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.

Light-induced nitric oxide release in the skin beyond UVA and blue light: Red & near-infrared wavelengths.

Nitric oxide (NO) is omnipresent in the body and synthesized by 3 isoenzymes (nNOS, eNOS and iNOS), all detected in human skin. NO can be stored in a pool of compounds readily converted to NO following skin irradiation by UVR and blue light. This non-enzymatic (without NOS involvement) photolytic reaction mobilizes cutaneous stores of NO derivatives to the bloodstream, lowering blood pressure. However, with the likelihood of skin deleterious effects caused by UVR/blue light, safer wavelengths in the red/near-infrared (NIR) spectrum are becoming potential contenders to release cutaneous NO, possibly via NOS temperature-dependent effects. The use of red/NIR light to mobilize NO stores from the body's largest organ (the skin) is auspicious. This review focuses on UVR, blue, red, and NIR spectra and their capacity to release NO in human skin. PubMed and Google Scholar were used as article databases to find relevant publications related to this particular field.

Investigating epidemiologic trends and the geographic distribution of patients with anal squamous cell carcinoma throughout Canada.

Background: Anal cancer is a rare disease, constituting 0.5% of new cancer cases in the United States. The most common subtype is squamous cell carcinoma (scc). Studies in several developed nations have reported on an increasing incidence of anal cancer in recent decades, and various risk factors pertaining to the pathogenesis of the disease have been identified, including infection with the human papillomavirus, tobacco use, and immunosuppression. The epidemiology and distribution of anal scc throughout Canada remain poorly understood, however. Methods: Using 3 population-based cancer registries, a retrospective analysis of demographic data across Canada for 1992-2010 was performed. The incidence and mortality for anal scc was examined at the levels of provinces, cities, and the forward sortation area (FSA) component (first 3 characters) of postal codes. Results: During 1992-2010, 3720 individuals were diagnosed with anal scc in Canada; 64% were women. The overall national incidence rate was 6.3 cases per million population per year, with an average age at diagnosis of 60.4 years. The incidence increased over time, with significantly higher incidence rates documented in British Columbia and Nova Scotia (9.3 cases per million population each). Closer examination revealed clustering of cases in various urban centres and self-identified lgbtq communities in Toronto, Montreal, and Vancouver. Discussion: This study provides, for the first time, a comprehensive analysis of the burden of anal scc in Canada, identifying susceptible populations and shedding light onto novel avenues of research to lower the incidence of anal cancer throughout the country.

Epidemiology of adult and pediatric Burkitt lymphoma in Canada: sequelae of the HIV epidemic.

Background: Although the pathogenesis and epidemiology of endemic Burkitt lymphoma (bl) have been extensively studied, the epidemiologic landscape of sporadic and immunodeficiency-associated bl in North America remains poorly understood. Methods: We used 3 distinct population-based cancer registries to retrospectively study bl incidence and mortality in Canada. Data for patient sex; age at the time of diagnosis; and reporting province, city, and forward sortation area (fsa, the first three characters of a postal code) were analyzed. Results: During 1992-2010, 1420 patients with bl in Canada were identified (incidence rate: 2.40 cases per million patient-years), of which 71.1% were male patients. Mean age at diagnosis was 55.5 ± 20.8 years. A bimodal incidence by age distribution was seen in both sexes, with pediatric- and adult-onset peaks. An analysis based on fsas identified select communities with statistically higher rates of adult bl. Several of those fsas were located within the 3 major metropolitan areas (Montreal, Vancouver, Toronto) and within self-identified lgbtq communities. The fsas with a higher socioeconomic status score were associated with lower rates of bl. Conclusions: Current results highlight the geographic and historic pattern of bl in Canada. The human immunodeficiency virus remains an important risk factor for adult bl.

Analysis of the Healthy Subject Response to Prolonged Contact with Tuberculosis Patients.

Prolonged contact of healthy subjects with Mycobacterium tuberculosis can change their blood formula and immune status, thus reflecting adaptive reactions to constant antigenic load. The peripheral blood analysis of health care workers in a tuberculosis hospital demonstrates changes in cell populations which prevent development of tuberculosis, in particular, CD4+ Т cells and CD3+ Т cells. It is shown that the number of the memory CD4+ Т cells specific to M.tuberculosis antigens which produce interferon gamma depends on the duration of work contact with tuberculosis patients. The use of health care workers' blood characteristics as a control for tuberculosis patients is discussed.

