Optical recordings of organellar membrane potentials and the components of membrane conductance in lysosomes.

The eukaryotic cell is highly compartmentalized with organelles. Owing to their function in transporting metabolites, metabolic intermediates and byproducts of metabolic activity, organelles are important players in the orchestration of cellular function. Recent advances in optical methods for interrogating the different aspects of organellar activity promise to revolutionize our ability to dissect cellular processes with unprecedented detail. The transport activity of organelles is usually coupled to the transport of charged species; therefore, it is not only associated with the metabolic landscape but also entangled with membrane potentials. In this context, the targeted expression of fluorescent probes for interrogating organellar membrane potential (Ψorg) emerges as a powerful approach, offering less-invasive conditions and technical simplicity to interrogate cellular signalling and metabolism. Different research groups have made remarkable progress in adapting a variety of optical methods for measuring and monitoring Ψorg. These approaches include using potentiometric dyes, genetically encoded voltage indicators, hybrid fluorescence resonance energy transfer sensors and photoinduced electron transfer systems. These studies have provided consistent values for the resting potential of single-membrane organelles, such as lysosomes, the Golgi and the endoplasmic reticulum. We can foresee the use of dynamic measurements of Ψorg to study fundamental problems in organellar physiology that are linked to serious cellular disorders. Here, we present an overview of the available techniques, a survey of the resting membrane potential of internal membranes and, finally, an open-source mathematical model useful to interpret and interrogate membrane-bound structures of small volume by using the lysosome as an example.

Comparative Analysis of Fluorescent Labeling Techniques for Tracking Canine Amniotic Stem Cells.

The utmost aim of regenerative medicine is to promote the regeneration of injured tissues using stem cells. Amniotic mesenchymal stem cells (AmMSCs) have been used in several studies mainly because of their easy isolation from amniotic tissue postpartum and immunomodulatory and angiogenic properties and the low level of rejection. These cells share characteristics of both embryonic/fetal and adult stem cells and are particularly advantageous because they do not trigger tumorigenic activity when injected into immunocompromised animals. The large-scale use of AmMSCs for cellular therapies would greatly benefit from fluorescence labeling studies to validate their tracking in future therapies. This study evaluated the fluorophore positivity, fluorescence intensity, and longevity of canine AmMSCs. For this purpose, canine AmMSCs from the GDTI/USP biobank were submitted to three labeling conditions, two commercial fluorophores [CellTrace CFSE Cell Proliferation kit - CTrace, and CellTracker Green CMFDA - CTracker (CellTracker Green CMFDA, CT, #C2925, Molecular Probes®; Life Technologies)] and green fluorescent protein (GFP) expression after lentiviral transduction, to select the most suitable tracer in terms of adequate persistence and easy handling and analysis that could be used in studies of domestic animals. Fluorescence was detected in all groups; however, the patterns were different. Specifically, CTrace and CTracker fluorescence was detected 6 h after labeling, while GFP was visualized no earlier than 48 h after transduction. Flow cytometry analysis revealed more than 70% of positive cells on day 7 in the CTrace and CTracker groups, while fluorescence decreased significantly to 10% or less on day 20. Variations between repetitions were observed in the GFP group under the present conditions. Our results showed earlier fluorescence detection and more uniform results across repetitions for the commercial fluorophores. In contrast, fluorescence persisted for more extended periods in the GFP group. These results indicate a promising direction for assessing the roles of canine AmMSCs in regenerative medicine without genomic integration.

Wide visible-range activatable fluorescence ZnSe:Eu3+/Mn2+@ZnS quantum dots: local atomic structure order and application as a nanoprobe for bioimaging.

