Recycling Bonded Nd-Fe-B Magnet Wastes by Chemical Reaction and Its Mechanism.

The difficulty of short-process bonded Nd-Fe-B magnet waste recycling lies in the effective removal of the cured polymer matrix while protecting the magnetic powder. In this study, the polymer matrix in bonded Nd-Fe-B magnet waste was destroyed using sodium hydroxide ethanol solution, and the effect of the recycling process on the magnetic powders was studied. The nonmagnetic polymer matrix was removed, while the magnetic phase was not destroyed. The carbon and oxygen contents of the recycled magnetic powders decreased by 92.96 and 89.30%, respectively, while the MS (saturation magnetization), Mr (remanence), and Hcj (coercivity) values of the recycled magnetic powders were 99.8, 98.5, and 95.9% of the original magnetic powders, respectively. The curing and decomposition processes of the polymer matrix were also analyzed. During the curing process, dicyandiamide and bisphenol A epoxy resin acted as bridges and skeletons, respectively, finally forming a thermosetting three-dimensional network structure. In the alkaline alcohol solution, the bridges and skeletons were destroyed by the free hydroxyl groups and free hydrogen radicals in ethanol, and small molecular products were dissolved in the solution.

Re-evaluation of the 18 non-human protein standards used to create the empirical statistical model for decoy library searching.

The Empirical Statistical Model (ESM) for decoy library searching fused the expected amino acid sequence of 18 non-human protein standards to a human decoy library. The ESM assumed a priori the standards were pure such that only the 18 nominal proteins were true positive, all other proteins were false positive, there was no overlap in the peptides of non-human proteins versus human proteins, and that the score distribution of individual peptides would resolve true positive from false positive results or noise. The results of random and independent sampling by LC-ESI-MS/MS indicated that the fundamental assumptions of the ESM were not in good agreement with the actual purity of the commercial test standards and so the method showed a 99.7% false negative rate. The ESM for decoy library searching apparently showed poor agreement with SDS-PAGE using silver staining, goodness of fit of MS/MS spectra by X!TANDEM, FDR correction by Benjamini and Hochberg, or comparison to the observation frequency of null random MS/MS spectra, that all confirmed the standards contain hundreds of proteins with a low FDR of primary structural identification. The protein observation frequency increased with abundance and the log10 precursor intensity distributions were Gaussian and nearly ideal for relative quantification.

Re-evaluation of the rabbit myosin protein standard used to create the empirical statistical model for decoy library searching.

A Rabbit myosin standard, like that used to create the empirical statistical model, was randomly and independently sampled by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The rabbit myosin protein standard appeared pure by SDS-PAGE and CBBR staining but showed many other proteins by silver staining. The LC-MS intensity from myosin and IgG samples were above the 99% safe limit of detection and quantification computed from 36 blank LC-ESI-MS/MS runs. The myosin contained ≤406 Gene Symbols, open reading frames or loci where 79 protein types showed ≥3 peptides from X!TANDEM. Myosins, actin, troponin, other proteins showed 95%-100% homology between the rabbit versus the human decoy library. The myosin protein complex from STRING was true positive compared to random or noise spectra MS/MS with a low type I error (p-value) and low FDR (q-value) computed in R. SDS-PAGE, Western blot, comparison to random and noise MS/MS spectra, X!TANDEM p-values, FDR corrected q-values, and STRING all agreed that the error rate of LC-ESI-MS/MS with a quadrupole ion trap is far below that assumed a priori by the design of the empirical statistical model for decoy library searching.

Identification of differentially regulated secretome components during skeletal myogenesis.

Myogenesis is a well-characterized program of cellular differentiation that is exquisitely sensitive to the extracellular milieu. Systematic characterization of the myogenic secretome (i.e. the ensemble of secreted proteins) is, therefore, warranted for the identification of novel secretome components that regulate both the pluripotency of these progenitor mesenchymal cells, and also their commitment and passage through the differentiation program. Previously, we have successfully identified 26 secreted proteins in the mouse skeletal muscle cell line C2C12 (1). In an effort to attain a more comprehensive picture of the regulation of myogenesis by its extracellular milieu, quantitative profiling employing stable isotope labeling by amino acids in cell culture was implemented in conjunction with two parallel high throughput online reverse phase liquid chromatography-tandem mass spectrometry systems. In summary, 34 secreted proteins were quantified, 30 of which were shown to be differentially expressed during muscle development. Intriguingly, our analysis has revealed several novel up- and down-regulated secretome components that may have critical biological relevance for both the maintenance of pluripotency and the passage of cells through the differentiation program. In particular, the altered regulation of secretome components, including follistatin-like protein-1, osteoglycin, spondin-2, and cytokine-induced apoptosis inhibitor-1, along with constitutively expressed factors, such as fibulin-2, illustrate dynamic changes in the secretome that take place when differentiation to a specific lineage occurs.

