Empirical gradient threshold technique for automated segmentation across image modalities and cell lines.

New microscopy technologies are enabling image acquisition of terabyte-sized data sets consisting of hundreds of thousands of images. In order to retrieve and analyze the biological information in these large data sets, segmentation is needed to detect the regions containing cells or cell colonies. Our work with hundreds of large images (each 21,000×21,000 pixels) requires a segmentation method that: (1) yields high segmentation accuracy, (2) is applicable to multiple cell lines with various densities of cells and cell colonies, and several imaging modalities, (3) can process large data sets in a timely manner, (4) has a low memory footprint and (5) has a small number of user-set parameters that do not require adjustment during the segmentation of large image sets. None of the currently available segmentation methods meet all these requirements. Segmentation based on image gradient thresholding is fast and has a low memory footprint. However, existing techniques that automate the selection of the gradient image threshold do not work across image modalities, multiple cell lines, and a wide range of foreground/background densities (requirement 2) and all failed the requirement for robust parameters that do not require re-adjustment with time (requirement 5). We present a novel and empirically derived image gradient threshold selection method for separating foreground and background pixels in an image that meets all the requirements listed above. We quantify the difference between our approach and existing ones in terms of accuracy, execution speed, memory usage and number of adjustable parameters on a reference data set. This reference data set consists of 501 validation images with manually determined segmentations and image sizes ranging from 0.36 Megapixels to 850 Megapixels. It includes four different cell lines and two image modalities: phase contrast and fluorescent. Our new technique, called Empirical Gradient Threshold (EGT), is derived from this reference data set with a 10-fold cross-validation method. EGT segments cells or colonies with resulting Dice accuracy index measurements above 0.92 for all cross-validation data sets. EGT results has also been visually verified on a much larger data set that includes bright field and Differential Interference Contrast (DIC) images, 16 cell lines and 61 time-sequence data sets, for a total of 17,479 images. This method is implemented as an open-source plugin to ImageJ as well as a standalone executable that can be downloaded from the following link: https://isg.nist.gov/.

Segmenting time-lapse phase contrast images of adjacent NIH 3T3 cells.

We present a new method for segmenting phase contrast images of NIH 3T3 fibroblast cells that is accurate even when cells are physically in contact with each other. The problem of segmentation, when cells are in contact, poses a challenge to the accurate automation of cell counting, tracking and lineage modelling in cell biology. The segmentation method presented in this paper consists of (1) background reconstruction to obtain noise-free foreground pixels and (2) incorporation of biological insight about dividing and nondividing cells into the segmentation process to achieve reliable separation of foreground pixels defined as pixels associated with individual cells. The segmentation results for a time-lapse image stack were compared against 238 manually segmented images (8219 cells) provided by experts, which we consider as reference data. We chose two metrics to measure the accuracy of segmentation: the 'Adjusted Rand Index' which compares similarities at a pixel level between masks resulting from manual and automated segmentation, and the 'Number of Cells per Field' (NCF) which compares the number of cells identified in the field by manual versus automated analysis. Our results show that the automated segmentation compared to manual segmentation has an average adjusted rand index of 0.96 (1 being a perfect match), with a standard deviation of 0.03, and an average difference of the two numbers of cells per field equal to 5.39% with a standard deviation of 4.6%.

Kinetics of the reactions of hypochlorous acid and amino acid chloramines with thiols, methionine, and ascorbate.

Thiol oxidation by hypochlorous acid and chloramines is a favorable reaction and may be responsible for alterations in regulatory or signaling pathways in cells exposed to neutrophil oxidants. In order to establish the mechanism for such changes, it is necessary to appreciate whether these oxidants are selective for different thiols as compared with other scavengers. We have measured rate constants for reactions of amino acid chloramines with a range of thiols, methionine, and ascorbate, using a combination of stopped-flow and competitive kinetics. For HOCl, rate constants are too fast to measure directly by our system and values relative to reduced glutathione were determined by competition with methionine. For taurine chloramine, the rate constants for reaction with 5-thio-2-nitrobenzoic acid, GSH, methionine, and ascorbate at pH 7.4 were 970, 115, 39, and 13 M(-1) s(-1), respectively. Values for 10 thiols varied by a factor of 20 and showed an inverse relationship to the pK(a) of the thiol group. Rate constants for chloramines of glycine and N-alpha-acetyl-lysine also showed these relationships. Rates increased with decreasing pH, suggesting a mechanism involving acid catalysis. For hypochlorous acid, rates of reaction with 5-thio-2-nitrobenzoic acid, GSH, cysteine, and most of the other thiols were very similar. Relative reactivities varied by less than 5 and there was no dependence on thiol pK(a). Chloramines have the potential to be selective for different cellular thiols depending on their pK(a). For HOCl to be selective, other factors must be important, or its reactions could be secondary to chloramine formation.

