Non-thermal disruption of ß-adrenergic receptor-activated Ca2+ signalling and apoptosis in human ES-derived cardiomyocytes by microwave electric fields at 2.4 GHz.

The ubiquity of wireless electronic-device connectivity has seen microwaves emerge as one of the fastest growing forms of electromagnetic exposure. A growing evidence-base refutes the claim that wireless technologies pose no risk to human health at current safety levels designed to limit thermal (heating) effects. The potential impact of non-thermal effects of microwave exposure, especially in electrically-excitable tissues (e.g., heart), remains controversial. We exposed human embryonic stem-cell derived cardiomyocytes (CM), under baseline and beta-adrenergic receptor (ß-AR)-stimulated conditions, to microwaves at 2.4 GHz, a frequency used extensively in wireless communication (e.g., 4G, Bluetooth™ and WiFi). To control for any effect of sample heating, experiments were done in CM subjected to matched rates of direct heating or CM maintained at 37 °C. Detailed profiling of the temporal and amplitude features of Ca2+ signalling in CM under these experimental conditions was reconciled with the extent and spatial clustering of apoptosis. The data show that exposure of CM to 2.4 GHz EMF eliminated the normal Ca2+ signalling response to ß-AR stimulation and provoked spatially-clustered apoptosis. This is first evidence that non-thermal effects of 2.4 GHz microwaves might have profound effects on human CM function, responsiveness to activation, and survival.

Functional imaging of a model unicell: Spironucleus vortens as an anaerobic but aerotolerant flagellated protist.

Advances in optical microscopy are continually narrowing the chasm in our appreciation of biological organization between the molecular and cellular levels, but many practical problems are still limiting. Observation is always limited by the rapid dynamics of ultrastructural modifications of intracellular components, and often by cell motility: imaging of the unicellular protist parasite of ornamental fish, Spironucleus vortens, has proved challenging. Autofluorescence of nicotinamide nucleotides and flavins in the 400-580 nm region of the visible spectrum, is the most useful indicator of cellular redox state and hence vitality. Fluorophores emitting in the red or near-infrared (i.e., phosphors) are less damaging and more penetrative than many routinely employed fluors. Mountants containing free radical scavengers minimize fluorophore photobleaching. Two-photon excitation provides a small focal spot, increased penetration, minimizes photon scattering and enables extended observations. Use of quantum dots clarifies the competition between endosomal uptake and exosomal extrusion. Rapid motility (161 µm/s) of the organism makes high resolution of ultrastructure difficult even at high scan speeds. Use of voltage-sensitive dyes determining transmembrane potentials of plasma membrane and hydrogenosomes (modified mitochondria) is also hindered by intracellular motion and controlled anesthesia perturbs membrane organization. Specificity of luminophore binding is always questionable; e.g. cationic lipophilic species widely used to measure membrane potentials also enter membrane-bounded neutral lipid droplet-filled organelles. This appears to be the case in S. vortens, where Coherent Anti-Stokes Raman Scattering (CARS) micro-spectroscopy unequivocally images the latter and simultaneous provides spectral identification at 2840 cm-1. Secondary Harmonic Generation highlights the highly ordered structure of the flagella.

Connect and Conquer: Collectivized Behavior of Mitochondria and Bacteria.

The connectedness of signaling components in network structures is a universal feature of biologic information processing. Such organization enables the transduction of complex input stimuli into coherent outputs and is essential in modulating activities as diverse as the cooperation of bacteria within populations and the dynamic organization of mitochondria within cells. Here, we highlight some common principles that underpin collectivization in bacteria and mitochondrial populations and the advantages conferred by such behavior. We discuss the concept that bacteria and mitochondria act as signal transducers of their localized metabolic environments to bring about energy-dependent clustering to modulate higher-order function across multiple scales.

Bioluminescence of Vibrio fischeri: bacteria respond quickly and sensitively to pulsed microwave electric (but not magnetic) fields.