Quantitative statistical analysis of standard and human blood proteins from liquid chromatography, electrospray ionization, and tandem mass spectrometry.

It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic parent peptide and fragment ion m/z and intensity data in the mascot generic files from LC-ESI-MS/MS of purified standard proteins, and human blood protein fractionated by partition chromatography, were parsed into a Structured Query Language (SQL) database and were matched with protein and peptide sequences provided by the X!TANDEM algorithm. The many parent and/or fragment ion intensity values were log transformed, tested for normality, and analyzed using the generic Statistical Analysis System (SAS). Transformation of both parent and fragment intensity values by logarithmic functions yielded intensity distributions that closely approximate the log-normal distribution. ANOVA models of the transformed parent and fragment intensity values showed significant effects of treatments, proteins, and peptides, as well as parent versus fragment ion types, with a low probability of false positive results. Transformed parent and fragment intensity values were compared over all sample treatments, proteins or peptides by the Tukey-Kramer Honestly Significant Difference (HSD) test. The approach provided a complete and quantitative statistical analysis of LC-ESI-MS/MS data from human blood.

Mass spectrometry of peptides and proteins from human blood.

It is difficult to convey the accelerating rate and growing importance of mass spectrometry applications to human blood proteins and peptides. Mass spectrometry can rapidly detect and identify the ionizable peptides from the proteins in a simple mixture and reveal many of their post-translational modifications. However, blood is a complex mixture that may contain many proteins first expressed in cells and tissues. The complete analysis of blood proteins is a daunting task that will rely on a wide range of disciplines from physics, chemistry, biochemistry, genetics, electromagnetic instrumentation, mathematics and computation. Therefore the comprehensive discovery and analysis of blood proteins will rank among the great technical challenges and require the cumulative sum of many of mankind's scientific achievements together. A variety of methods have been used to fractionate, analyze and identify proteins from blood, each yielding a small piece of the whole and throwing the great size of the task into sharp relief. The approaches attempted to date clearly indicate that enumerating the proteins and peptides of blood can be accomplished. There is no doubt that the mass spectrometry of blood will be crucial to the discovery and analysis of proteins, enzyme activities, and post-translational processes that underlay the mechanisms of disease. At present both discovery and quantification of proteins from blood are commonly reaching sensitivities of ∼1 ng/mL.

Quantitative iTRAQ analysis of retinal ganglion cell degeneration after optic nerve crush.

Retinal ganglion cells (RGCs) are central nervous system (CNS) neurons that transmit visual information from the retina to the brain. Apoptotic RGC degeneration causes visual impairment that can be modeled by optic nerve crush. Neuronal apoptosis is also a salient feature of CNS trauma, ischemia (stroke), and diseases of the CNS such as Alzheimer's, Parkinson's, multiple sclerosis, and amyotrophic lateral sclerosis. Optic nerve crush induces the apoptotic cell death of ∼ 70% of RGCs within the first 14 days after injury. This model is particularly attractive for studying adult neuron apoptosis because the time-course of RGC death is well established and axon regeneration within the myelinated optic nerve can be concurrently evaluated. Here, we performed a large scale iTRAQ proteomic study to identify and quantify proteins of the rat retina at 1, 3, 4, 7, 14, and 21 days after optic nerve crush. In total, 337 proteins were identified, and 110 were differentially regulated after injury. Of these, 58 proteins were upregulated (>1.3 ×), 46 were downregulated (<0.7 ×), and 6 showed both positive and negative regulation over 21 days, relative to normal retinas. Among the differentially expressed proteins, Thymosin-ß4 showed an early upregulation at 3 days, the time-point that immediately precedes the induction of RGC apoptosis after injury. We examined the effect of exogenous Thymosin-ß4 administration on RGC death after optic nerve injury. Intraocular injections of Thymosin-ß4 significantly increased RGC survival by ∼ 3-fold compared to controls and enhanced axon regeneration after crush, demonstrating therapeutic potential for CNS insults. Overall, our study identified numerous proteins that are differentially regulated at key time-points after optic nerve crush, and how the temporal profiles of their expression parallel RGC death. This data will aid in the future development of novel therapeutics to promote neuronal survival and regeneration in the adult CNS.

