Automatic segmentation of lysosomes and analysis of intracellular pH with Radachlorin photosensitizer and FLIM.

We report application of the fluorescence lifetime imaging microscopy (FLIM) for analysis of distributions of intracellular acidity using a chlorin-e6 based photosensitizer Radachlorin. An almost two-fold increase of the photosensitizer fluorescence lifetime in alkaline microenvironments as compared to acidic ones allowed for clear distinguishing between acidic and alkaline intracellular structures. Clusterization of a phasor plot calculated from fits of the FLIM raw data by two Gaussian distributions provided accurate automatic segmentation of lysosomes featuring acidic contents. The approach was validated in colocalization experiments with LysoTracker fluorescence in living cells of four established lines. The dependence of photosensitizer fluorescence lifetime on microenvironment acidity allowed for estimation of pH inside the cells, except for the nuclei, where photosensitizer does not penetrate. The developed method is promising for combined application of the photosensitizer for both photodynamic treatment and diagnostics.

Analysis of Radachlorin localization in living cells by fluorescence lifetime imaging microscopy.

Intracellular localization of photosensitizer molecules is influential on cell death pathway at photodynamic treatment and is thus an important aspect in achieving enhanced efficacy of photodynamic therapy. In this paper we performed thorough studies of the distribution of Radachlorin photosensitizer in three established cell lines: HeLa, A549, and 3T3 with fluorescence lifetime imaging microscopy through the analysis of lifetime distributions. Experiments carried out in Radachlorin solutions in phosphate buffered saline revealed the pronounced dependence of the fluorescence quantum yield and lifetime on solution pH. This finding was used for analysis of lifetime images of living cells and their phasor plot representations and allowed us to suggest that Radachlorin localized predominantly in lysosomes, known to have acidic pH values. Experiments on co-localization of Radachlorin fluorescence lifetimes and LysoTracker fluorescence intensity supported this suggestion. The results obtained show that the inhomogeneity of fluorescence quantum yield within a cell can be significant due to lower pH values in lysosomes than in other intracellular compartments. This finding suggests that the actual amount of accumulated Radachlorin can be underestimated if being evaluated solely by comparison of fluorescence intensities.

Comparative analysis of Radachlorin accumulation, localization, and photobleaching in three cell lines by means of holographic and fluorescence microscopy.

In this paper we compare the response of cells of established lines of different origin: HeLa, A549 and 3T3 to photodynamic treatment with Radachlorin photosensitizer. The analysis was performed on different aspects of the treatment procedure including photosensitizer accumulation, localization and photobleaching in cells and post-treatment dynamics of changes in cellular morphology at different treatment doses. It was shown that in the three cell lines Radachlorin accumulated in lysosomes to much greater extent than in mitochondria. The cells' response to treatment was analyzed by identification of their death pathways and evaluation of average phase shift dynamics using digital holographic microscopy. The analysis performed on the three cell lines allowed us to evaluate treatment doses specific for each pathway in each line. Among the three lines HeLa cells were found to be the most susceptible to treatment while 3T3 cells the most resistant. The comparison of these results with the data on Radachlorin accumulation, localization and photobleaching rates showed that the observed higher sensitivity of HeLa cells to photodynamic treatment correlated with higher photosensitizer uptake and more intensive photobleaching while lower sensitivity of 3T3 cells correlated with lower uptake and less intensive photobleaching.

In vitro monitoring of photoinduced necrosis in HeLa cells using digital holographic microscopy and machine learning.

Digital holographic microscopy supplemented with the developed cell segmentation and machine learning and classification algorithms is implemented for quantitative description of the dynamics of cellular necrosis induced by photodynamic treatment in vitro. It is demonstrated that the developed algorithms operating with a set of optical, morphological, and physiological parameters of cells, obtained from their phase images, can be used for automatic distinction between live and necrotic cells. The developed classifier provides high accuracy of about 95.5% and allows for calculation of survival rates in the course of cell death.

Quantitative assessment of changes in cellular morphology at photodynamic treatment in vitro by means of digital holographic microscopy.

