Rapid kinetic analysis of multichannel records by a simultaneous fit to all dwell-time histograms

A method is presented for rapidly extracting single-channel transition rate constants from patch-clamp recordings containing signals from several channels. The procedure is based on a simultaneous fit of the observed dwell-time distributions for all conductance levels, using a maximum likelihood approach. This algorithm allows estimation of single-channel rate constants in cases where more advanced methods may be impractical because of their extremely long computational time. A correction is included for the limited time resolution of the recording system, according to theory developed by Roux and Sauvé (Biophys. J. 48:149-158, 1985), by accounting for the impact of undetected transitions on the dwell-time distributions, and by introducing an improved practical implementation of a fixed dead time for the case of more than one channel. This feature allows application of the method to noisy data, after filtering. A computer program implementing the method is tested successfully on a variety of simulated multichannel current traces.     

The transducer model for contrast detection and discrimination: formal relations, implications, and an empirical test

The transducer function mu for contrast perception describes the nonlinear mapping of stimulus contrast onto an internal response. Under a signal detection theory approach, the transducer model of contrast perception states that the internal response elicited by a stimulus of contrast c is a random variable with mean mu(c). Using this approach, we derive the formal relations between the transducer function, the threshold-versus-contrast (TvC) function, and the psychometric functions for contrast detection and discrimination in 2AFC tasks. We show that the mathematical form of the TvC function is determined only by mu, and that the psychometric functions for detection and discrimination have a common mathematical form with common parameters emanating from, and only from, the transducer function mu and the form of the distribution of the internal responses. We discuss the theoretical and practical implications of these relations, which have bearings on the tenability of certain mathematical forms for the psychometric function and on the suitability of empirical approaches to model validation. We also present the results of a comprehensive test of these relations using two alternative forms of the transducer model: a three-parameter version that renders logistic psychometric functions and a five-parameter version using Foley's variant of the Naka-Rushton equation as transducer function. Our results support the validity of the formal relations implied by the general transducer model, and the two versions that were contrasted account for our data equally well.     

The role of temporal generalization in a temporal discrimination task

Two experiments trained rats to discriminate two or three stimulus durations using a temporal discrimination task. A standard peak shift effect was observed when training was administered with short versus long signals in Experiment 1. Both discrimination accuracy scores and the generalization gradients revealed that shorter intervals were discriminated more accurately, which may be due to the scalar property of timing. In Experiment 2, three signals (short, medium, and long) were associated with three different responses, or two of the intervals were associated with one response (short and long or short and medium) and the other interval with a different response. Here, the short/medium versus long discrimination was learned most readily of the three tasks. The results of both experiments indicated a strong contribution of learning of individual durations combined with scalar generalization gradients, but Experiment 2 indicated that categorical encoding of durations may have also been operating.     

Spatial and temporal analysis of disease occurrence for detection of clustering

Data on disease occurrence often consist of the number of cases recorded in a set of regions during each of several time periods. In this paper a method of analysis of such data is proposed which allows one to distinguish and test for spatial clustering, temporal clustering, and space-time clustering of cases. Departures of case numbers from expectation are partitioned into region and time period main effects and region-by-time interactions. Spatial and temporal relationships among regions and time periods are incorporated by pooling estimates over neighborhoods. In addition to providing tests for clustering, the analysis identifies the contributions to clustering made by individual neighborhoods. Data on Creutzfeldt-Jakob disease in France are used to illustrate the approach.     

Testing for microscopic reversibility in the gating of maxi K+ channels using two-dimensional dwell-time distributions.

An assumption usually made when developing kinetic models for the gating of ion channels is that the transitions among the various states involved in the gating obey microscopic reversibility. If this assumption is incorrect, then the models and estimated rate constants made with the assumption would be in error. This paper examines whether the gating of a large conductance Ca-activated K+ channel in skeletal muscle is consistent with microscopic reversibility. If microscopic reversibility is obeyed, then the number of forward and backward transitions per unit time for each individual reaction step will, on average, be identical and, consequently, the gating must show time reversibility. To look for time reversibility, two-dimensional dwell-time distributions of the durations of open and closed intervals were obtained from single-channel current records analyzed in the forward and in the backward directions. Two-dimensional dwell-time distributions of pairs of open intervals and of pairs of closed intervals were also analyzed to extend the resolution of the method to special circumstances in which intervals from different closed (or open) states might have similar durations. No significant differences were observed between the forward and backward analysis of the two-dimensional dwell-time distributions, suggesting time reversibility. Thus, we find no evidence to indicate that the gating of the maxi K+ channel violates microscopic reversibility.     