Chi-square comparison of tryptic peptide-to-protein distributions of tandem mass spectrometry from blood with those of random expectation.

Proteomics uses tandem mass spectrometers and correlation algorithms to match peptides and their fragment spectra to amino acid sequences. The replication of multiple liquid chromatography experiments with electrospray ionization of peptides and tandem mass spectrometry (LC-ESI-MS/MS) produces large sets of MS/MS spectra. There is a need to assess the quality of large sets of experimental results by statistical comparison with that of random expectation. Classical frequency-based statistics such as goodness-of-fit tests for peptide-to-protein distributions could be used to calculate the probability that an entire set of experimental results has arisen by random chance. The frequency distributions of authentic MS/MS spectra from human blood were compared with those of false positive MS/MS spectra generated by a computer, or instrument noise, using the chi-square test. Here the mechanics of the chi-square test to compare the results in toto from a set of LC-ESI-MS/MS experiments with those of random expectation is detailed. The chi-square analysis of authentic spectra demonstrates unambiguously that the analysis of blood proteins separated by partition chromatography prior to tryptic digestions has a low probability that the cumulative peptide-to-protein distribution is the same as that of random or noise false positive spectra.

Peptide-to-protein distribution versus a competition for significance to estimate error rate in blood protein identification.

The simplest model-that authentic tandem mass spectrometry (MS/MS) spectra are no different from noise, random spectra, or false-positive results-may be directly examined by chi-square comparison of the peptide-to-protein distribution. The peptide-to-protein distribution of a set of 4151 redundant blood proteins identified by X!TANDEM indicated that there is a low probability that the authentic data were the same as noise, random spectra, or false-positive correlations (P<0.0001). In contrast, a competition for significance failed to distinguish approximately 90% of authentic blood proteins from those of noise, random spectra, or false-positive results (P<0.01) and apparently incurred a large type II error (false negative). The chi-square test of peptide-to-protein frequency distributions was found to be an efficient means to distinguish authentic data from false-positive results. Frequency-based statistics unambiguously demonstrated that proteins can be identified by liquid chromatography-electrospray ionization-MS/MS from human blood with acceptable confidence. Thus, the chi-square fit of the peptide-to-protein distribution could distinguish authentic data from random or false-positive data, but the score distribution method could not separate real results from false results.

Precipitation and selective extraction of human serum endogenous peptides with analysis by quadrupole time-of-flight mass spectrometry reveals posttranslational modifications and low-abundance peptides.

The endogenous peptides of human serum may have regulatory functions, have been associated with physiological states, and their modifications may reveal some mechanisms of disease. In order to correlate levels of specific peptides with disease alongside internal standards, the polypeptides must first be reliably extracted and identified. Endogenous blood peptides can be effectively enriched by precipitation of the serum with organic solvents followed by selective extraction of peptides using aqueous solutions modified with organic solvents. Polypeptides on filter paper were assayed with Coomasie brilliant blue binding. The polypeptides were resolved by detergent tricine polyacrylamide electrophoresis and visualized by diamine silver staining. Peptides in the extracts were collected by C18 and analyzed by matrix-assisted laser desorption/ionization and liquid chromatography-electrospray ionization-tandem mass spectrometry (MS/MS) quadrupole time-of-flight MS/MS. Peptides were resolved as multiple isotopic peaks in MS mode with mass deviation of 0.1 Da or less and similar accuracy for fragments. The sensitivity of MS and MS/MS analysis was estimated to be in the picomolar range or less. The peptide composition of the extracts was dependent on solvent formulation. Multiple peptides from apolipoproteins, complement proteins, coagulation factors, and many others were identified by X!Tandem with high mass accuracy of peptide ions and fragments from collision-induced dissociation. Many previously unreported posttranslational modifications of peptides including phosphorylations, oxidations, glycosylations, and others were detected with high mass accuracy and may be of clinical importance. About 4,630 redundant peptides were identified with 99% confidence separately, and together some 1,251 distinct proteins were identified with 99% confidence or greater using the Paragon algorithm.