Temporal dependence of changes in the morphological characteristics of cells of two cultured lines of cancer origin, HeLa and A549, induced by photodynamic treatment with Radachlorin photosensitizer, have been monitored using digital holographic microscopy during first two hours after short-term irradiation. The observed post-treatment early dynamics of the phase shift in the transmitted wavefront indicated several distinct scenarios of cell behavior depending upon the irradiation dose. In particular the phase shift increased at low doses, which can be associated with apoptosis, while at high doses it decreased, which can be associated with necrosis. As shown, the two cell types responded differently to similar irradiation doses. Although the sequence of death scenarios with the increase of the irradiation dose was the same, each scenario was realized at substantially different doses. These findings suggest that the average phase shift of the transmitted wavefront can be used for quantitative non-invasive cell death characterization. The conclusions made were cofirmed by commonly used test assays using confocal fluorescent microscopy.

Digital holographic microscopy in label-free analysis of cultured cells' response to photodynamic treatment.

A methodology providing noninvasive monitoring and evaluation of the effect of photodynamic treatment on live cells in vitro is presented. Variations in morphological characteristics of cells in the course and after treatment are recorded by means of digital holographic microscopy. High-precision measurements of phase shift gained by probe radiation in HeLa and human endometrial mesenchymal stem cell cultures demonstrate for the first time changes of their volume occurred in response to treatment.

[THE DIFFICULTY IN DIAGNOSING OF RARE FORMS OF CONGENITAL DISORDERS OF AMINO ACID METABOLISM IN INFANTS (HEREDITARY TYROSINEMIA)].

Diagnosis of amino acid metabolism disorders according to the clinics without laboratory diagnosis is almost impossible in infants with a history of neonatal and/or premorbid background and multi-organ failure. Mortality due to hereditary tyrosinemia type I is greater than 90%.

Near-barrier fusion of the 8B + 58Ni proton-halo system.

Fusion cross sections were measured for the exotic proton-halo nucleus 8B incident on a 58Ni target at several energies near the Coulomb barrier. This is the first experiment to report on the fusion of a proton-halo nucleus. The resulting excitation function shows a striking enhancement with respect to expectations for normal projectiles. Evidence is presented that the sum of the fusion and breakup yields saturates the total reaction cross section.

Nonautonomous solitons in external potentials.

Novel soliton solutions for the nonautonomous nonlinear Schrödinger equation models with linear and harmonic oscillator potentials substantially extend the concept of classical solitons and generalize it to the plethora of nonautonomous solitons that interact elastically and generally move with varying amplitudes, speeds, and spectra adapted both to the external potentials and to the dispersion and nonlinearity variations. The nonautonomous soliton concept can be applied to different physical systems, from hydrodynamics and plasma physics to nonlinear optics and matter waves, and offer many opportunities for further scientific studies.

Acridine orange accumulation in acid organelles of normal and vacuolated frog skeletal muscle fibres.

The spatial distribution of acid membrane organelles and their relationships with normal and vacuolated transverse tubules has been studied in living frog skeletal muscle fibres using confocal microscopy. Acridine orange (AO) was used to evaluate acid compartments, while a lipophilic styryl dye, RH 414, was employed to stain the membranes of the T-system. AO accumulated in numerous spherical granules located near the poles of nuclei and between myofibrils where they were arranged in short parallel rows, triplets or pairs. AO granules could be divided into three groups: green (monomeric AO), red (aggregated AO), and mixed green/red. As demonstrated by lambda-scanning, most granules were mixed. Double staining of muscle fibres with AO and RH 414 revealed almost all AO granules located near the transverse tubules. Vacuolation of the T-system was induced by glycerol loading and subsequent removal. The close juxtaposition of AO granules and the T-system was preserved in vacuolated fibres. The lumens of vacuoles did not accumulate AO. It is concluded that AO granules represent an accumulation of AO in lysosome-related organelles and fragmented Golgi apparatus and a possible functional role of the spatial distribution of such acidic compartments is discussed.