Incidence and mortality trends and geographic patterns of follicular lymphoma in Canada.

Background: Follicular lymphoma (fl) is the most common indolent lymphoma and the 2nd most common non-Hodgkin lymphoma, accounting for 10%-20% of all lymphomas in the Western world. Epidemiologic and geographic trends of fl in Canada have not been investigated. Our study's objective was to analyze incidence and mortality rates and the geographic distribution of fl patients in Canada for 1992-2010. Methods: Demographic and geographic patient data for fl cases were obtained using the Canadian Cancer Registry, the Registre québécois du cancer, and the Canadian Vital Statistics database. Incidence and mortality rates and 95% confidence intervals were calculated per year and per geographic area. Rates were plotted using linear regression models to assess trends over time. Overall data were mapped using Microsoft Excel mapping software (Redmond, WA, U.S.A.) to identify case clusters across Canada. Results: Approximately 22,625 patients were diagnosed with fl during 1992-2010. The age-standardized incidence rate of this malignancy in Canada was 38.3 cases per million individuals per year. Geographic analysis demonstrated that a number of Maritime provinces and Manitoba had the highest incidence rates, and that the provinces of Nova Scotia and Quebec had the highest mortality rates in the nation. Regional data demonstrated clustering of fl within cities or regions with high herbicide use, primary mining, and a strong manufacturing presence. Conclusions: Our study provides a comprehensive overview of the fl burden and its geographic distribution in Canada. Regional clustering of this disease in concentrated industrial zones strongly suggests that multiple environmental factors might play a crucial role in the development of this lymphoma.

The first representatives of tetranuclear gold(i) complexes of P,N-containing cyclophanes.

The first representatives of the tetranuclear gold(i) complexes of P,N-containing cyclophanes with two 1,5-diaza-3,7-diphosphacyclooctane rings incorporated into the macrocyclic core have been obtained. The complexation leads to a change in ligand conformations so that the diazadiphosphacyclooctane fragments of the complexes adopt twist-chair conformations, and two of the four gold(i) ions are located over and under the partially collapsed macrocyclic cavity. The complexes demonstrate moderate solid-state green emission.

Covalent self-assembly of the specific RSSR isomer of 14-membered tetrakisphosphine.

The first representative of the specific RSSR isomer of 14-membered tetrakisphosphine has been obtained instead of the RRRR/SSSS isomer predicted according to the empirical rule formulated recently. The geometry of the obtained 14-P4N2 is preorganized for the dicopper complex formation with the unique coordination mode in the row of P4N2 corands.

Enantioselective cellular uptake of chiral semiconductor nanocrystals.

The influence of the chirality of semiconductor nanocrystals, CdSe/ZnS quantum dots (QDs) capped with L- and D-cysteine, on the efficiency of their uptake by living Ehrlich Ascite carcinoma cells is studied by spectral- and time-resolved fluorescence microspectroscopy. We report an evident enantioselective process where cellular uptake of the L-Cys QDs is almost twice as effective as that of the D-Cys QDs. This finding paves the way for the creation of novel approaches to control the biological properties and behavior of nanomaterials in living cells.

[The Role of Extracellular Calcium Ions Reduction in T Cell Activation in Human Peripheral Blood].

Calcium plays a fundamental role in many essential live functions. It is well-known that many infections can lead to hypocalcaemia in humans. The importance of extracellular calcium concentration ([Ca2+]o) in T-cell activation is well recognized, but [Ca2+]o levels necessary for lymphocyte activation have not been investigated in detail. Whole blood from healthy donors was stimulated by immobilized anti-CD3 plus soluble co-stimulation anti-CD28 mAb and activation parameters have been analyzed. Interleukins-2 (IL-2), JL-4, IL-17A, interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production and CD4+CD69+, CD4+CD25+, CD4+(CYTOKINE)+ T cells were used as activation parameters. To change current [Ca2+]o values in PB we used EGTA. No EGTA influence on resting T cells was shown. Dependences of T-cell activation parameters on various [Ca2+]o were studied. These data indicate that extracellular Ca2+ chelation by EGTA leads to non-linear dependences of T-cell activation parameters on [Ca2+]o values. Obtained dependence of the number of CD4+CD69+ T cells from EGTA can be represented as a nonlinear curve with two distinct peaks. Cytokines production by activated Th1, Th2 and Th17 cells and CD4+(CYTOKINE)+ T cells have been analyzed in dependence on EGTA. The first peak is close to the norm-pathology border. The second peak is in the pathological [Ca2+]o values of PB. However, the local minimum between them is entirely in the "pathological" range. Obtained dependences can be represented as a nonlinear curve with two distinct peaks. All dependences shape have similar curve describing the influence of [EGTA] values on activated CD4+CD69+ T cells. Physiological significance of obtained data discussed.