Enhanced activity of inositol-1,4,5-trisphosphate receptors in atrial myocytes of atrial fibrillation patients.

The roles of inositol-1,4,5-trisphosphate receptors (IP3Rs) in arrhythmia are not fully understood, especially in human beings. Recently, the reported upregulated expression of IP3Rs in atrial myocytes of atrial fibrillation (AF) subjects suggested that IP3Rs might be associated with AF. To directly understand the roles of IP3Rs in AF, we have investigated the IP3R-dependent Ca2+ events as well as the cross-talk between IP3Rs and ryanodine receptors (RyRs) in permeabilized atrial myocytes of AF and normal sinus rhythm (NSR) patients by Ca2+ imaging. In the presence of tetracaine, IP(3)R-dependent Ca2+ events in AF atrial myocytes showed increased frequency, delayed termination and broadened width, compared with NSR myocytes. Moreover, when RyRs were not inhibited, IP3 or adenophostin induced an outburst of RyR-dependent spontaneous Ca2+ sparks with the altered spatial-temporal characteristics. The activation of IP3Rs also enhanced Ca2+ waves. These effects on RyR-dependent Ca2+ signaling were significantly stronger in AF myocytes than in NSR cells and were completely blocked by 2-aminoethoxydiphenyl borate. Thus, our results suggested not only an enhanced activity of IP3Rs but also an elevated cross-talk between IP3R- and RyR-mediated Ca2+ signaling in atrial myocytes of human AF patients, a reflection of altered function of IP3Rs in AF.

Capture of an activated receptor complex from the surface of live cells by affinity receptor chromatography.

Cell surface receptors and their associated signaling pathways on the plasma membrane are key targets in understanding cellular responses. However, the isolation and identification of receptor complexes has been elusive. The Fc receptor was captured from the surface of live cells using microbeads coated with the receptor's cognate ligand, gamma globulin (IgG), and analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS) alongside several controls. Live-cell affinity receptor chromatography (LARC) resulted in a partially nonredundant list of 288 proteins that were specific to the Fc receptor complex. The proteins identified were in close agreement with previously determined factors in the Fc receptor complex as demonstrated by genetic and biochemical methods and revealed novel complex members. Confocal microscopy was used to confirm recruitment of SRC, SYK, PLC, PKC, PI3K, SHIP, TEC, CDC42, RAP, PAK, GAP, GEF, GRP, and CRK to the receptor complex upon activation by the same ligand microbeads. The expression of mutants and silencing RNA against specific isoforms were used to demonstrate a functional role for novel members of the Fc receptor complex, including RHOG (RAS homologue member G), p115 RhoGEF (protein of 115-kDa RAS homologue guanine exchange factor), and CRKL (CRK-like). The recruitment of AKT pleckstrin homology (PH) domain green fluorescent protein (GFP) was used to quantify the production of phosphorylated inositol at the activated receptor complex. We conclude that it is feasible to capture an activated receptor complex from the surface of live cells using ligand-coated microbeads for identification of members of a receptor complex or pathway by LC-MS/MS.

Ryanodine receptor-mediated Ca2+ events in atrial myocytes of patients with atrial fibrillation.

BACKGROUND: Many alterations in sarcoplasmic reticulum Ca(2+) handling proteins in atrial myocytes have been associated with atrial fibrillation (AF) in clinical patients, whereas the functional consequences of these alterations mostly remain unclear. METHODS AND RESULTS: To know whether or not ryanodine receptor (RyR)-mediated intracellular Ca(2+) events in AF atrial myocytes are affected by protein alterations, we investigated spontaneous Ca(2+) sparks and Ca(2+) waves in intact and permeabilized atrial myocytes of AF patients (n(patients) = 21) and normal sinus rhythm (NSR) patients (n(patients) = 22) by laser scanning confocal microscopy. It was found that the frequency, amplitude and rise time of Ca(2+) sparks were comparable between AF and NSR groups, while full width and full duration at half maximum intensity significantly increased in the AF group. Along with these changes, the frequency of small and global Ca(2+) waves increased in AF atrial myocytes. CONCLUSIONS: Our results clearly indicated that the spatiotemporal properties but not the frequency of Ca(2+) sparks were affected in AF atrial myocytes. In addition, the frequency of Ca(2+) waves increased. This profile of the alterations in RyR-mediated Ca(2+) events in AF atrial myocytes was different from previous studies. The underlying mechanisms, as well as possible reasons for this discrepancy, were discussed.

