Reliability of parameter estimates from models applied to respiratory impedance data

Many previous studies have fit lumped parameter models to respiratory input (Zin) and transfer (Ztr) impedance data. For frequency ranges higher than 4-32 Hz, a six-element model may be required in which an airway branch (with a resistance and inertance) is separated from a tissue branch (with a resistance, inertance, and compliance) by a shunt compliance. A sensitivity analysis is applied to predict the effects of frequency range on the accuracy of parameter estimates in this model obtained from Zin or Ztr data. Using a parameter set estimated from experimental data between 4 and 64 Hz in dogs, both Zin and Ztr were simulated from 4 to 200 Hz. Impedance sensitivity to each parameter was also calculated over this frequency range. The simulation predicted that for Zin a second resonance occurs near 80 Hz and that the impedance is considerably more sensitive to several of the parameters at frequencies surrounding this resonance than at any other frequencies. Also, unless data is obtained at very high frequencies (where the model is suspect), Zin data provides more accurate estimates than Ztr data. After adding random noise to the simulated Zin data, we attempted to extract the original parameters by using a nonlinear regression applied to three frequency ranges: 4-32, 4-64, and 4-110 Hz. Estimated parameters were substantially incorrect when using only 4- to 32-Hz or 4- to 64-Hz data, but nearly correct when fitting 4- to 110-Hz data. These results indicate that respiratory system parameters can be more accurately extracted from Zin than Ztr, and to make physiological inferences from parameter estimates based on Zin impedance data in dogs, the data must include frequencies surrounding the second resonance.     

Automatic analysis of flow cytometric DNA histograms from irradiated mouse male germ cells

An automatic procedure for recovering the DNA content distribution of mouse irradiated testis cells from flow cytometric histograms is presented. First, a suitable mathematical model is developed, to represent the pattern of DNA content and fluorescence distribution in the sample. Then a parameter estimation procedure, based on the maximum likelihood approach, is constructed by means of an optimization technique. This procedure has been applied to a set of DNA histograms relative to different doses of 0.4-MeV neutrons and to different time intervals after irradiation. In each case, a good agreement between the measured histograms and the corresponding fits has been obtained. The results indicate that the proposed method for the quantitative analysis of germ cell DNA histograms can be usefully applied to the study of the cytotoxic and mutagenic action of agents of toxicological interest such as ionizing radiations.     

Application of bromodeoxyuridine incorporation measurements to the determination of cell distribution within the S phase of the cell cycle

Flow cytofluorimetric measurement of incorporated bromodeoxyuridine, using a double-stained cell population, allows the determination of the distribution of cells along the cell cycle. We have developed a simple computer program for the direct treatment of 64 x 64 channel histograms. This analysis appears to provide interesting data about the distribution of cells in the various phases of the cell cycle, namely the S phase. Two examples have been chosen to illustrate possible fields for the application of such a program. Comparison of two cell lines such as friend murine erythroleukemia cells (MELC) and fibroblasts FR3T3 cells has shown that this analysis can be used for cell-cycle characterization of a given cell line. The program also allows the differential analysis of cell distribution along the cell cycle as a function of a given parameter. This possibility has been applied to study the variation of cell-cycle parameters as a function of the time of induced differentiation of MELC and reveals changes in the distribution of the cells along the various phases of the cell cycle, namely in the S phase.     

On-line three-parameter data uptake, analysis, and display device for flow cytometry and other applications

Multiparameter flow cytometric measurements are of growing interest in the study of complex features of biological cells. With state of the art instrumentation, three-parameter (3-P) data handling is relatively complicated and time consuming and the display methods are not satisfactory. As an alternative, an interactive 3-P analyzing module, Cytomic 123 is described, which displays 3-P fields during and immediately after data uptake in the form of a cubic array of 32,768 channels. The fields can be randomly rotated by hardware and software. The event frequencies in the field are primarily visualized by brightness modulation of the display dots. Additionally, the display of the field may be confined to user selected ranges of event frequencies, which may also be superposed to mixed frequency displays. A set of preprogrammed functions is available for the following tasks: (a) uptake of 3-P histograms combined with on-line control of the transducer pulses, (b) automatic uptake of a series of 2-P time correlated histograms in the cube, (c) generation and numerical evaluation of sections and projections of cube histograms, (d) interactive generation and evaluation of spatial subfields for integration, or as sorting matrix by successive erosion of section planes, or reprojection of projection windows, and (e) isometric display of sections and projections and exchange of data sets with other Cytomic modules or other data systems, especially the Cytomic 12 module, whose 2-P capabilities can be used. The module is built with low cost Z80 microprocessor eurocards. A standard oscilloscope serves as a display unit.     

Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.     

Transmission of single and multiple viral variants in primary HIV-1 subtype C infection

To address whether sequences of viral gag and env quasispecies collected during the early post-acute period can be utilized to determine multiplicity of transmitted HIV's, recently developed approaches for analysis of viral evolution in acute HIV-1 infection [1,2] were applied. Specifically, phylogenetic reconstruction, inter- and intra-patient distribution of maximum and mean genetic distances, analysis of Poisson fitness, shape of highlighter plots, recombination analysis, and estimation of time to the most recent common ancestor (tMRCA) were utilized for resolving multiplicity of HIV-1 transmission in a set of viral quasispecies collected within 50 days post-seroconversion (p/s) in 25 HIV-infected individuals with estimated time of seroconversion. The decision on multiplicity of HIV infection was made based on the model's fit with, or failure to explain, the observed extent of viral sequence heterogeneity. The initial analysis was based on phylogeny, inter-patient distribution of maximum and mean distances, and Poisson fitness, and was able to resolve multiplicity of HIV transmission in 20 of 25 (80%) cases. Additional analysis involved distribution of individual viral distances, highlighter plots, recombination analysis, and estimation of tMRCA, and resolved 4 of the 5 remaining cases. Overall, transmission of a single viral variant was identified in 16 of 25 (64%) cases, and transmission of multiple variants was evident in 8 of 25 (32%) cases. In one case multiplicity of HIV-1 transmission could not be determined. In primary HIV-1 subtype C infection, samples collected within 50 days p/s and analyzed by a single-genome amplification/sequencing technique can provide reliable identification of transmission multiplicity in 24 of 25 (96%) cases. Observed transmission frequency of a single viral variant and multiple viral variants were within the ranges of 64% to 68%, and 32% to 36%, respectively.     

A practical graphical method for estimating the fraction of cells in S in DNA histograms from clinical tumor samples containing aneuploid cell populations

A graphical method for the analysis of unperturbed DNA histograms is presented in which the area of the normalized histogram subtended by the fraction of cells in S is represented by a trapezoid whose dimensions are dependent on features common to all such histograms. The technique takes measurement variability into account. This method was applied to a variety of synthetic DNA histograms. Overall, calculated values for the fraction of cells in S correlated well with actual values. This method was applied to 36 diploid cases of non-Hodgkin lymphoma; results correlated well with those obtained by a computer-based method. The results of the graphical-method were also highly reproducible between different observers. The graphical method can be used in the presence of aneuploid cell populations. Techniques for calculating S fractions in the presence of aneuploidy in clinical samples are described. These techniques were applied to synthetic histograms of mixed diploid and aneuploid populations. Calculated values correlated well with actual values.     

Isolated Coxiella burnetii synthesizes DNA during acid activation in the absence of host cells

Two populations of Coxiella burnetii were isolated from fibroblast tissue cultures and examined for their ability to synthesize DNA when incubated in a defined medium. Both the populations released by mechanical lysis of heavily infected host cells, as well as those recovered from the tissue culture medium, incorporated H3 32PO4 into DNA. Incorporation occurred at pH 4.5 but not at pH 7.0, and proceeded for 12-15 h. When incorporation of [3H]thymidine was studied, only the organisms obtained by mechanical lysis of host cells were active. Those which had been released by natural means into the tissue culture medium, and then recovered for study, did not incorporate precursor thymidine but were extremely active in protein biosynthesis. In mechanically released organisms, thymidine incorporation was inhibited immediately by rifamycin (40 microM) and hydroxyurea (10 mM), but it was not affected by chloramphenicol (310 microM) until 4 h after addition of the drug. Incorporation of H3 32PO4 by both populations of organisms was also inhibited by rifamycin, chloramphenicol and hydroxyurea, but the time sequence of inhibition differed. Southern hybridization utilizing 32P-labelled DNA suggested that both populations synthesized authentic chromosomal DNA sequences, as well as QpH1 plasmid DNA, during acid activation of metabolism.     

