Cytometry of apoptosis. Historical perspective and new advances

Characteristic changes in cell morphology paralleled by the appearance of a multitude of molecular and biochemical markers occur during apoptosis. These changes vary depending on the cell type, mechanism of induction of apoptosis, and the time-window at which the process of apoptosis is analyzed. By virtue of the capability of rapid measurement of individual cells the flow- and imaging-cytometry become preferred technologies to detect, identify and record incidence of apoptosis in large cell populations. It also provided a valuable tool to investigate molecular mechanisms in field of necrobiology. This review outlines the progress in development of the most commonly used cytometric methods probing cells death based on analysis of fragmentation of DNA, activation of caspases, analysis of mitochondrial potential, alterations in plasma membrane structure and other features that characterize programmed cell death. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later

Integrated microfluidic technology for sub-lethal and behavioral marine ecotoxicity biotests

Changes in behavioral traits exhibited by small aquatic invertebrates are increasingly postulated as ethically acceptable and more sensitive endpoints for detection of water-born ecotoxicity than conventional mortality assays. Despite importance of such behavioral biotests, their implementation is profoundly limited by the lack of appropriate biocompatible automation, integrated optoelectronic sensors, and the associated electronics and analysis algorithms. This work outlines development of a proof-of-concept miniaturized Lab-on-a-Chip (LOC) platform for rapid water toxicity tests based on changes in swimming patterns exhibited by Artemia franciscana (Artoxkit MTM) nauplii. In contrast to conventionally performed end-point analysis based on counting numbers of dead/immobile specimens we performed a time-resolved video data analysis to dynamically assess impact of a reference toxicant on swimming pattern of A. franciscana. Our system design combined: (i) innovative microfluidic device keeping free swimming Artemia sp. nauplii under continuous microperfusion as a mean of toxin delivery; (ii) mechatronic interface for user-friendly fluidic actuation of the chip; and (iii) miniaturized video acquisition for movement analysis of test specimens. The system was capable of performing fully programmable time-lapse and video-microscopy of multiple samples for rapid ecotoxicity analysis. It enabled development of a user-friendly and inexpensive test protocol to dynamically detect sub-lethal behavioral end-points such as changes in speed of movement or distance traveled by each animal

Integrating microfluidic generation, handling and analysis of biomimetic giant unilamellar vesicles

The key roles played by phospholipids in many cellular processes, has led to the development of model systems, to explore both lipid–lipid and lipid–peptide interactions. Biomimetic giant unilamellar vesicles represent close facsimiles of in vivo cellular membranes, although currently their widespread use in research is hindered by difficulties involving their integration into high-throughput techniques, for exploring membrane biology intensively in situ. This paper presents an integrated microfluidic device for the production, manipulation and high-throughput analysis of giant unilamellar vesicles. Its utility is demonstrated by exploring the lipid interaction dynamics of the pore-forming antimicrobial peptide melittin, assessed through the release of fluorescent dyes from within biomimetic vesicles, with membrane compositions similar to mammalian plasma membranes

Miniaturized Embryo Array for Automated Trapping, Immobilization and Microperfusion of Zebrafish Embryos

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale

Gradient microfluidics enables rapid bacterial growth inhibition testing

Bacterial growth inhibition tests have become a standard measure of the adverse effects of inhibitors for a wide range of applications, such as toxicity testing in the medical and environmental sciences. However, conventional well-plate formats for these tests are laborious and provide limited information (often being restricted to an end-point assay). In this study, we have developed a microfluidic system that enables fast quantification of the effect of an inhibitor on bacteria growth and survival, within a single experiment. This format offers a unique combination of advantages, including long-term continuous flow culture, generation of concentration gradients, and single cell morphology tracking. Using Escherichia coli and the inhibitor amoxicillin as one model system, we show excellent agreement between an on-chip single cell-based assay and conventional methods to obtain quantitative measures of antibiotic inhibition (for example, minimum inhibition concentration). Furthermore, we show that our methods can provide additional information, over and above that of the standard well-plate assay, including kinetic information on growth inhibition and measurements of bacterial morphological dynamics over a wide range of inhibitor concentrations. Finally, using a second model system, we show that this chip-based systems does not require the bacteria to be labeled and is well suited for the study of naturally occurring species. We illustrate this using Nitrosomonas europaea, an environmentally important bacteria, and show that the chip system can lead to a significant reduction in the period required for growth and inhibition measurements (<4 days, compared to weeks in a culture flask)

Human surfactant protein D alters oxidative stress and HMGA1 expression to induce p53 apoptotic pathway in eosinophil leukemic cell line