The development of QDs-based fluorescent bionanoprobe for cellular imaging fundamentally relies upon the precise knowledge of particle-cell interaction, optical properties of QDs inside and outside of the cell, movement of a particle in and out of the cell, and the fate of particle. We reported engineering and physicochemical characterization of water-dispersible Eu3+/Mn2+ co-doped ZnSe@ZnS core/shell QDs and studied their potential as a bionanoprobe for biomedical applications, evaluating their biocompatibility, fluorescence behaviour by CytoViva dual mode fluorescence imaging, time-dependent uptake, endocytosis and exocytosis in RAW 264.7 macrophages. The oxidation state and local atomic structure of the Eu dopant studied by X-ray absorption fine structure (XAFS) analysis manifested that the Eu3+ ions occupied sites in both ZnSe and ZnS lattices for the core/shell QDs. A novel approach was developed to relieve the excitation constraint of wide bandgap ZnSe by co-incorporation of Eu3+/Mn2+ codopants, enabling the QDs to be excited at a wide UV-visible range. The QDs displayed tunable emission colors by a gradual increase in Eu3+ concentration at a fixed amount of Mn2+, systematically enhancing the Mn2+ emission intensity via energy transfer from the Eu3+ to Mn2+ ion. The ZnSe:Eu3+/Mn2+@ZnS QDs presented high cell viability above 85% and induced no cell activation. The detailed analyses of QDs-treated cells by dual mode fluorescence CytoViva microscopy confirmed the systematic color-tunable fluorescence and its intensity enhances as a function of incubation time. The QDs were internalized by the cells predominantly via macropinocytosis and other lipid raft-mediated endocytic pathways, retaining an efficient amount for 24 h. The unique color tunability and consistent high intensity emission make these QDs useful for developing a multiplex fluorescent bionanoprobe, activatable in wide-visible region.

Detection of lipid peroxidation in dog spermatozoa with the fluorescent dye C11-BODIPY581/591.

C11-BODIPY581/591 is a fluorescent probe that has been successfully used to evaluate lipid peroxidation in different species, but it has not been completely studied in the dog. Thus, the aim of the present study was to assess lipid peroxidation of dog spermatozoa using C11-BODIPY581/591 and compare different positive controls of the technique. Twenty-four ejaculates were collected from 8 adult male dogs. Routine seminal characteristics were evaluated in raw semen. Lipid peroxidation evaluation was performed as described in other species. Samples were divided in three aliquots, exposed to UV radiation, incubated with hydrogen peroxide or left without treatment (control). Lipid peroxidation was significantly greater only in UV-exposed samples than in the control ones (91 ± 6% vs. 8.3 ± 3.5%, p Ë‚ .01). In conclusion, C11-BODIPY581/591 is useful to evaluate lipid peroxidation of dog spermatozoa and UV radiation is a good promoter of membrane oxidation, so irradiated samples can be used as a positive control of this technique.

Plasma Membrane Fluidity: An Environment Thermal Detector in Plants.

The lipid matrix in cell membranes is a dynamic, bidimensional array of amphipathic molecules exhibiting mesomorphism, which contributes to the membrane fluidity changes in response to temperature fluctuation. As sessile organisms, plants must rapidly and accurately respond to environmental thermal variations. However, mechanisms underlying temperature perception in plants are poorly understood. We studied the thermal plasticity of membrane fluidity using three fluorescent probes across a temperature range of -5 to 41 °C in isolated microsomal fraction (MF), vacuolar membrane (VM), and plasma membrane (PM) vesicles from Arabidopsis plants. Results showed that PM were highly fluid and exhibited more phase transitions and hysteresis, while VM and MF lacked such attributes. These findings suggest that PM is an important cell hub with the capacity to rapidly undergo fluidity modifications in response to small changes of temperatures in ranges spanning those experienced in natural habitats. PM fluidity behaves as an ideal temperature detector: it is always present, covers the whole cell, responds quickly and with sensitivity to temperature variations, functions with a cell free-energy cost, and it is physically connected with potential thermal signal transducers to elicit a cell response. It is an optimal alternative for temperature detection selected for the plant kingdom.

Calibration of mammalian skeletal muscle Ca2+ transients recorded with the fast Ca2+ dye Mag-Fluo-4.