Biological systems with intrinsic luminescent properties serve as powerful and noninvasive bioreporters for real-time and label-free monitoring of cell physiology. This study employs the bioluminescent marine bacterium Vibrio fischeri to investigate the effects of separated microwave electric (E) and magnetic (H) fields. Using a cylindrical TM010 mode aluminum resonant cavity, designed to spatially separate E and H fields of a pulsed microwave (2.45 GHz) input, we sampled at 100-ms intervals the 490-nm emission of bioluminescence from suspensions of the V. fischeri. E-field exposure (at 4.24 and 13.4 kV/m) results in rapid and sensitive responses to 100-ms pulses. H-field excitation elicits no measurable responses, even at 100-fold higher power input levels (equivalent to 183 A/m). The observed effects on bacterial light output partially correlate with measured E-field-induced temperature increases. In conclusion, the endogenous bioluminescence of V. fischeri provides a sensitive and noninvasive method to assess the biological effects of microwave fields.

Fluorescent functionalised naphthalimides and their Au(i)-NHC complexes for potential use in cellular bioimaging.

A series of cationic, dihydroimidazolinium-functionalized 1,8-naphthalimide fluorophores have been isolated as their hexafluorophosphate salts, [HL]PF6. These pro-ligands react with [AuCl(tht)] in the presence of base to form N-heterocyclic carbene (NHC) complexes, [AuCl(L)]. Two X-ray structures represent a pro-ligand and complex pairing: the latter reveals the two-coordinate linear geometry of the NHC-Au(i) species, as well as intermolecular interactions supported by both ligand π-π stacking and a weak aurophilic interaction of 3.3205(6) Å. The luminescence properties of the pro-ligands and complexes are dominated by the ICT character of the substituted fluorophore at ca. 500 nm, which is further modulated via functionalization at the 4-position of the naphthalimide. Cytotoxicity assessments were performed for all [HL]PF6 and [AuCl(L)] species against LOVO, MCF-7, A549 and PC3 cell lines; added lipophilicity seems to correlate with increased cytotoxicity. Confocal fluorescence microscopy was undertaken on a selected [HL]PF6 and [AuCl(L)] species and showed that the intracellular distribution is dependent upon the specific ligand structure. More detailed co-localisation studies show that selected examples present a predominant lysosomal staining pattern. FLIM studies exemplified the applicability of these probes, and secondly suggest that fluorescence lifetime could be used to provide information on the integrity of the complex and thus liberation of gold in a biological environment.

Redox Pathways as Drug Targets in Microaerophilic Parasites.

The microaerophilic parasites Entamoeba histolytica, Trichomonas vaginalis, and Giardia lamblia jointly cause hundreds of millions of infections in humans every year. Other microaerophilic parasites such as Tritrichomonas foetus and Spironucleus spp. pose a relevant health problem in veterinary medicine. Unfortunately, vaccines against these pathogens are unavailable, but their microaerophilic lifestyle opens opportunities for specifically developed chemotherapeutics. In particular, their high sensitivity towards oxygen can be exploited by targeting redox enzymes. This review focusses on the redox pathways of microaerophilic parasites and on drugs, either already in use or currently in the state of development, which target these pathways.

Avoid Excessive Oxygen Levels in Experiments with Organisms, Tissues and Cells.

O2 levels encountered in vivo in cells and tissues are almost always at least an order of magnitude less than atmospheric pO2 because of sensing, signalling and bioenergetic demand. Although deleterious reactions are minimized by protective mechanisms (residual toxic products scavenged and detoxified) ambient levels should be mimicked in experiments with whole organisms, their isolated organs, tissues or cells and also with cultures of cell lines. These are also important issues for microorganisms inhabiting low O2 niches within higher organisms and their cells. Here, we highlight the importance of optimization of micro-aerobic conditions for experimentation and the deleterious consequences of not doing so.

Water soluble, cyclometalated Pt(ii)-Ln(iii) conjugates towards novel bimodal imaging agents.

Facile conjugation of a luminescent cyclometalated Pt(II) complex with a DO3A-derived Gd(III) moiety yields a hybrid species with visible luminescence and enhanced relaxivity.

Motility of the diplomonad fish parasite Spironucleus vortens through thixotropic solid media.