Platform for Quantitative Evaluation of Spatial Intratumoral Heterogeneity in Multiplexed Fluorescence Images.

We introduce THRIVE (Tumor Heterogeneity Research Interactive Visualization Environment), an open-source tool developed to assist cancer researchers in interactive hypothesis testing. The focus of this tool is to quantify spatial intratumoral heterogeneity (ITH), and the interactions between different cell phenotypes and noncellular constituents. Specifically, we foresee applications in phenotyping cells within tumor microenvironments, recognizing tumor boundaries, identifying degrees of immune infiltration and epithelial/stromal separation, and identification of heterotypic signaling networks underlying microdomains. The THRIVE platform provides an integrated workflow for analyzing whole-slide immunofluorescence images and tissue microarrays, including algorithms for segmentation, quantification, and heterogeneity analysis. THRIVE promotes flexible deployment, a maintainable code base using open-source libraries, and an extensible framework for customizing algorithms with ease. THRIVE was designed with highly multiplexed immunofluorescence images in mind, and, by providing a platform to efficiently analyze high-dimensional immunofluorescence signals, we hope to advance these data toward mainstream adoption in cancer research. Cancer Res; 77(21); e71-74. ©2017 AACR.

Oligomeric interaction between ryanodine receptors: potential role in Ca(2+) release.

Receptor proteins in both eukaryotic and prokaryotic cells often form regular lattice or array in the membrane. Recent theoretical analyses indicate that such arrays may provide a novel mechanism for receptor signaling regulation in cells. The functional coupling between neighboring receptors could improve the signaling performance. The ryanodine receptors (RyR)/calcium release channels usually form 2-D regular lattice in the endoplasmic/sarcoplasmic reticulum membranes. Thus, RyR is a potentially good model to study the function of receptor 2-D array. In this article, we briefly review recent progresses in this research field, including RyR-RyR interaction, RyR array's function and working mechanisms. The investigations performed by new methods in our laboratory are summarized. We demonstrate that the RyR-RyR interaction is modulated by the functional states of RyRs. Accordingly, the mechanism of "dynamic coupling" of RyR array is proposed. Its possible role in RyR-mediated Ca(2+) release is discussed.

Effect of intermittent and continuous hypoxia on ryanodine receptors of rat heart.

Intermittent hypoxia (IH) adaptation has been shown to exert beneficial effects on the functions of hearts that had been subjected to insult by ischemia or ischemia/reperfusion. To understand whether calcium release channels/ryanodine receptors (RyRs) were involved, the effects of IH and continuous hypoxia (CH) on [3H]ryanodine binding to homogenates of rat hearts were investigated. Similar studies were performed on rat skeletal muscle. The main results on cardiac muscle were as follows: 1) Ischemia for up to 45 min in normal rat hearts had no obvious effect on the equilibrium ryanodine binding constant (K(d)), while the maximum number of ryanodine binding sites (B(max)) was affected in a time-dependent manner. B(max) was significantly increased with 15 min ischemia, which then returned to control levels upon prolonging the ischemia to 30 min. After 45 min ischemia, a small decrease of B(max) was observed. 2) IH adaptation for up to 28 days did not change B(max), but a significant decrease of B(max) was apparent after longer IH adaptation or after CH exposure. Although B(max) was not altered by 30 min ischemia, 30 min reperfusion following 30 min ischemia induced an evident decrease of B(max). After either IH or CH adaptation, the ischemia/reperfusion- induced decrease of B(max) was abolished. 3) Several effects on K(d) of ischemia and ischemia/reperfusion, with and without IH or CH adaptation, were observed. The most distinct and consistent finding was that a clear increase of K(d) was induced by ischemia or ischemia/reperfusion in CH adapted rats. [3H]Ryanodine binding to homogenates of rat skeletal muscle was also affected by IH and CH adaptation. In contrast to that found in cardiac muscle, a decrease of B(max) in skeletal muscle appeared only after CH adaptation. The physiological significance of these effects is discussed.