BACKGROUND: Mag-Fluo-4 is increasingly employed for studying Ca2+ signaling in skeletal muscle; however, the lack of information on the Ca2+-Mag-Fluo-4 reaction limits its wider usage. METHODS: Fluorescence and isothermal titration calorimetry (ITC) experiments were performed to determine the binding stoichiometry (n) and thermodynamics (enthalpy (ΔH) and entropy (ΔS) changes), as well as the in vitro and in situ Kd of the Ca2+-Mag-Fluo-4 reaction. Rate constants (kon, koff), fluorescence maximum (Fmax), minimum (Fmin), and the dye compartmentalization were also estimated. Experiments in cells used enzymatically dissociated flexor digitorum brevis fibres of C57BL6, adult mice, loaded at room temperature for 8 min, with 6 µM Mag-Fluo-4, AM, and permeabilized with saponin or ionomycin. All measurements were done at 20 °C. RESULTS: The in vitro fluorescence assays showed a binding stoichiometry of 0.5 for the Ca2+/Mag-Fluo-4 (n = 5) reaction. ITC results (n = 3) provided ΔH and ΔS values of 2.3 (0.7) kJ/mol and 97.8 (5.9) J/mol.K, respectively. The in situ Kd was 1.652 × 105µM2(n = 58 fibres, R2 = 0.99). With an Fmax of 150.9 (8.8) A.U. (n = 8), Fmin of 0.14 (0.1) A.U. (n = 10), and ΔF of Ca2+ transients of 8.4 (2.5) A.U. (n = 10), the sarcoplasmic [Ca2+]peak reached 22.5 (7.8) µM. Compartmentalized dye amounted to only 1.1 (0.7)% (n = 10). CONCLUSIONS: Two Mag-Fluo-4 molecules coalesce around one Ca2+ ion, in an entropy-driven, very low in situ affinity reaction, making it suitable to reliably track the kinetics of rapid muscle Ca2+ transients. GENERAL SIGNIFICANCE: Our results may be relevant to the quantitative study of Ca2+ kinetics in many other cell types.

In vivo uptake of a fluorescent conjugate of melanin-concentrating hormone in the rat brain.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide synthesized by posterior hypothalamic and incerto-hypothalamic neurons that project throughout the central nervous system. The MCHergic system modulates several important functions such as feeding behavior, mood and sleep. MCH exerts its biological functions through interaction with the MCHR-1 receptor, the only functional receptor present in rodents. The internalization process of MCHR-1 triggered by MCH binding was described in vitro in non-neuronal heterologous systems with over-expression of MCHR-1. Reports of in vivo MCHR-1 internalization dynamics are scarce, however, this is an important process to explore based on the critical functions of the MCHergic system. We had previously determined that 60 min after intracerebroventricular (i.c.v.) microinjections of MCH conjugated with fluorophore rhodamine (R-MCH), the dorsal and median raphe nucleus presented R-MCH positive labeled neurons. In the present work, we further studied the in vivo uptake process focusing on the distribution and time-dependent pattern of R-MCH positive cells 10, 20 and 60 min (T10, T20 and T60, respectively) after i.c.v. microinjection of R-MCH. We also explored this uptake process to see whether it was receptor- and clathrin-dependent and examined the phenotype of R-MCH positive cells and their proximity to MCHergic fibers. We found a great number of R-MCH positive cells with high fluorescence intensity in the lateral septum, nucleus accumbens and hippocampus at T20 and T60 (but not at T10), while a lower number with low intensity was observed in the dorsal raphe nucleus. At T20, in rats pre-treated with a MCHR-1 antagonist (ATC-0175) or with phenylarsine oxide (PAO), a clathrin endocytosis inhibitor, a robust decrease (> 50 %) of R-MCH uptake occurred in these structures. The R-MCH positive cells were identified as neurons (NeuN positive, GFAP negative) and some MCHergic fibers run in the vicinities of them. We concluded that neurons localized at structures that were close to the ventricular surfaces could uptake R-MCH in vivo through a receptor-dependent and clathrin-mediated process. Our results support volume transmission of MCH through the cerebrospinal fluid to reach distant targets. Finally, we propose that R-MCH would be an effective tool to study MCH-uptake in vivo.

Efficiency of CellROX deep red® and CellROX orange® fluorescent probes in identifying reactive oxygen species in sperm samples from high and low fertility bulls.