Co-morbidity of infectious and addictive diseases in St. Petersburg and the Leningrad Region, Russia.

The Russian health care system is organized around specific diseases, with relatively little focus on integration across specialties to address co-morbidities. This organizational structure presents new challenges in the context of the recent epidemics of injection drug use (IDU) and HIV. This paper uses existing and new data to examine the prevalence of reported new cases of drug dependence (heroin) and HIV over time as well as associations between drug dependence and alcoholism, hepatitis B and C, and tuberculosis in the City of St. Petersburg and the Leningrad region. We found a sharp rise in reported cases of IDU beginning in 1991 and continuing until 2002/2003, followed by a sharp rise in newly reported cases of HIV. These rises were followed by a drop in new cases of HIV and drug addiction in 2002/2003 and a drop in the proportion of HIV-positive individuals with IDU as a risk factor. Infection with hepatitis B and C were common, especially among injection drug users (38 and 85%, respectively), but also in alcoholics (7 and 14%). Tuberculosis was more common in alcoholics (53%) than in persons with alcoholism and drug dependence (10%), or with drug dependence alone (4%). Though these data have many limitations, they clearly demonstrate that drug dependence and/or alcoholism, HIV, hepatitis, and tuberculosis frequently co-occur in St. Petersburg and the Leningrad Region. Prevention and treatment services across medical specialties should be integrated to address the wide range of issues that are associated with these co-morbidities.

Sensitivity of lysosomal enzymes to the plant alkaloid sanguinarine: comparison with other SH-specific agents.

The influence of the benzo[c]phenanthridine alkaloid sanguinarine on some lysosomal enzyme activities was investigated. Sanguinarine inhibits lysosomal hydrolases in homogenates of cultured mouse fibroblasts. After incubation of mouse fibroblasts in culture with 100 microM sanguinarine an approximately 50% decrease in the activities of N-acetyl-beta,D-glucosaminidase (NAGA), beta-galactosidase (GAL), arylsulfatase and acid lipase was observed. Because the biological activity of sanguinarine might arise from the interaction of its iminium cation with enzyme thiol groups, we compared its effect on NAGA, GAL and acid phosphatase (AcP) activities with the effects of SH-specific reagents p-chloromercuribenzoic acid (CPMA) and N-ethylmaleimide (NEM). Treatment of lysosomal fractions with millimolar concentrations of sanguinarine induces a dose-dependent inhibition of the enzymes; for example, 0.6 mM sanguinarine causes approximately a 40% decrease in AcP and NAGA activities. NEM has similar effects, and increasing the preincubation temperature from 0 degrees C to 37 degrees C intensifies the inhibition due to both agents. CPMA also inhibits the activity of GAL (IC50 0.7 microM), AcP (IC50 12.5 microM) and NAGA (IC50 6.8 microM) in a dose-dependent manner but is more potent than sanguinarine or NEM. Comparative analysis of the primary structures of these enzymes using the program BLAST reveals the presence of highly conserved cysteine residues, which confirms the importance of thiol-groups for their activities. Thus, both the experimental observations obtained in this study and the literature data imply a significant role of redox-based mechanisms in regulating lysosomal functional activity.

A simple mechanism for co-dependence on two activators at an Escherichia coli promoter.

The Escherichia coli melAB promoter is co-dependent upon two transcription activators, MelR and the cyclic AMP receptor protein, CRP. In this study we demonstrate positive co-operativity between the binding of MelR and CRP at the melAB promoter, which provides a simple mechanism for its co-dependence. MelR binds to four sites, centred at positions -42.5, -62.5, -100.5 and -120.5 relative to the melAB transcription start point. When MelR is pre-bound, CRP is able to bind to a target located between MelR at positions -62.5 and -100.5. This increases the occupation of the two downstream sites for MelR, which is essential for transcription activation. We have identified residues within activating region 1 (AR1) of CRP that are important in transcription activation of the melAB promoter. At simple CRP-dependent promoters, the surface of CRP containing these residues is involved in contacting the RNA polymerase alpha subunit. Our results show that, at the melAB promoter, the surface of CRP containing AR1 contacts MelR rather than RNA polymerase. Thus, MelR and CRP activate transcription by a novel mechanism in which they bind co-operatively to adjacent sites and form a bacterial enhanceosome.