Protamine-agarose non-charged alkyl derivatives of agarose in the purification of rat-liver phosphoprotein phosphatases.

1. Protamine-agarose and hydrophobic interaction chromatography were found to be effective in the purification of phosphoprotein phosphatase(s) (phosphoprotein phosphohydrolase, EC 3.1.3.16) of rat-liver. The phosphoprotein phosphatase of rat-liver cytosol were first resolved into three fractions, termed A, B and C, in order of elution from DEAE-cellulose. Whereas all fractions displayed activity towards [32P]phosphoprotamine, only fractions B and C displayed appreciable activity towards [32P]phosphopyruvate kinase. Since fraction B exhibited the most properties and the highest recovery of enzymatic activity towards [32P]phosphoprotamine and [32P]phosphopyruvate kinase, it was selected for further purification. The method developed involves sequential chromatography of fraction B on Sephadex G-200, protamineagarose, histone-agarose and then again on Sephadex G-200 as a final step. A 400-fold enrichment in the phosphoprotamine phosphatase activity of fraction B was obtained. Purified fraction B also displayed substantial phosphatase activity towards [32P]phosphopyruvate kinase and [32P]phosphohistones. An apparent molecular weight of about 250 000 was estimated for purified fraction B on a calibrated Sephadex G-200 column. The present data indicate that rat-liver cytosol contains multiple forms of phosphoprotein phosphatases and suggest a technique which might be applied for the further purification of at least fraction B. 2. In a separate approach, a combination of pentyl-agarose and protamineagarose chromatography was shown to be a conbenient method for the enrichment (up to 20-fold of phosphoprotein phosphatase activity from crude liver extracts.     

Purification and characterization of glycogen phosphorylase B from skeletal muscle of the mullet Liza ramada: amino acid sequence of the phosphorylation site

1. Skeletal muscle glycogen phosphorylase b has been purified from Liza ramada (mullet). 2. The Mr of the purified enzyme subunit was found to be 97,000. By gel filtration a relative Mr of 190,000 was found. 3. Proteolytic digestion of 32P-phosphorylated mullet phosphorylase gave a [32P]-labelled peptide which is observed to contain Ser, its sequence being -Gln-Ile-Ser-Val-Pro-. 4. During 'in vitro' phosphorylation of mullet phosphorylase, 32P was incorporated in different protein bands resolved by isoelectric focusing. The degree of radioactivity associated with each one changed with the incubation time.     

Variance within homogeneous phytoplankton populations, II: Analysis of clonal cultures

In part I of this series of articles, a framework was presented for interpreting histograms of volume or fluorescence as measured by a flow cytometer on homogeneous phytoplankton populations. In this paper, the analytical framework is applied to flow cytometric histograms from laboratory experiments involving clonal phytoplankton cultures. The density function derived in part I was modified to include a third parameter representing a linear shift of the origin. This modified density function was fitted to chlorophyll fluorescence histograms for populations believed to be asynchronous (grown in continuous light) and also to histograms from populations grown on a 14:10 (h:h) light/dark cycle. Near-synchronous subpopulations sorted from an asynchronous population were also analyzed. In populations in which underlying assumptions (asynchronous divisions, constant growth) are valid, curve fits provide estimates of the inherent variability among cells at age 0. The implication of fitting the density function to populations in which these assumptions are not valid is discussed.     