Investigation of a series of nutrient-supplemented thixotropic gels at successive dilutions that impede the trajectories of a highly vigorous motile flagellated protist, Spironucleus vortens, provides insights into both its swimming characteristics and a means for its immobilization. The progress of movement of this organism through the solidified growth medium was monitored by the in situ reductive production of a formazan chromophore from a dissolved tetrazolium salt. The physical properties of the gels were measured using an Anton Paar rheometer. The test parameters and measurements included: angular frequency, complex viscosity, complex shear modulus, shear rate and rotational recovery. These rheological characteristics affected the forward velocity of the organism through the gels, during and after multiple resetting, information potentially useful for determination of the dynamic characteristics of flagellar movement and propulsion rates of the organism. Application to separation of single cells, individuals of distinct sizes or the differing species from mixed cultures of motile and non-motile organisms or less actively swimming species was evident. These applications can be used when isolating the parasite from the intestinal contents of its host or from faecal pellets.

Comparative biochemistry of Giardia, Hexamita and Spironucleus: Enigmatic diplomonads.

The diplomonad genera are here represented by three highly diverse species, both free-living (Hexamita inflata), and parasitic (Spironucleus vortens and Giardia intestinalis). All three are moderately aerotolerant flagellates, inhabiting environments where O2 tensions are low and fluctuating. Many diplomonads are opportunistic pathogens of avian, terrestrial and aquatic animals. Hexamitids inhabit deep waters and sediments of lakes and marine basins, S. vortens commonly infects the intestinal tract of ornamental fish, particularly of cichlids and cyprinids, and G. intestinalis, the upper intestinal tracts of humans as well as domestic and farm animals. Despite these very different habitats, their known physiological and biochemical characteristics are similar, but they do differ in significant respects as their lifestyles and life cycles demand. They have efficient O2 scavenging systems, and are highly effective at countering rapid O2 fluctuations, or clustering away from its source (except for G. intestinalis when attached to the jejunal villi). Their core metabolic pathways (glycolysis using pyrophosphate), incomplete tricarboxylic acid cycle (lacking α-ketoglutarate dehydrogenase), and amino acid metabolism (with an alternative energy-generating arginine dihydrolase pathway as a possibility in some cases), largely conform to those of other protists inhabiting low-O2 environments. Mitochondrial evolutionary reduction to give hydrogenosomes as seen in Spironucleus spp. has proceeded further to its minimal state in the mitosomes of G. intestinalis. Understanding of essential redox reactions and the maintentence of redox state, especially in the infective encysted stage of G. intestinalis provide increasing possibilities for parasite control. To this aim a plethora of new synthetic chemicals and natural products (especially those from garlic, Allium sativum) show promise as replacements for the highly effective (but potentially toxic to higher organisms) 5-nitroimidazoles (e.g., metronidazole) in the treatment and/or prevention of dimplomonad infection in humans and animals.

Mitochondria-derived organelles in the diplomonad fish parasite Spironucleus vortens.

In some eukaryotes, mitochondria have become modified during evolution to yield derived organelles (MDOs) of a similar size (hydrogenosomes), or extremely reduced to produce tiny cellular vesicles (mitosomes). The current study provides evidence for the presence of MDOs in the highly infectious fish pathogen Spironucleus vortens, an organism that produces H2 and is shown here to have no detectable cytochromes. Transmission electron microscopy (TEM) reveals that S. vortens trophozoites contain electron-dense, membranous structures sometimes with an electron-dense core (200 nm-1 µm), resembling the hydrogenosomes previously described in other protists from habitats deficient in O2. Confocal microscopy establishes that these organelles exhibit autofluorescence emission spectra similar to flavoprotein constituents previously described for mitochondria and also present in hydrogenosomes. These organelles possess a membrane potential and are labelled by a fluorescently labeled antibody against Fe-hydrogenase from Blastocystis hominis. Heterologous antibodies raised to mitochondrial proteins frataxin and Isu1, also exhibit a discrete punctate pattern of localization in S. vortens; however these labelled structures are distinctly smaller (90-150 nm) than hydrogenosomes as observed previously in other organisms. TEM confirms the presence of double-membrane bounded organelles of this smaller size. In addition, strong background immunostaining occurs in the cytosol for frataxin and Isu1, and labelling by anti-ferredoxin antibody is generally distributed and not specifically localized except for at the anterior polar region. This suggests that some of the functions traditionally attributed to such MDOs may also occur elsewhere. The specialized parasitic life-style of S. vortens may necessitate more complex intracellular compartmentation of redox reactions than previously recognized. Control of infection requires biochemical characterization of redox-related organelles.