Detection of reactive oxygen species (ROS) is of great interest in semen analysis since their excess is detrimental to sperm function and male fertility. Fluorescence microscopy has achieved attention for providing broad possibilities of sperm evaluations and also for presenting substantial accessibility. In this context, this study investigated the efficiency of CellROX Deep Red® and Orange® probes in detecting ROS in bovine sperm cells and assessed their relationship with sperm fertility potential. First, 16 ejaculates were assigned in three treatments: T0 (no ROS production induced), T1x (ROS production induced once) and T2x (ROS production induced twice). Samples were incubated with Red and Orange probes and percentages of cells producing ROS were evaluated using fluorescence microscopy. Coefficient of determination was 0.61 for Red and 0.56 for Orange. Afterwards, frozen-thawed semen samples from high and low fertility bulls were evaluated regarding percentages of cells producing ROS detected by Red and Orange. Higher levels of ROS assessed by Red were detected in low fertility bovine samples. In conclusion, CellROX Red® and Orange® are both efficient in detecting ROS in bovine spermatozoa. Furthermore, higher sperm ROS detection by CellROX Red® might be associated with low fertility samples.

Plant extracts and betulin from Ligaria cuneifolia inhibit P-glycoprotein function in leukemia cells.

Overexpression of P-glycoprotein (P-gp), which is linked to multidrug resistance (MDR), is one of the underlying obstacles to the success of chemotherapy as it reduces the efficacy of anticancer drugs and the side effects of these increase as a result of any increased dose to achieve the therapeutic effect. To identify agents with P-gp inhibitory properties, ethanol extracts from 80 plants were screened for their ability to increase intracellular doxorubicin-associated fluorescence, and the extract of Ligaria cuneifolia was found to be the most effective. Its bioassay-guided isolation yielded the pentacyclic triterpene betulin as active agent. This efficiently inhibited P-gp mediated efflux, as demonstrated by the enhancement of the intracellular accumulation of doxorubicin and rhodamine 123 from 1.56 µM in the P-gp overexpressing MDR leukemia cell, Lucena 1. Betulin was also able to render Lucena 1 sensitive to Dox from 0.39 µM. The docking studies revealed that betulin tightly binds to a key region of the TMDs, with a binding mode overlapping one main site of doxorubicin and, more interestingly, emulating the same contacts as tariquidar, as revealed by the per-residue energetic analysis from molecular dynamics simulations. MTT assay using peripheral blood mononuclear cells and hemolysis assay showed that betulin is devoid of toxicity. These findings provide important evidence that betulin may be a safe and promising entity to be further investigated to develop agents able to overcome P-gp-mediated MDR, resulting in a more effective and less toxic chemotherapy.

Monitoring Lactate Dynamics in Individual Macrophages with a Genetically Encoded Probe.

Lactate, the product of aerobic glycolysis, plays a dual role as fuel and intercellular signal in inflammation, immune evasion, and tumor progression. The production of lactate by macrophages has been associated with their polarization and function. Here we describe imaging protocols to characterize the metabolism of cultured human macrophages using a genetically encoded fluorescent sensor-specific for lactate. By superfusing cultures with increasing lactate concentrations and pharmacological inhibitors, it is possible to estimate the kinetic parameters of monocarboxylate transporter 4 (MCT4) and lactate production. Practical advice is given regarding sensor expression, imaging, and data analysis. The spatiotemporal resolution of this technique is amenable to the study of fast events at the single-cell level in different immune and other cell types.

Characterisation by Excitation-Emission Matrix Fluorescence Spectroscopy of Pigments in Mucus Secreted of Earthworm Eisenia foetida Exposed to Lead.

The earthworm exposed to toxics shows physiological responses as: avoidance and mucus secretion. Heavy metals are particularly toxic to earthworms and the mucus secretion has been considered as a defence mechanism against undesirable substance. The chromophores present in the mucus secretion of Eisenia foetida have been poorly studied. Mucus secretion of E. foetida was induced by PbCl2. High PbCl2 concentrations provoked abundant mucus secretion which showed fluorescence when illuminated by UV light. Dialysis membrane separation, UV Visible and Excitation-Emission Matrix Fluorescence (EEM) spectroscopy were used to characterise the fluorescent pigments. EEM spectroscopy analysis of the mucus secretion signalled three excitation-emission peaks at: 310/380 nm, 370/520 nm and 440/520 nm. Two fluorophores were separated by dialysis. One of them matched the fluorescent compound riboflavin excitation-emission profile; the other is a protein with a peak 290/350 nm. Native-PAGE electrophoresis was conducted to assess the riboflavin-biding ability of the coelomic fluid protein produced by Eisenia foetida showing a high riboflavin-biding ability.