Repetitive excitation of bursts of action potentials in the rat hippocampus following single shock stimulation

1. Post-stimulus time (PST) histograms of rat hippocampal cells were recorded in vivo following single-shock stimulation of the fornix. 2. The PST histograms displayed a series of peaks of decreasing amplitude, similar to damped oscillatory responses previously recorded in cats and rabbits. 3. The effect of increased background activity was investigated by recording histograms with concurrent pulse train stimulation of the contralateral hippocampus. The histograms showed a decreased latency to the onset of the second peak. 4. Damped oscillatory activity seen in the in vivo rat preparation could not be elicited in the in vitro rat slice preparation. Thus species differences cannot account for the absence in slice studies of this type of damped oscillatory activity. 5. We conclude that the level of spontaneous activity is one factor contributing to the genesis of multiple peaks in histograms in the in vivo preparation.     

高分子量RGD-蛛丝蛋白重组体的构建、高密度发酵及纯化

蜘蛛丝是自然界综合性能优良的天然蛋白质纤维之一,因其具有良好的生物相容性和可降解性在生物医学领域具有潜在的应用前景。在本室已经构建的RGD-蜘蛛拖丝蛋白基因16多聚体基础上,通过首尾相连、倍加等方法进一步多聚化,得到RGD-蜘蛛拖丝蛋白基因32和64多聚体,分别将这两种多聚体与原核高效表达载体pET-30a( )连接,转化大肠杆菌BL21(DE3)pLysS,得到的32多聚体表达重组子命名为pNSR32,64多聚体表达重组子命名为pNSR64。通过酶切、琼脂糖电泳鉴定及对目的片段的测序均与理论值相符。将32和64多聚体基因序列注册GenBank,序列号分别为DQ469929和DQ837297。重组体pNSR32和pNSR64经IPTG诱导表达,SDS-PAGE图谱显示表达产物分子量分别为102kD和196.6kD,与天然蛛丝蛋白分子量接近并与理论值相吻合。高分子量的蛛丝蛋白在原核生物成功实现高效表达,在国内外尚未见报道。在此基础上对pNSR32工程菌进行高密度发酵,建立了简单高效的目的蛋白纯化工艺。     

A simple and highly sensitive method for sequence determination of 32P-labeled oligonucleotides

A new procedure is described for the sequence determination of oligonucleotides produced by digestion of RNA with pancreatic RNase A. The oligonucleotide is treated with spleen exonuclease and all intermediates are resolved by thin-layer chromatography on polyethyleneimine plates. On the basis of the increase in mobility it can be decided for each successive step whether a Gp- or an Ap-residue has been removed by reference to a calibration grid. The method is very simple and can easily be applied to a large number of samples. An amount of ³²P-radioactivity corresponding to 40 dpm/nucleotide is sufficient for analysis.     

Error analysis in flow microfluorometry

Sources of error in a typical algorithm for the analysis of single flow-microfluorometric histograms are identified. A new statistical model for such data is presented, by means of which the error sources are quantitatively investigated. These theoretical investigations lead to three practical observations: A more detailed characterization of the fluorescence dispersion process is needed for a more refined algorithm. Levels of dispersion typically experienced are such that from a single histogram the distribution of cells within S-phase cannot be finely resolved; but the crude distribution of cells among the three phases G1, S, and G2-M may be accurately estimated. If currently typical levels of dispersion can be halved, then the S-phase distribution can be finely resolved.     

LogViewer: A Software Tool to Visualize Quality Control Parameters to Optimize Proteomics Experiments using Orbitrap and LTQ-FT Mass Spectrometers

Visualization tools that allow both optimization of instrument''s parameters for data acquisition and specific quality control (QC) for a given sample prior to time-consuming database searches have been scarce until recently and are currently still not freely available. To address this need, we have developed the visualization tool LogViewer, which uses diagnostic data from the RAW files of the Thermo Orbitrap and linear trap quadrupole-Fourier transform (LTQ-FT) mass spectrometers to monitor relevant metrics. To summarize and visualize the performance on our test samples, log files from RawXtract are imported and displayed. LogViewer is a visualization tool that allows a specific and fast QC for a given sample without time-consuming database searches. QC metrics displayed include: mass spectrometry (MS) ion-injection time histograms, MS ion-injection time versus retention time, MS² ion-injection time histograms, MS² ion-injection time versus retention time, dependent scan histograms, charge-state histograms, mass-to-charge ratio (M/Z) distributions, M/Z histograms, mass histograms, mass distribution, summary, repeat analyses, Raw MS, and Raw MS². Systematically optimizing all metrics allowed us to increase our protein identification rates from 600 proteins to routinely determine up to 1400 proteins in any 160-min analysis of a complex mixture (e.g., yeast lysate) at a false discovery rate of <1%. Visualization tools, such as LogViewer, make QC of complex liquid chromotography (LC)-MS and LC-MS/MS data and optimization of the instrument''s parameters accessible to users.     