Unexpected properties of NADP-dependent secondary alcohol dehydrogenase (ADH-1) in Trichomonas vaginalis and other microaerophilic parasites.

Our previous observation that NADP-dependent secondary alcohol dehydrogenase (ADH-1) is down-regulated in metronidazole-resistant Trichomonas vaginalis isolates prompted us to further characterise the enzyme. In addition to its canonical enzyme activity as a secondary alcohol dehydrogenase, a pronounced, so far unknown, background NADPH-oxidising activity in absence of any added substrate was observed when the recombinant enzyme or T. vaginalis extract were used. This activity was strongly enhanced at low oxygen concentrations. Unexpectedly, all functions of ADH-1 were efficiently inhibited by coenzyme A which is a cofactor of a number of key enzymes in T. vaginalis metabolism, i.e. pyruvate:ferredoxin oxidoreductase (PFOR). These observations could be extended to Entamoeba histolytica and Tritrichomonas foetus, both of which have a homologue of ADH-1, but not to Giardia lamblia which lacks an NADP-dependent secondary alcohol dehydrogenase. Although we could not identify the substrate of the observed background activity, we propose that ADH-1 functions as a major sink for NADPH in microaerophilic parasites at low oxygen tension.

Contribution of spores to the ability of Clostridium difficile to adhere to surfaces.

Clostridium difficile is the commonest cause of hospital-acquired infection in the United Kingdom. We characterized the abilities of 21 clinical isolates to form spores; to adhere to inorganic and organic surfaces, including stainless steel and human adenocarcinoma cells; and to germinate. The composition of culture media had a significant effect on spore formation, as significantly more spores were produced in brain heart infusion broth (Student's t test; P = 0.018). The spore surface relative hydrophobicity (RH) varied markedly (14 to 77%) and was correlated with the ability to adhere to stainless steel. We observed no correlation between the ribotype and the ability to adhere to steel. When the binding of hydrophobic (DS1813; ribotype 027; RH, 77%) and hydrophilic (DS1748; ribotype 002; RH, 14%) spores to human gut epithelial cells at different stages of cell development was examined, DS1813 spores adhered more strongly, suggesting the presence of surface properties that aid attachment to human cells. Electron microscopy studies revealed the presence of an exosporium surrounding DS1813 spores that was absent from spores of DS1748. Finally, the ability of spores to germinate was found to be strain and medium dependent. While the significance of these findings to the disease process has yet to be determined, this study has highlighted the importance of analyzing multiple isolates when attempting to characterize the behavior of a bacterial species.

Biologically compatible, phosphorescent dimetallic rhenium complexes linked through functionalized alkyl chains: syntheses, spectroscopic properties, and applications in imaging microscopy.

A range of luminescent, dimetallic complexes based upon the rhenium fac-tricarbonyl diimine core, linked by aliphatic chains of varying lengths and functionality, have been synthesized and their photophysical properties examined. Each complex displays characteristic (3)M(Re)L(diimine)CT emission in aerated acetonitrile solution, with long lifetimes in the range of 129-248 ns and corresponding quantum yields in the range 3.2-8.0%. In aqueous solution, as opposed to acetonitrile, the complexes generally show a small hypsochromic shift in λ(em) and an extension of the (3)MLCT lifetime, attributed to a hydrophobically driven association of the alkyl chains with the rhenium-bound diimine units. In live cell imaging experiments using MCF7 cells the complexes all show good uptake by non-energy dependent mechanisms without endosomal entrainment, and with varying propensity to localize in organelles. The degrees of uptake and localization properties are discussed in terms of the length and chemical nature of the linkers, and in terms of the likely interactions between these and the various cellular components encountered.

A 'Sleeping Trojan Horse' which transports metal ions into cells, localises in nucleoli, and has potential for bimodal fluorescence/PET imaging.

A rhenium polypyridine-based molecular vessel is membrane impermeant when empty, but, upon loading with metal ions, the cationic form is taken up by MCF-7 cells, localising in nucleoli. The luminescence of the vessel and its copper binding ability suggest potential as a bimodal fluorescence/PET imaging agent.