[Program for the thorough complex examination of individuals who liquidated the Chernobyl power accident consequences]. / Programma uglublennogo kompleksnogo obsledovaniia uchastnikov likvidatsii posledstvii avarii na CHAES.

A program for thorough complex examination of individuals who liquidated Chernobyl power accident consequences provides objective diagnostic information and early diagnosis of both general and oncologic diseases. The program covers examination in regional Medical Centers giving highly qualified medical care for the liquidators. Regional and county hospitals can participate in the program, concerning also individuals exposed to radiation during other radiation accidents.

A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1).

A simple and reproducible microtiter plate assay for measuring superoxide dismutase (SOD) activity is described. Water-soluble tetrazolium, the sodium salt of 4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-be nzene disulfonate, was used as a detector of superoxide radical generated by xanthine oxidase and hypoxanthine, in the presence of a range of concentrations of superoxide dismutase. A major advantage of the assay is that one reaction mixture is prepared and aliquotted into wells, avoiding pipetting errors and variable xanthine oxidase activity between samples. Inclusion of standardized SOD solution in each run enables inter-assay comparability without requiring a constant superoxide generation rate under all occasions. The assay is applicable for chloroform-ethanol red cell extracts as well as tissue homogenates without high-speed centrifugation. Fifty percent inhibition of formazan formation was achieved at 2.4+/-0.1 ng of Cu, ZnSOD per well with the coefficient of variation 4.2%.

Design and optimization of a diode-pumped fiber-coupled yb:er glass waveguide laser.

An adaptive simulated annealing optimization algorithm is used to derive laser rate equation and waveguiding models with which the best design for a diode-pumped fiber-coupled, Yb:Er glass waveguide laser can be determined. Material parameters that correspond to commercially available laser-glass and diode-pump sources are used in this study. Given a continuous-wave 300-mW pump at 977 nm, approximately 48 mW of power at 1540 nm can be coupled into the LP(01) mode of an optical fiber. Fabrication and alignment tolerance analyses are presented.

Ion-exchanged waveguide lasers in Er3+/Yb3+ codoped silicate glass.

We investigated an Er(3+)/Yb(3+) codoped silicate glass as a host material for waveguide lasers operating near 1.5 microm. Spectroscopic properties of the glass are reported. Waveguide lasers were fabricated by K(+)-ion exchange from a nitrate melt. The waveguides support a single transverse mode at 1.5 microm. An investigation of the laser performance as a function of the Yb:Er ratio was performed, indicating an optimal ratio of approximately 5:1. Slope efficiencies of as great as 6.5% and output powers as high as 19.6 mW at 1.54 microm were realized. The experimental results are compared with a waveguide laser model that is used to extract the Er(3+) upconversion coefficients and the Yb(3+)-Er(3+) cross-relaxation coefficients. The results indicate the possibility of obtaining high-performance waveguide lasers from a durable silicate host glass.

Superoxide radical production by sponges Sycon sp.

Using the catechol Tiron as an O2-. scavenger, we showed that sea sponges (Sycon sp.) produce superoxide radicals in sea water at a high rate without any stimuli added. The rate of O2-. outflow from sponges to their water surroundings reaches a value of 0.5 nmol/min per sponge at pH 6.5. The generation of O2-. was inhibited by Cu,Zn-superoxide dismutase, and restored by the addition of KCN. We also confirmed the abiotic production of O2-. in sea water, detected earlier with a different method by Petasne and Zika [Nature 325 (1987) 516-518].

Comparison of Cu,Zn-superoxide dismutases of clawed frogs of the genus Xenopus.

Isoenzyme profiles and thermal stability of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) of clawed frogs of genus Xenopus (X. laevis and X. borealis) were compared. Earlier the presence of an unusually thermolabile mutant Cu,Zn-SOD as well as normal enzyme was shown in X. laevis. Having confirmed the data, we further show that X. borealis contains only the thermolabile Cu,Zn-SOD. This suggested that a mutation in the Cu,Zn-SOD gene and genome duplication have occurred in the immediate predecessor of X. laevis. Later on the parent Cu,Zn-SOD gene was lost, leaving only the mutated form of Cu,Zn-SOD in X. borealis.