UP element-dependent transcription at the Escherichia coli rrnB P1 promoter: positional requirements and role of the RNA polymerase alpha subunit linker.

The UP element stimulates transcription from the rrnB P1 promoter through a direct interaction with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). We investigated the effect on transcription from rrnB P1 of varying both the location of the UP element and the length of the alpha subunit interdomain linker, separately and in combination. Displacement of the UP element by a single turn of the DNA helix resulted in a large decrease in transcription from rrnB P1, while displacement by half a turn or two turns totally abolished UP element-dependent transcription. Deletions of six or more amino acids from within the alpha subunit linker resulted in a decrease in UP element-dependent stimulation, which correlated with decreased binding of alphaCTD to the UP element. Increasing the alpha linker length was less deleterious to RNA polymerase function at rrnB P1 but did not compensate for the decrease in activation that resulted from displacing the UP element. Our results suggest that the location of the UP element at rrnB P1 is crucial to its function and that the natural length of the alpha subunit linker is optimal for utilisation of the UP element at this promoter.

Transcription activation at the Escherichia coli melAB promoter: the role of MelR and the cyclic AMP receptor protein.

MelR is a melibiose-triggered transcription activator that belongs to the AraC family of transcription factors. Using purified Escherichia coli RNA polymerase and a cloned DNA fragment carrying the entire melibiose operon intergenic region, we have demonstrated in vitro open complex formation and activation of transcription initiation at the melAB promoter. This activation is dependent on MelR and melibiose. These studies also show that the cyclic AMP receptor protein (CRP) interacts with the melAB promoter and increases MelR-dependent transcription activation. DNAase I footprinting has been exploited to investigate the location of MelR-and CRP-binding sites at the melAB promoter. We showed previously that MelR binds to two identical 18 bp target sequences centred at position -100.5 (Site 1) and position -62.5 (Site 2). In this work, we show that MelR additionally binds to two other related 18 bp sequences: Site 1', centred at position -120.5, located immediately upstream of Site 1, and Site R, at position -238.5, which overlaps the transcription start site of the divergent melR promoter. MelR can bind to Site 1', Site 1, Site 2 and Site R, in both the absence and the presence of melibiose. However, in the presence of melibiose, MelR also binds to a fifth site (Site 2', centred at position -42.5) located immediately downstream of Site 2, and overlapping the -35 region of the melAB promoter. Additionally, although CRP is unable to bind to the melAB promoter in the absence of MelR, in the presence of MelR, it binds to a site located between MelR binding Site 1 and Site 2. Thus, tandem-bound MelR recruits CRP to the MelR. We propose that expression from the melAB promoter has an absolute requirement for MelR binding to Site 2'. Optimal expression of the melAB promoter requires Sites 1', Site 1, Site 2 and Site 2'; CRP acts as a 'bridge' between MelR bound at Sites 1' and 1 and at Sites 2 and 2', increasing expression from the melAB promoter. In support of this model, we show that improvement of the base sequence of Site 2' removes the requirement for Site 1' and Site 1, and short circuits the effects of CRP.

Repression of the Escherichia coli melR promoter by MelR: evidence that efficient repression requires the formation of a repression loop.

The Escherichia coli MelR protein is a transcription activator that, in the presence of melibiose, activates expression of the melAB operon by binding to four sites located just upstream of the melAB promoter. MelR is encoded by the melR gene, which is expressed from a divergent transcript that starts 237 bp upstream of the melAB promoter transcript start point. In a recent study, we have identified a fifth DNA site for MelR that overlaps the melR promoter transcript start and -10 region. Here we show that MelR binding to this site can downregulate expression from the melR promoter; thus, MelR autoregulates its own expression. Optimal repression of the melR promoter is observed in the absence of melibiose and requires one of the four other DNA sites for MelR at the melAB promoter. The two MelR binding sites required for this optimal repression are separated by 177 bp. We suggest that, in the absence of melibiose, MelR forms a loop between these two sites. We argue that, in the presence of melibiose, this loop is broken as the melAB promoter is activated. However, in the presence of melibiose, the melR promoter can still be partially repressed by MelR binding to the site that overlaps the transcript start and -10 region. Parallels with the Escherichia coli araC-araBAD regulatory region are discussed.