The Accumulation of Diacylglycerol and Activation of Protein Kinase C by Caffeine Treatment in Frog Skeletal Muscle.

Our previous work has shown that high K(+) exposure could induce an accumulation of diacylglycerol (DAG) and the activation of protein kinase C (PKC) in frog skeletal muscle. To elucidate its mechanism, the effect of caffeine on the pathway of DAG/PKC signal transduction was investigated. Caffeine could induce DAG accumulation and PKC activation in a dose- and time-dependent manner. The accumulation of DAG induced by caffeine was significantly different from that produced by high K(+) exposure, although the intracellular calcium was raised to a similar peak. In addition, differences in the activation of PKC were shown between caffeine and high K(+) exposure. These differences suggest that the myoplasmic calcium increase produced by high K(+) exposure is not a sole factor for the activation of DAG/PKC signal transduction in frog skeletal muscle, and the high K(+)-induced depolarization may play some roles.

The Accumulation of Diacylglycerol and Activation of Protein Kinase C Induced by High K(+) Exposure in the Frog Skeletal Muscle.

The effects of high K(+) exposure on the accumulation of diacylglycerol (DAG) and on the activation of protein kinase C (PKC) have been studied in frog skeletal muscle. The endogenous DAG increased by treatment with 80 mM K(+) for different lengths of time. Correspondingly, high K(+) exposure could induce a translocation of PKC. For instance, The membrane-associated PKC activity (pmol/min.g muscle) was increased from the control value of 523mmplus;37 to 725mmplus;33 and 858mmplus;59 after 80 mM K(+) exposure for 2 and 60 minutes, while the cytosolic PKC activity was reduced from 543mmplus;35 to 307mmplus; mmplus;20 and 254mmplus;46, respectively. The accumulation of DAG and the activation of PKC induced by high K(+) exposure suggest a possible role of the pathway of DAG/PKC in the regulation of the excitation-contraction coupling in skeletal muscle fibres.

Atomic force microscopy study of the rabbit skeletal muscle ryanodine receptors in different functional states.

Atomic force microscope was applied to investigate the effect of extrinsic phospholipid on the structure of rabbit skeletal muscle ryanodine receptor/calcium release channel (RyR1). In addition, in the presence of extrinsic phospholipid, the height and elasticity of the RyR1s in different functional states were also measured. The results indicate: (i) most of the RyR1s showed a normal structure only in the presence of extrinsic phospholipid; (ii) treatment of the RyR1s with AMP and Ca(2+) together could increase their Young's Modulus but not change their apparent height; (iii) no detectable change in either height or Young's Modulus of the RyR1s appeared, if the RyR1s were treated with other activators or inhibitors.

How Many Templates Does It Take for a Good Segmentation?: Error Analysis in Multiatlas Segmentation as a Function of Database Size.

This paper proposes a novel formulation to model and analyze the statistical characteristics of some types of segmentation problems that are based on combining label maps / templates / atlases. Such segmentation-by-example approaches are quite powerful on their own for several clinical applications and they provide prior information, through spatial context, when combined with intensity-based segmentation methods. The proposed formulation models a class of multiatlas segmentation problems as nonparametric regression problems in the high-dimensional space of images. The paper presents a systematic analysis of the nonparametric estimation's convergence behavior (i.e. characterizing segmentation error as a function of the size of the multiatlas database) and shows that it has a specific analytic form involving several parameters that are fundamental to the specific segmentation problem (i.e. chosen anatomical structure, imaging modality, registration method, label-fusion algorithm, etc.). We describe how to estimate these parameters and show that several brain anatomical structures exhibit the trends determined analytically. The proposed framework also provides per-voxel confidence measures for the segmentation. We show that the segmentation error for large database sizes can be predicted using small-sized databases. Thus, small databases can be exploited to predict the database sizes required ("how many templates") to achieve "good" segmentations having errors lower than a specified tolerance. Such cost-benefit analysis is crucial for designing and deploying multiatlas segmentation systems.