ANS Interacts with the Ca2+-ATPase Nucleotide Binding Site.

The binding of 8-anilino-1-naphthalene sulfonate (ANS) to the nucleotide binding domain (N-domain) of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) was studied. Molecular docking predicted two ANS binding modes (BMI and BMII) in the nucleotide binding site. The molecular interaction was confirmed as the fluorescence intensity of ANS was dramatically increased when in the presence of an engineered recombinant N-domain. Molecular dynamics simulation showed BMI (which occupies the ATP binding site) as the mode that is stable in solution. The above was confirmed by the absence of ANS fluorescence in the presence of a fluorescein isothiocyanate (FITC)-labeled N-domain. Further, the labeling of the N-domain with FITC was hindered by the presence of ANS, i.e., ANS was bound to the ATP binding site. Importantly, ANS displayed a higher affinity than ATP. In addition, ANS binding led to quenching the N-domain intrinsic fluorescence displaying a FRET pattern, which suggested the existence of a Trp-ANS FRET couple. Nonetheless, the chemical modification of the sole Trp residue with N-bromosuccinimide (NBS) discarded the existence of FRET and instead indicated structural rearrangements in the nucleotide binding site during ANS binding. Finally, Ca2+-ATPase kinetics in the presence of ANS showed a partial mixed-type inhibition. The Dixon plot showed the ANS-Ca2+-ATPase complex as catalytically active, hence supporting the existence of a functional dimeric Ca2+-ATPase in sarcoplasmic reticulum vesicles. ANS may be used as a molecular platform for the development of more effective inhibitors of Ca2+-ATPase and appears to be a new fluorescent probe for the nucleotide binding site. Graphical Abstract Molecular docking of ANS to the nucleotide binding site of Ca2+-ATPase. ANS fluorescence increase reveals molecular interaction.

Binding of rose bengal to lysozyme modulates photooxidation and cross-linking reactions involving tyrosine and tryptophan.

This work examined the hypothesis that interactions of Rose Bengal (RB2-) with lysozyme (Lyso) might mediate type 1 photoreactions resulting in protein cross-linking even under conditions favoring 1O2 formation. UV-visible spectrophotometry, isothermal titration calorimetry (ITC), and docking analysis were employed to characterize RB2--Lyso interactions, while oxidation of Lyso was studied by SDS-PAGE gels, extent of amino acid consumption, and liquid chromatography (LC) with mass detection (employing tryptic peptides digested in H218O and H2O). Docking studies showed five interaction sites including the active site. Hydrophobic interactions induced a red shift of the visible spectrum of RB2- giving a Kd of 4.8 µM, while data from ITC studies, yielded a Kd of 0.68 µM as an average of the interactions with stoichiometry of 3.3 RB2- per Lyso. LC analysis showed a high consumption of readily-oxidized amino acids (His, Trp, Met and Tyr) located at different and diverse locations within the protein. This appears to reflect extensive damage on the protein probably mediated by a type 2 (1O2) mechanism. In contrast, docking and mass spectrometry analysis provided evidence for the generation of specific intra- (Tyr23-Tyr20) and inter-molecular (Tyr23-Trp62) Lyso cross-links, and Lyso dimer formation via radical-radical, type 1 mechanisms.

Unravelling the modus-operandi of chromenylium-cyanine fluorescent probes: a case study.

Small-molecule fluorescent probes having optimized optical properties, such as high photostability and brightness, local microenvironment sensitivity and specific subcellular localizations, are increasingly available. Although the basis for designing efficient fluorophores for bioimaging applications is well established, implementing an improvement in a given photophysical characteristic always tends to compromise another optical property. This problem has enormous consequences for in vivo imaging, where ensuring a specific localization and precise control of the probe response is challenging. Herein we discuss a fluorescent probe, CC334, as a case study of the chromenylium-cyanine family that commonly exhibits highly complex photophysical schemes and highly interfered bioanalytical responses. By an exhaustive and concise analysis of the CC334 optical responses including detailed spectroscopic calibrations, steady-state microenvironment effects, ultrafast photophysics analysis and computational studies, we elucidate a new strategy to apply the probe in the singlet oxygen reactive oxygen species (1O2-ROS) monitoring using in vitro and in vivo models. The probe provides a new avenue for designing fluorescent probes to understand the dynamic behavior of subcellular environments.