This article is made available through the Brunel Open Access Publishing Fund. Copyright: © 2013 Mahajan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Surfactant protein D (SP-D), an innate immune molecule, has an indispensable role in host defense and regulation of inflammation. Immune related functions regulated by SP-D include agglutination of pathogens, phagocytosis, oxidative burst, antigen presentation, T lymphocyte proliferation, cytokine secretion, induction of apoptosis and clearance of apoptotic cells. The present study unravels a novel ability of SP-D to reduce the viability of leukemic cells (eosinophilic leukemic cell line, AML14.3D10; acute myeloid leukemia cell line, THP-1; acute lymphoid leukemia cell lines, Jurkat, Raji; and human breast epithelial cell line, MCF-7), and explains the underlying mechanisms. SP-D and a recombinant fragment of human SP-D (rhSP-D) induced G2/M phase cell cycle arrest, and dose and timedependent apoptosis in the AML14.3D10 eosinophilic leukemia cell line. Levels of various apoptotic markers viz. activated p53, cleaved caspase-9 and PARP, along with G2/M checkpoints (p21 and Tyr15 phosphorylation of cdc2) showed significant increase in these cells. We further attempted to elucidate the underlying mechanisms of rhSP-D induced apoptosis using proteomic analysis. This approach identified large scale molecular changes initiated by SPD in a human cell for the first time. Among others, the proteomics analysis highlighted a decreased expression of survival related proteins such as HMGA1, overexpression of proteins to protect the cells from oxidative burst, while a drastic decrease in mitochondrial antioxidant defense system. rhSP-D mediated enhanced oxidative burst in AML14.3D10 cells was confirmed, while antioxidant, N-acetyl-L-cysteine, abrogated the rhSP-D induced apoptosis. The rhSP-D mediated reduced viability was specific to the cancer cell lines and viability of human PBMCs from healthy controls was not affected. The study suggests involvement of SP-D in host’s immunosurveillance and therapeutic potential of rhSP-D in the eosinophilic leukemia and cancers of other origins.Department of Biotechnology, Indi

Conjugation with L, L-diphenylalanine Self-Assemblies Enhances In Vitro Antitumor Activity of Phthalocyanine Photosensitizer

We present the synthesis and characterization of new peptide conjugates obtained by hierarchical co-assembly of L,L-diphenylalanine (FF) and zinc phthalocyanine complexes (ZnPc) in water. Self-assembly capabilities under defined conditions were investigated by scanning electron microscopy, and photophysical properties were evaluated using UV-Vis and fluorescence spectroscopy. AFM observations demonstrated that these ZnPcs form different highly ordered arrays on the crystalline faces of the FF microplates and that surface roughness significantly changes with the presence of differently substituted phthalocyanine units. XRD assays showed that the overall molecular packing of the conjugates is organized according to a hexagonal symmetry, with ZnPcs hosted in the interstices of the peptide phase. In vitro photodynamic studies were conducted on human breast cancer MCF-7 cells to investigate both cellular uptake and cytotoxicity. It was shown that FF self-assemblies are not toxicity and enhance accumulation of ZnPc in MCF-7 cells, improving apoptotic cell death upon irradiation. Our findings demonstrate enhancement of ZnPc antitumor efficiency by FF conjugates and a proof-of-concept for new photosensitizer carriers based on peptide conjugates

Applications of microalgal biofilms for wastewater treatment and bioenergy production

Background: Microalgae have shown clear advantages for the production of biofuels compared with energy crops. Apart from their high growth rates and substantial lipid/triacylglycerol yields, microalgae can grow in wastewaters (animal, municipal and mining wastewaters) efficiently removing their primary nutrients (C, N, and P), heavy metals and micropollutants, and they do not compete with crops for arable lands. However, fundamental barriers to the industrial application of microalgae for biofuel production still include high costs of removing the algae from the water and the water from the algae which can account for up to 30–40% of the total cost of biodiesel production. Algal biofilms are becoming increasingly popular as a strategy for the concentration of microalgae, making harvesting/dewatering easier and cheaper. Results: We have isolated and characterized a number of natural microalgal biofilms from freshwater, saline lakes and marine habitats. Structurally, these biofilms represent complex consortia of unicellular and multicellular, photosynthetic and heterotrophic inhabitants, such as cyanobacteria, microalgae, diatoms, bacteria, and fungi. Bioflm #52 was used as feedstock for bioenergy production. Dark fermentation of its biomass by Enterobacter cloacae DT-1 led to the production of 2.4 mol of H2/mol of reduced sugar. The levels and compositions of saturated, monosaturated and polyunsaturated fatty acids in Bioflm #52 were target-wise modifed through the promotion of the growth of selected individual photosynthetic inhabitants. Photosynthetic components isolated from different biofilms were used for tailoring of novel biofilms designed for (i) treatment of specifc types of wastewaters, such as reverse osmosis concentrate, (ii) compositions of total fatty acids with a new degree of unsaturation and (iii) bio-focculation and concentration of commercial microalgal cells. Treatment of different types of wastewaters with biofilms showed a reduction in the concentrations of key nutrients, such as phosphates, ammonia, nitrates, selenium and heavy metals. Conclusions: This multidisciplinary study showed the new potential of natural bioflms, their individual photosynthetic inhabitants and assembled new algal/cyanobacterial bioflms as the next generation of bioenergy feedstocks which can grow using wastewaters as a cheap source of key nutrient

Assessment of biocompatibility of 3D printed photopolymers using zebrafish embryo toxicity assays

3D printing has emerged as a rapid and cost-efficient manufacturing technique to enable the fabrication of bespoke, complex prototypes. If the technology is to have a significant impact in biomedical applications, such as drug discovery and molecular diagnostics, the devices produced must be biologically compatible to enable their use with established reference assays and protocols. In this work we demonstrate that we can adapt the Fish Embryo Test (FET) as a new method to quantify the toxicity of 3D printed microfluidic devices. We assessed the biocompatibility of four commercially available 3D printing polymers (VisiJetCrystal EX200, Watershed 11122XC, Fototec SLA 7150 Clear and ABSplus P-430), through the observation of key developmental markers in the developing zebrafish embryos. Results show all of the photopolymers to be highly toxic to the embryos, resulting in fatality, although we do demonstrate that postprinting treatment of Fototec 7150 makes it suitable for zebrafish culture within the FET