Cu,Zn-superoxide dismutase gene dosage and cell resistance to oxidative stress: a review.

It is well established that superoxide dismutase (SOD) is the irresplaceable enzyme for aerobic lifestyle. Our understanding of its role has made strides recently as the result of gene transfection approach. Available data on consequences of Cu,Zn-SOD gene transfection in cell resistance to oxygen toxicity are reviewed. There are data that increasing only Cu,Zn-SOD can be toxic, and the balance between Cu,Zn-SOD and peroxide-removing enzymes is supposed to be of prime importance in the antioxidant defence. Role of Cu,Zn-SOD deregulation in carcinogenesis is discussed.

Interaction of reactive oxygen species with DNA. A review.

Data on direct and indirect effects of reactive oxygen species on DNA are reviewed. The classification of the types of DNA oxidative damage and repair enzymes including glycosylases and endo- and exonucleases is presented. The regulatory function of reactive oxygen species and their role in carcinogenesis are discussed.

Nuclear DNA damage during NAD(P)H oxidation by membrane redox chains.

Nuclear DNA damage, as the result of active oxygen formation by NAD(P)H-dependent redox chains, was studied. Isolated rat liver nuclei were incubated in the presence of NAD(P)H and iron chelators. Nuclear DNA damage was analyzed by electrophoresis in alkaline agarose. DNA damage after the addition of electron donors alone or with FeCl3 or DFO-Fe3+ was not visualized. Dramatic decay of high molecular weight DNA was observed with EDTA-Fe3+ or DTPA-Fe3+ in the presence of NAD(P)H. SOD did not prevent DNA damage, whereas catalase was protective. DNA damage was revealed after the addition of cumene hydroperoxide with EDTA-Fe3+, and it was sharply increased in the presence of NADPH. It is suggested that alkoxyl radicals in addition to hydroxyl radicals are involved in DNA damage during NAD(P)H oxidation in the presence of iron chelators, which can be reduced by membrane redox chains.

[The role of oxygen radicals formed during function of the membrane redox chain, in damage of nuclear DNA]. / Rol' kislorodnykh radikalov, obrazuiushchikhsia pri funktsionirovanii membrannykh redoks-tsepei, v povrezhdenii iadernoi DNK.

Nuclear DNA damage resulting from incubation of isolated rat liver nuclei in the presence of NADPH or NADH and ethylenediamine tetraacetic acid, or diethylenetriamine pentaacetic acid, or desferoxamine complexes with Fe3+, has been studied. The degree of DNA damage was assessed by agarose electrophoresis. The observed damage was prevented by catalase but not superoxide dismutase. The mechanism of oxidative damage of nuclear DNA during membrane redox chains functioning is discussed.

[Oxidative stress as a criterion in environmental assessment]. / Okislitel'nyi stress kak kriterii otsenki okruzhaiushchei sredy.

Environmental changes affect the active oxygen generation and thereby involve the oxidative stress. Estimation of the active oxygen metabolism as a method of biotesting is discussed. The results of measurement of specific superoxide dismutase suggest that this approach may be used for environmental assessment.

Oxidant carcinogenesis and antioxidant defense.

Growth promotion by oxidants is observed with cultured human and mouse fibroblasts as well as epidermal cells. It is expected to play a role in inflammation, fibrosis, and tumorigenesis. Indeed, oxidants trigger (patho)physiological reactions that resemble those induced by growth and differentiation factors. For example, active oxygen activates protein kinases, causes DNA breakage, and induces the growth competence-related protooncogenes c-fos and c-myc. The cellular antioxidant defenses affect the consequences of oxidant exposure. Transfectants of mouse epidermal cells that overproduce Cu,Zn-superoxide dismutase (SOD) were sensitized to the toxic effects of an extracellular burst of O2-. plus H2O2, whereas overproducers of catalase (CAT) were protected. Transfection of SOD overproducers with CAT corrected their hypersensitivity. Inducibility of the protooncogene c-fos by oxidants was diminished in SOD and CAT overproducers, albeit probably for different reasons. It is concluded that a fine balance of the multiple components of the antioxidant defense determines the growth response of cells to oxidative stress. In studies of the mechanism of the transcriptional induction of c-fos by oxidants, we identified the joint DSE-AP1 elements (dyad symmetry element, DSE) as major enhancer motifs in the 5'-upstream regulatory sequences of c-fos. Oxidants also increased the de novo synthesis of protein factors that bind to the fos-AP1 enhancer motif. Protein kinase and ADPR transferase inhibitors suppressed the transcriptional induction of c-fos as well as the increase in factor binding to fos-AP1. We conclude that protein phosphorylation and protein polyADP-ribosylation are required for the transcriptional induction of c-fos and the synthesis of protein factors that bind to fos-AP1. It is likely that the FOS and JUN proteins are among these factors and that they participate in the regulation of c-fos expression by oxidants.