Water-soluble fluorescent red colorant production by Talaromyces amestolkiae.

The replacement of synthetic colors in food products by natural alternatives has been boosted by consumers willing to pay more for healthier products. However, the success of microbial colorants depends not only on its acceptability on the market but also its production costs. Talaromyces species can produce water-soluble red colorants induced by glucose and monosodium glutamate (MSG). In this study, the influence of several conditions was evaluated to produce natural red colorants by submerged culture of Talaromyces amestolkiae. Under optimal conditions (g/L: glucose 10, MSG 25, MgSO4 0.012, FeSO4 0.01, CaCl2 0.015; and initial pH of 5.0), a 30-fold increase in the production was achieved, reaching a red colorant production of 13.44 UA500nm. Depending on the initial pH, colorants with different hues and chroma values were obtained. Deep yellow colorants were derived from neutral and basic pH, while deep red colors were derived from acidic pH. The fluorescence spectrum of culture broth obtained before and after complexation with salts presented red colorants with yellow fluorescence spectra. The information generated in this study would be useful for the formulation of industrial media for large-scale cultivation of T. amestolkiae, which have the potential to produce Talaromyces fermented colorants for use in health foods and pharmaceutics.

Cannabinoids Induce Cell Death and Promote P2X7 Receptor Signaling in Retinal Glial Progenitors in Culture.

Development of progenitors in the embryonic retina is modulated by signaling molecules, and cannabinoid receptors are highly expressed in the early developing retina. Here, we investigated whether the CB1/CB2 receptor agonist WIN 5212-2 (WIN) modulated the proliferation, viability, and calcium responses in chick embryo retinal progenitors in culture. A decline in [3H]-thymidine incorporation was observed when cultures were incubated with 0.5-1.0 µM WIN, an effect that was mimicked by URB602 and URB597, inhibitors of the monoacylglycerol lipase and fatty acid amide hydrolase, respectively. A reduction in the number of proliferating cell nuclear antigen-positive nuclei was also noticed in WIN-treated cultures, suggesting that activation of cannabinoid receptors decreases the proliferation of cultured retinal progenitors. WIN (0.5-5.0 µM), but not capsaicin, decreased retinal cell viability, an effect that was blocked by CB1 and CB2 receptor antagonists and by the P2X7 receptor antagonist A438079, implicating this nucleotide receptor in the cannabinoid-mediated cell death. Treatment with WIN also induced an increase in mitochondrial superoxide and P2X7 receptor-mediated uptake of sulforhodamine B in the cultured cells. While a high proportion of cultured cells responded to glutamate, GABA, and 50 mM KCl with intracellular calcium shifts, very few cells responded to the activation of P2X7 receptors by ATP. Noteworthy, while decreasing the number of cells responding to glutamate, GABA, and KCl, treatment of the cultures with WIN induced a significant increase in the number of cells responding to 1 mM ATP, suggesting that activation of cannabinoid receptors primes P2X7 receptor calcium signaling in retinal progenitors in culture.

Reduced Liver Lipid Peroxidation in Subcellular Fractions Is Associated with a Hypometabolic State in Rats with Portacaval Anastomosis.

