef fish. Anatomical measurements of the eyes allowed calculations of visual acuity. Changes in the refractive state were observed in approximately 75% of the prey capturing strikes, preceding the strikes by 30 ms. These changes were rare between strikes or when prey was absent. Anatomical measurements indicated that the number of photo-detection units in a retinal image greatly exceeded the minimal number needed to detect prey. We conclude that nocturnal vision in A. annularis is sufficiently sensitive to allow accommodation during prey capture.
The purposes of this study are to provide an estimation of the incidence of transport injuries in a defined local community in Nicaragua by using the capture-recapture method, and to compare results using this method when data at different levels of severity are utilized. Two sources of injury data were used to monitor injuries: hospital data (inpatient and outpatient) and traffic police records. Characteristics available for matching included name, age, sex, and date of occurrence. The methodology of capture-recapture was used to estimate the ascertainment degree of both sources of data and the estimate mortality and incidence rates. Estimates were calculated both when all hospital data were taken into account (inpatient and outpatient combined) and when only inpatient records were matched against police records.First, including police records and all hospital data, the mortality and morbidity estimates were 35.5/100000 and 43.7/1000 per year, respectively. Second, when outpatients were excluded from the analysis, the corresponding estimates were 28.6/100000 and 7.5/1000, respectively. In non-fatal cases, the ascertainment-corrected coverage through police records was 2.6% and through hospital surveillance 19.0% when both inpatients and outpatients were included. In fatal cases, the corresponding rates were 56.1 and 22.8%, respectively. The combined data set pointed out pedestrians and cyclists as the main risk groups. Most fatalities were due to head injuries. Our results show that neither police records nor hospital records nor the aggregate database provided acceptable coverage of transport-related injuries. Combining police and hospital data by means of capture-recapture analysis produces more valid estimates, but caution must be given to the issue of severity heterogeneity between the two sources.