The balance between Cu,Zn-superoxide dismutase and catalase affects the sensitivity of mouse epidermal cells to oxidative stress.

Oxidants are toxic, but at low doses they can stimulate rather than inhibit the growth of mammalian cells and play a role in the etiology of cancer and fibrosis. The effect of oxidants on cells is modulated by multiple interacting antioxidant defense systems. We have studied the individual roles and the interaction of Cu,Zn-superoxide dismutase (SOD) and catalase (CAT) in transfectants with human cDNAs of mouse epidermal cells JB6 clone 41. Since only moderate increases in these enzymes are physiologically meaningful, we chose the following five clones for in-depth characterization: CAT 4 and CAT 12 with 2.6-fold and 4.2-fold increased catalase activities, respectively, SOD 15 and SOD 3 with 2.3-fold and 3.6-fold increased Cu,Zn-SOD activities, respectively, and SOCAT 3 with a 3-fold higher catalase activity and 1.7-fold higher Cu,Zn-SOD activity than the parent JB6 clone 41. While the increases in enzyme activities were moderate, the human cDNAs were highly expressed in the transfectants. As demonstrated for the clone SOD 15, this discordance between message concentrations and enzyme activities may be due to the low stability of the human Cu,Zn-SOD mRNA in the mouse recipient cells. According to immunoblots the content of Mn-SOD was unaltered in the transfectants. While the activities of glutathione peroxidase were comparable in all strains, the concentrations of reduced glutathione (GSH) were significantly lower in SOD 3 and SOD 15. This decrease in GSH may reflect a chronic prooxidant state in these Cu,Zn-SOD overproducers.(ABSTRACT TRUNCATED AT 250 WORDS)

[Proteolytic degradation of nuclear matrix proteins in the rat liver, Zajdela's hepatoma and hepatoma 22A in the presence of ATP]. / Proteoliticheskii raspad belkov iadernogo matriksa pecheni krysy, gepatomy Zaidela i gepatomy 22A v prisutstvii ATF.

An intense proteolytic degradation of both proteins and phosphoproteins has been observed in isolated nuclear matrices from rat liver, Zajdela Hepatoma and Hepatoma 22a, incubated with NP-40, DTT and gamma-[32P] ATP being most intense in Hepatoma 22a. Practically all phosphoproteins of Hepatoma 22a nuclear matrix degraded. This implies either an extremely high proteolytic activity in the preparation or the presence of a specific to phosphoproteins protease absent from rat liver and Zajdela Hepatoma nuclear matrices.

Effect of carminomycin on the viability of fibroblasts from patients with regular trisomy 21.

The sensitivity of three human fibroblast lines, trisomic with respect to chromosome 21, to an anthracycline antibiotic carminomycin was compared with that of a normal fibroblast line using a 51Cr release assay. It was found that for an intermediate antibiotic concentration (10 microM) the sensitivity of trisomic fibroblasts, of increased content of Cu,Zn-superoxide dismutase was lower. These results suggest a role for superoxide-mediated membrane damage in the cytotoxic action of anthracycline antibiotics.

Superoxide generation by the respiratory chain of tumor mitochondria.

O2-. generation by the succinate oxidase segment of the respiratory chain of mitochondria and submitochondrial particles from hepatoma 22a and hepatoma Zajdela has been studied with the use of the Tiron method. In the presence of succinate, superoxide generation is induced by antimycin, 2-n-4-hydroxyquinoline N-oxide or funiculosin, and is inhibited by mucidin, myxothiazol or cyanide. The rate of O2-. generation in the antimycin-inhibited state is maximal at the [succinate]/[fumarate] ratio of 1:10 and diminishes at more positive and more negative redox potentials. These characteristics of O2-.-generation are the same as observed earlier in submitochondrial particles from normal tissues. Accordingly, the mechanism of superoxide production is suggested to be the same in tumor and normal mitochondria, namely, autoxidation of the unstable ubisemiquinone in the ubiquinol-oxidizing centre o of cytochrome bc1 complex. With respect to the rate of O2-. generation, the hepatoma mitochondrial membranes are approximately twice as active as bovine heart submitochondrial particles and exceed those from rat liver by more than one order of magnitude.