A surgical connection between portal and inferior cava veins was performed to generate an experimental model of high circulating ammonium and hepatic hypofunctioning. After 13 weeks of portacaval anastomosis (PCA), hyperammonemia and shrinkage in the liver were observed. Low glycemic levels accompanied by elevated levels of serum alanine aminotransferase were recorded. However, the activity of serum aspartate aminotransferase was reduced, without change in circulating urea. Histological and ultrastructural observations revealed ongoing vascularization and alterations in the hepatocyte nucleus (reduced diameter with indentations), fewer mitochondria, and numerous ribosomes in the endoplasmic reticulum. High activity of hepatic caspase-3 suggested apoptosis. PCA promoted a marked reduction in lipid peroxidation determined by TBARs in liver homogenate but specially in the mitochondrial and microsomal fractions. The reduced lipoperoxidative activity was also detected in assays supplemented with Fe2+. Only discreet changes were observed in conjugated dienes. Fluorescent probes showed significant attenuation in mitochondrial membrane potential, reactive oxygen species (ROS), and calcium content. Rats with PCA also showed reduced food intake and decreased energy expenditure through indirect calorimetry by measuring oxygen consumption with an open-flow respirometric system. We conclude that experimental PCA promotes an angiogenic state in the liver to confront the altered blood flow by reducing the prooxidant reactions associated with lower metabolic rate, along with significant reduction of mitochondrial content, but without a clear hepatic dysfunction.

Comparison of JC-1 and MitoTracker probes for mitochondrial viability assessment in stored canine platelet concentrates: A flow cytometry study.

Mitochondria perform crucial roles in many biochemical processes, and mitochondrial depolarization is an early sign of platelet apoptosis. The mitochondrial membrane potential is usually evaluated through JC-1 probe, but it can also be assessed with MitoTracker probes. Our aim was to evaluate mitochondrial viability in stored canine platelet concentrates (PCs) with the fluorescent probes JC-1 and MitoTracker. Platelets from 22 canine PCs were stained with JC-1 and MitoTracker probes on days 1, 3, and 5 of storage. Data on metabolic parameters were also collected for correlation studies. Results of JC-1 and MitoTracker revealed a decrease in mitochondrial membrane potential in day 5 of storage compared to days 1 and 3, providing evidence of mitochondrial depolarization, a finding that was confirmed by the data on metabolic parameters. MitoTracker probes also added information regarding platelet swelling. In conclusion, MitoTracker probes offered a more complete mitochondrial analysis in the evaluation of stored canine PCs. © 2018 International Society for Advancement of Cytometry.

Using Flow Cytometry Analysis in Plant Tissue Culture Derived Plants.

Somaclonal variation (SC) in plants regenerated from tissue culture, via organogenesis or somatic embryogenesis, is frequently associated with abnormalities in the content of deoxyribonucleic acid (DNA), viz., aneuploidy and polyploidy. Flow cytometry (FCM) using the nucleic acid-specific fluorochrome propidium iodide has proven to be a rapid, simple, and reproducible technique for assessment of DNA content and ploidy variation occurring in plant tissue cultures. Here an outline of the sample preparation of suspension with intact nuclei by the two-step standard method, and FCM analysis of DNA ploidy stability in plants regenerated from embryogenic cell suspension (ECS) of banana Musa acuminata, AAA, cv. Grand Naine (Cavendish subgroup) using an internal standard is described.

A hybrid mathematical modeling approach of the metabolic fate of a fluorescent sphingolipid analogue to predict cancer chemosensitivity.

Sphingolipid (SL) metabolism is a complex biological system that produces and transforms ceramides and other molecules able to modulate other cellular processes, including survival or death pathways key to cell fate decisions. This signaling pathway integrates several types of stress signals, including chemotherapy, into changes in the activity of its metabolic enzymes, altering thereby the cellular composition of bioactive SLs. Therefore, the SL pathway is a promising sensor of chemosensitivity in cancer and a target hub to overcome resistance. However, there is still a gap in our understanding of how chemotherapeutic drugs can disturb the SL pathway in order to control cellular fate. We propose to bridge this gap by a systems biology approach to integrate i) a dynamic model of SL analogue (BODIPY-FL fluorescent-sphingomyelin analogue, SM-BOD) metabolism, ii) a Gaussian mixture model (GMM) of the fluorescence features to identify how the SL pathway senses the effect of chemotherapy and iii) a fuzzy logic model (FLM) to associate SL composition with cell viability by semi-quantitative rules. Altogether, this hybrid model approach was able to predict the cell viability of double experimental perturbations with chemotherapy, indicating that the SL pathway is a promising sensor to design strategies to overcome drug resistance in cancer.