[The Analysis for Probable Reasons of Cd4+ T-Cell Activation Non-Linear Dependence on Extra Cellular Calcium Ion Concentration in Human Peripheral Blood in vitro].

The analysis for probable reasons of CD4+ T-cell activation non-linear dependence on [Ca2+]o in HPB in vitro is the general aim of current work. At the beginning we pursued the analysis of receptor-dependent (the mixture of monoclonal antibodies (mAbs) to CD3 and CD28 molecules) and receptor-independent (phorbol-myristate-acetate and ionomycin mixture) means to activate T cells in vitro with different [Ca2+]o in HPB. The key role of intracellular T-cell signaling systems in activated T cells in their non-similar sensitivity to calcium ions in the blood was shown. The analysis of differentiation next stages of CD4+ T-cell activation in vitro relatively [Ca2+]o in PHB demonstrates the key role of the earliest induction stages in non-similar sensitivity to calcium ions in CD4+ T-cell activation in vitro. According to the pursued analysis; the non-similar sensitivity of CD4+ T-cell in vitro to activation is in no-way connected with pace differences on the primary stages of activation process. The comparison of CD4+ memory T cells with their naive T-cell precursors in the cell activation process in hypocalcemia conditions was made in the separate experimental series. The 1st maximum consists in average of 85% CD4+CD45R0high CD69+ memory T cells. Naive CD4+CD45RAlowCD69+ T cells constitute the remainder 15%. The 2nd maximum almost completely consists of CD4+CD45R0+CD69+ memory T cells. The ratio between CD4+CD69+ T cell maximums depends on donor ages and represents linear dependence with R = -0.981. The most probable candidate on the role of CD4+ T cell, being capable of activation in hypocalcemia conditions, are memory T lymphocytes, being resistant to ionomycin action (I R) subset. To check this assumption the mononuclear cells and their IR-fraction were prepared from donor PB. Then the mononuclear cells and their IR-fraction were activated by mAbs mixture at different [EGTA] values. For IR-fraction, enriched with CD4+CD45RA-CD45R0+ memory T cells, slightly seen 1st maximum and drastic 2nd maximum in the "pathology" [Ca2+] region was observed. Most likely, namely, at the 2nd maximum, there is IR CD4+CD45RA-CD45R0+ memory T cell majority, having changed intracellular calcium signaling system, to be activated. Hence, the existence for CD4+ T cell activation two maximums in hypocalcemia conditions is connected with the presence of two subsets of CD4+ memory T cells, differing in their calcium-dependent intracellular signaling system, in HPB.

Structural and spectral characterization of novel non-centrosymmetric 2,4-dintrobenzene derivative.

A novel organic compound 9-(2,4-dinitrophenyloxy)-3,3,8-trimethyl-1,5-dihydro-[1,3]dioxepino[5,6-c]pyridine (DNPAP) has been synthesized and a nonlinear optical (NLO) crystal has been grown by slow evaporation method. Compound was subjected to different characterization analyses in order to find out its suitability for optoelectronic applications. Single crystal and powder X-ray diffraction analyses show that DNPAP crystallizes in the orthorhombic space group Pca21. The range of optical absorption was ascertained by recording UV­Vis spectrum. The second harmonic generation (SHG) test has shown that DNPAP possesses 6.4-times higher NLO efficiency compared to KDP.

Endemic human blastomycosis in Quebec, Canada, 1988-2011.

Blastomycosis is a systemic fungal infection found in various parts of the world. A review of literature for Quebec, Canada revealed only few case reports with the most recent one dating back to 1993. However, whether Quebec represents an important endemic region for blastomycosis in North America is unknown. In this work we reviewed 158 cases of human blastomycosis documented in Quebec during 1988-2011 using microbiological records available from the provincial public health laboratory. The estimated annual incidence of blastomycosis in the province is was ~0·133 cases per 100 000 individuals with the highest rates of 0·79 and 0·46 cases per 100 000 recorded in South-eastern and South-western Quebec. Moreover, the annual incidence rate significantly increased over the past 20 years. This study for the first time establishes Quebec as an important endemic region for Blastomyces dermatitidis.