Histochemical study on the maturation of human megakaryocytes using microfluorometry.

A microcytofluorometrical DNA measurement was basically studied and was applied to single megakaryocytes previously identified on a Wright-Giemsa stained smear. The smear was first photographed and the location of each megakaryocyte was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained with 4',6-diamidino-2-phenylindole (DAPI) reagent (pH 7.4) at 4 degrees C. Nuclear blue fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA with the coefficient of variation (CV) of 3.6% when stained for 30 min. After 30 min DAPI staining, the DNA measurement was microcytofluorometrically performed in single megakaryocytes which had been morphologically classified into 4 groups on the basis of cytoplasmic maturation, Bessis' classification, assessed on Wright-Giemsa-stained bone-marrow smears from normal human beings. The histograms of the cells did not show any difference in DNA ploidy distribution among the classes: that is, the DNA histograms disclosed ploidy distribution from 4 N to 64 N with the largest population of 16 N. These findings suggest that nuclear DNA synthesis is completed before platelet production starts. This method is useful for comparing the morphological features and DNA content of single megakaryocytes.     

Error analysis in flow microfluorometry

Sources of error in a typical algorithm for the analysis of single flow-microfluorometric histograms are identified. A new statistical model for such data is presented, by means of which the error sources are quantitatively investigated. These theoretical investigations lead to three practical observations: A more detailed characterization of the fluorescence dispersion process is needed for a more refined algorithm. Levels of dispersion typically experienced are such that from a single histogram the distribution of cells within S-phase cannot be finely resolved; but the crude distribution of cells among the three phases G₁, S, and G₂-M may be accurately estimated. If currently typical levels of dispersion can be halved, then the S-phase distribution can be finely resolved.     

Use of a multiple S1 nuclease protection assay to monitor changes in RNA levels for type 1 phosphatase and several proto-oncogenes in response to insulin.

Changes in insulin-regulated gene expression occur in a time- and tissue-dependent fashion. To monitor these changes we have adapted the S1 nuclease protection assay to allow simultaneous estimation of multiple RNA species in a single sample by using synthetic oligonucleotides of various lengths as probes for specific RNA species, which can then be resolved by electrophoresis. The multiple S1 nuclease protection assay was used to assess the influence of insulin on the RNA concentrations of 12 different genes in human skeletal muscle. Estimates obtained by this assay were comparable with those obtained by Northern analysis. RNA levels for proto-oncogene c-src displayed a transient 4-fold increase, whereas RNA levels for type 1 protein phosphatase were suppressed by 50% during the same time period. RNAs corresponding to known insulin-responsive genes such as c-fos, c-myc, c-Ha-ras, and c-src displayed rapid and transient 2-4-fold increases between 30 and 60 min as detected by either Northern analysis or the multiple S1 nuclease protection assay. In addition, RNA levels for the insulin receptor, Glut-4, Glut-3, and c-jun were apparently unaffected by exposure of the cells to insulin.     

A device for evaluation of the dominant component of the EEG activity.

A setup composed of a bioanalyser and additional electronic circuits is described allowing to measure the instantaneous frequency and amplitude of the dominant component of EEG activity. Besides recording the above values on paper by means of the EEG apparatus, functions of probability density of both the amplitude and the frequency of EEG activity are estimated on-line during preadjusted time intervals, and displayed on the screen of the bioanalyser. The mutual relationship between instantaneous EEG amplitude and frequency can be expressed in two-dimensional histograms displayed on a memory scope. The system described is suitable for laboratories having no possibility to use general purpose computer on-line.