Development of high‐resolution DNA barcodes for Dioscorea species discrimination and phylogenetic analysis

The genus Dioscorea is widely distributed in tropical and subtropical regions, and is economically important in terms of food supply and pharmaceutical applications. However, DNA barcodes are relatively unsuccessful in discriminating between Dioscorea species, with the highest discrimination rate (23.26%) derived from matK sequences. In this study, we compared genic and intergenic regions of three Dioscorea chloroplast genomes and found that the density of SNPs and indels in intergenic sites was about twice and seven times higher than that of SNPs and indels in the genic regions, respectively. A total of 52 primer pairs covering highly variable regions were designed and seven pairs of primers had 80%–100% PCR success rate. PCR amplicons of 73 Dioscorea individuals and assembled sequences of 47 Dioscorea SRAs were used for estimating intraspecific and interspecific divergence for the seven loci: The rpoB‐trnC locus had the highest interspecific divergence. Automatic barcoding gap discovery (ABGD), Poisson tree processes (PTP), and generalized mixed Yule coalescence (GMYC) analysis were applied for species delimitation based on the seven loci and successfully identified the majority of species, except for species in the Enantiophyllum section. Phylogenetic analysis of 51 Dioscorea individuals (28 species) showed that most individuals belonging to the same species tended to cluster in the same group. Our results suggest that the variable loci derived from comparative analysis of plastid genome sequences could be good DNA barcode candidates for taxonomic analysis and species delimitation.     

The resolution and discrimination of images in the visual system

We analysed the conformity of optic and retinal cones anatomy factors by the two-point test. Obtained by F. Campbell and R. W. Gubish, the point spread function has a width of about 1 arc min. Cones sizes are equal to 0.5 arc min in the fovea. Functional pixel consists of 3-5 cones under the point spread function. Such an organisation in very useful in decreasing the samping noise of receptors. We carried out psychophysical investigations to show a consensus among the optic, receptors', and neuronal levels. In experiments we studied changes of the two-point pattern perception in respect to the points separation, measured the orientation threshold of small size stimuli. Data were compared with optical point-spread function, the hexagonal mosaic of cones, and line spread function of spatial elements, which form spatial frequency channels at the cortical level.     

Acquisition, extinction and temporal discrimination in human conditioned avoidance

Two experiments were conducted with the aim of designing a videogame for the study of human conditioned avoidance. Participants had to destroy enemy spaceships with the goal of increasing the score in a counter. Coloured signals might announce the launching of a bomb that could hit participant's spaceship producing a 30 points decrease in participant's score. Three groups of participants were trained in discriminating between a warning signal (W) and a safety signal (S) in Experiment 1. Instrumental group could avoid the loss of points by hiding the spaceship before the offset of W. Participants in the Yoked group received the same treatment received by their instrumental partners, regardless of their behaviour. In the Pavlovian group, W was always followed by the loss of points, regardless of participant's behaviour. Discrimination between W and S was better in the Instrumental groups than in the Yoked and Pavlovian control groups. Experiment 2 found extinction of avoidance when the warning signal was not followed by the bomb. Temporal discrimination was found within the participants that received the instrumental contingency in both experiments, with higher avoidance response towards the end of the warning signal. Temporal discrimination disappeared after extinction in Experiment 2.     

Methods for increasing the resolution of two-dimensional protein electrophoresis

A two-dimensional gel elctrophoresis protocol has been developed which provides for a 1.5-to 3-fold increase in the resolution of proteins compared to other frequently used methods. The major variations from previous protocols include increased pore size in the isoelectric focusing gels; cholamidopropyldimethylhydroxypropanesulfonate, a zwitterionic detergent, replaces most of the Nonidet P-40, a nonionic detergent, in the isoelectric focusing gels; no equilibration step is employed between the first and second dimensional separation. The use of a stacking gel in the second dimension has been eliminated; a more efficient and evenly distributed cooling system has been designed for the molecular mass separation, allowing faster migration with higher current. Finally, the crosslinker diacrylylpiperazine is employed which improves protein separation and detection with ammoniacal silver staining. Silver-stained two-dimensional gel electrophoretograms of human plasma and hamster brain tissues and autoradiographs of rat liver cells are compared to the results obtained from previous methods.     