Mutations that increase the activity of the promoter of the Escherichia coli melibiose operon improve the binding of MelR, a transcription activator triggered by melibiose.

MelR is an Escherichia coli transcription factor that activates expression of the melAB operon in response to the presence of melibiose in the environment. MelR stimulates transcription initiation at the melAB promoter by binding to four sites centered at positions -120.5, -100.5, -62.5, and -42.5 upstream of the transcript start point. In a previous study, we described a spontaneous mutant that exhibited increased melAB expression. Sequence analysis showed that this mutant carries five consecutive base changes at positions -49, -50, -51, -52, and -53 upstream of the melAB transcript start. Here we show that these changes improve MelR binding to the target site centered at position -42.5 at the melAB promoter and that this improvement is responsible for increased promoter activity. Thus, the activity of the melAB promoter is fixed by the occupation by MelR of a DNA site that overlaps the -35 hexamer: MelR appears to be a typical class II-type transcription activator.

The effect of oxidative stress on structural transitions of human erythrocyte ghost membranes.

Differential scanning microcalorimetry was used to study the effect of oxidative stress induced by cumene hydroperoxide (CHP) and Fe2+ on structural transitions of membranes of human erythrocyte ghosts. The CHP homolysis was shown to cause: (a) reduction of the intensity of all structural transitions with the disappearance of B1- and D-transitions; (b) decrease in the enthalpy of oxidized membrane denaturation; (c) negative slope of thermograms; (d) anomalous growth of heat absorption by membranes above 72 degreesC. All these changes occurred until the ratio Fe2+/CHP/membranes<0.02:0.05:1 was reached, i.e., prior to the moment of maximal level of TBA-RS in membrane ghosts. We interpret changes in the character of heat absorption by oxidized membranes as perturbations in the structural organization and interactions inside the spectrin-actin-protein 4.1 domains, the spectrin-protein 4.2 domain, as well as inside the domain of spectrin-ankyrin-cdB3 and the domain formed by the msdB3. These perturbations are associated mainly with the decrease in the concentration of native protein in the domains because of oxidative aggregation of proteins, as evidenced by SDS electrophoresis of oxidized membranes. Preincubation of membranes with tocopherol did not block the aggregation of proteins in electrophoresis and the decrease in the intensity of structural transitions, whereas it blocked completely the formation of TBA-RS, changes in the thermogram slope and the sharp rise in the heat absorption above 72 degreesC. This proves that these processes are determined by the thermotropic properties of the oxidized lipid bilayer of membranes and also provides evidence that the degradation of PUFA of phospholipids modifies both the structure of protein domains and the physical properties of the lipid bilayer of membranes.

Transcription activation at promoters carrying tandem DNA sites for the Escherichia coli cyclic AMP receptor protein: organisation of the RNA polymerase alpha subunits.

We have constructed a family of promoters carrying tandem DNA sites for the Escherichia coli cyclic AMP receptor protein (CRP), with one of the sites centred between base-pairs 41 and 42 upstream from the transcription start site, and the second site located further upstream. In vivo activity measurements show that the activity of these promoters is completely dependent on CRP and that, depending on the precise location, CRP bound at the upstream site increases transcription activation. Hydroxyl radical footprinting was exploited to investigate the binding of CRP and RNA polymerase holoenzyme (RNAP) to these promoters. The study shows that the C-terminal domains of the RNAP alpha subunits bind adjacent to the upstream CRP and that their precise positioning depends on the location of upstream-bound CRP. The C-terminal domains of the RNAP alpha subunits interact with both the upstream and downstream-bound CRP via activating region 1 of CRP.