A simple photoactivation and image analysis module for visualizing and analyzing axonal transport with high temporal resolution

We describe a strategy for analyzing axonal transport of cytosolic proteins (CPs) using photoactivatable GFP-PAGFP-with modifications of standard imaging components that can be retroactively fitted to a conventional epifluorescence microscope. The photoactivation and visualization are nearly simultaneous, allowing studies of proteins with rapidly mobile fractions. Cultured hippocampal neurons are transfected with PAGFP-tagged constructs, a discrete protein population within axons is photoactivated, and then the activated population is tracked by live imaging. We show the utility of this method in analyzing axonal transport of CPs that have inherent diffusible pools and distinguish this transport modality from passive diffusion and vesicle transport. The analytical tools used to quantify the motion are also described. Aside from the time needed for preparation of neuronal cultures/transfection, the experiment takes 2-3 h, during which time several axons can be imaged and analyzed. These methods should be easy to adopt by most laboratories and may also be useful for monitoring CP movement in other cell types.     

Adenine and guanine 8CH exchange in nucleic acids: resolution and measurement by Raman optical multichannel analysis

Deuterium exchange of 8C protons of adenine and guanine in nucleic acids is conveniently monitored by laser Raman spectrophotometry, and the average exchange rate so determined [kA + kG] can be exploited as a dynamic probe of the secondary structure of DNA or RNA [J. M. Benevides and G. J. Thomas, Jr. (1985) Biopolymers 24, 667-682]. The present work describes a rapid Raman procedure, based upon optical multichannel analysis, which permits discrimination of the different 8CH exchange rates, kA of adenine and kG of guanine, in a single experimental protocol. For this procedure, simultaneous measurements are made of the intensity decay or frequency shift in separately resolved Raman bands of adenine and guanine, each of which is sensitive only to 8C deuteration of its respective purine. Resolution of the rates kA and kG is demonstrated for the mononucleotide mixtures, 5'-rAMP + 5'-rGMP and 5'-dAMP + 5'-dGMP, for the polynucleotides poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC), for calf thymus DNA, and for the 17 base-pair operator OR3. We show that the different exchange rates of adenine and guanine, in nucleotide mixtures and in DNA, may also be calculated independently from intensity decay of the composite 1481-cm-1 band, comprising overlapped adenine and guanine components, over a time domain that encompasses two distinct regimes: (1) a relatively more rapid exchange of guanine, and (2) a concurrent slower exchange of adenine. Both methods developed here yield consistent results. We find, first, that exchange of guanine is approximately twofold more rapid than that of adenine when both purines are present in the same structure and solvent environment, presumably a consequence of the greater basicity of the 7N site of guanine. Second, we find that adenine suffers greater retardation of exchange than guanine when both purines are incorporated into a "classical" B-DNA secondary structure, such as that of calf thymus DNA. This finding suggests different microenvironments at the 7N-8C loci of adenine and guanine in aqueous B-DNA. We also confirm that adenine residues of B-form poly(dA-dT).poly(dA-dT) exchange much more slowly than those of other B-DNA sequences, implying a secondary structure for the alternating-AT sequence with unusual stereochemistry in the major groove. The greater resistance of adenine than guanine to 8CH exchange in the B-DNA secondary structure is more evident in high molecular weight calf thymus DNA and in the alternating AT and GC copolymer duplexes than in the smaller 17 base-pair operator OR3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of prefeeding, intercomponent-interval food, and extinction on temporal discrimination and pacemaker rate

This experiment investigated the effects of nonpharmacological disruption on temporal discrimination. Pigeons responded on a multiple schedule composed of fixed interval, color-matching, and temporal-discrimination components. The effects of three different disruptors (prefeeding, intercomponent-interval food, and extinction) were assessed. All disruptors decreased response rates during the fixed interval. Prefeeding and intercomponent-interval food had unsystematic effects on response patterning during the fixed interval, whereas extinction increased the relative response rate in the initial portions of the fixed interval. Accuracy of color matching was decreased by prefeeding and was not systematically affected by intercomponent-interval food and extinction. In the temporal-discrimination component, all disruptors flattened the psychophysical functions relating proportion long responses to sample duration. This result indicates a general disruption of temporal discrimination. In addition, parameter estimates derived from the behavioral theory of timing indicated all disruptors decreased pacemaker rate, a result consistent with the predictions of the theory. These results highlight the similarities between disruption of temporal discrimination by pharmacological and nonpharmacological manipulations.     

The myth of automated, high-throughput two-dimensional gel analysis

Many software packages have been developed to process and analyze 2-D gel images. Some programs have been touted as automated, high-throughput solutions. We tested five commercially available programs using 18 replicate gels of a rat brain protein extract. We determined computer processing time, approximate spot editing time, time required to correct spot mismatches, as well as total processing time. We also determined the number of spots automatically detected, number of spots kept after manual editing, and the percentage of automatically generated correct matches. We also determined the effect of increasing the number of replicate gels on spot matching efficiency for two of the programs. We found that for all programs tested, less than 3% of the total processing time was automated. The remainder of the time was spent in manual, subjective editing of detected spots and computer generated matches. Total processing time for 18 gels varied from 22 to 84 h. The percentage of correct matches generated automatically varied from 1 to 62%. Increasing the number of gels in an experiment dramatically reduced the percentage of automatically generated correct matches. Our results demonstrate that these 2-D gel analysis programs are not automatic or rapid, and also suggest that matching accuracy decreases as experiment size increases.     

Antibody array analysis with label-based detection and resolution of protein size

Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from Western blotting and resolved protein size as an additional parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble Western blots with bands of antibody reactivity across the size separation range (670-10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label were measured by high-speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal references of specificity. Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins, and cyclin-dependent kinase inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes.     

The power of respect in (and for) the study of stigma and discrimination

This review essay of Getting Respect: Responding to Stigma and Discrimination in the United States, Brazil, and Israel explores how the politics of respectability is employed in vastly different ways as a response to encounters with stigma and discrimination. For such victimized people, the manner and timing of its employment rests in the particular nature of the social and political contexts circumscribing their lives. Differences remain in whether people interpret certain interactions as validating responses as well as in the kinds of responses they ultimately produce. Taking these and other factors into account, the employment of the politics of respectability should not be regarded solely as the self-affirmation of the moral legitimacy of the victimized. Rather, that form of response is predicated on the recognition of the kinds of agency that may (or may not) be available given the large-scale social power dynamics that circumscribe micro-level acts of discrimination and stigmatization.     

Apoptosis and necrosis: detection, discrimination and phagocytosis.

Three major morphologies of cell death have been described: apoptosis (type I), cell death associated with autophagy (type II) and necrosis (type III). Apoptosis and cell death associated with autophagy can be distinguished by certain biochemical events. However, necrosis is characterized mostly in negative terms by the absence of caspase activation, cytochrome c release and DNA oligonucleosomal fragmentation. A particular difficulty in defining necrosis is that in the absence of phagocytosis apoptotic cells become secondary necrotic cells with many morphological features of primary necrosis. In this review, we present a selection of techniques that can be used to identify necrosis and to discriminate it from apoptosis. These techniques rely on the following cell death parameters: (1) morphology (time-lapse and transmission electron microscopy and flow fluorocytometry); (2) cell surface markers (phosphatidylserine exposure versus membrane permeability by flow fluorocytometry); (3) intracellular markers (oligonucleosomal DNA fragmentation by flow fluorocytometry, caspase activation, Bid cleavage and cytochrome c release by western blotting); (4) release of extracellular markers in the supernatant (caspases, HMGB-1 and cytokeratin 18). Finally, we report on methods that can be used to examine interactions between dying cells and phagocytes. We illustrate a quantitative method for detecting phagocytosis of dying cells by flow fluorocytometry. We also describe a recently developed approach based on the use of fluid phase tracers and different kind of microscopy, transmission electron and fluorescence microscopy, to characterize the mechanisms used by phagocytes to internalize dying cells.