Biophysical modeling of in vitro and in vivo processes underlying regulated photoprotective mechanism in cyanobacteria.

Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed  that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10-5 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.

Comparative analysis of mutant plants impaired in the main regulatory mechanisms of photosynthetic light reactions - From biophysical measurements to molecular mechanisms.

Chlorophyll (chl) fluorescence emission by photosystem II (PSII) and light absorption by P700 reaction center chl a of photosystem I (PSI) provide easy means to probe the function of the photosynthetic machinery. The exact relationship between the measured optical variables and the molecular processes have, however, remained elusive. Today, the availability of mutants with distinct molecular characterization of photosynthesis regulatory processes should make it possible to gain further insights into this relationship, yet a systematic comparative analysis of such regulatory mutants has been missing. Here we have systematically compared the behavior of Dual-PAM fluorescence and P700 variables from well-characterized photosynthesis regulation mutants. The analysis revealed a very convincing relationship between the given molecular deficiency in the photosynthetic apparatus and the original fluorescence and P700 signals obtained by using varying intensities of actinic light and by applying a saturating pulse. Importantly, the specific information on the underlying molecular mechanism, present in these authentic signals of a given photosynthesis mutant, was largely nullified when using the commonly accepted parameters that are based on further treatment of the original signals. Understanding the unique relationship between the investigated molecular process of photosynthesis and the measured variable is an absolute prerequisite for comprehensive interpretation of fluorescence and P700 measurements. The data presented here elucidates the relationships between the main regulatory mechanisms controlling the photosynthetic light reactions and the variables obtained by fluorescence and P700 measurements. It is discussed how the full potential of optical photosynthesis measurements can be utilized in investigation of a given molecular mechanism.

Oxidative mechanisms of biological activity of low-intensity radiofrequency radiation.

This review aims to cover experimental data on oxidative effects of low-intensity radiofrequency radiation (RFR) in living cells. Analysis of the currently available peer-reviewed scientific literature reveals molecular effects induced by low-intensity RFR in living cells; this includes significant activation of key pathways generating reactive oxygen species (ROS), activation of peroxidation, oxidative damage of DNA and changes in the activity of antioxidant enzymes. It indicates that among 100 currently available peer-reviewed studies dealing with oxidative effects of low-intensity RFR, in general, 93 confirmed that RFR induces oxidative effects in biological systems. A wide pathogenic potential of the induced ROS and their involvement in cell signaling pathways explains a range of biological/health effects of low-intensity RFR, which include both cancer and non-cancer pathologies. In conclusion, our analysis demonstrates that low-intensity RFR is an expressive oxidative agent for living cells with a high pathogenic potential and that the oxidative stress induced by RFR exposure should be recognized as one of the primary mechanisms of the biological activity of this kind of radiation.

Effect of incubating egg exposure to magnetic field on the biophysical blood properties of newly-hatched chicks.

Due to widespread of human exposure to electromagnetic fields, there has been increasing public concern about the potential health risks from low-frequency electromagnetic fields; ELF-EMF. The magnetic fields (MFs) affects functions of the living organisms, such as DNA synthesis and ion transportation through the cell membranes. In the present work, the effects of short-term exposure to magnetic fields (MFs) prior to incubation were investigated on the biophysical blood properties of chicks hatched from layer-type breeder eggs. The eggs were exposed to a MF of 0.75 mT at 50 Hz for 20, 40 and 60 min before incubation. This study was performed by measuring the dielectric relaxation of hemoglobin (Hb) molecules and the membrane solubility of red blood cells (RBCs) using the non-ionic detergent octylglucoside. Exposure of the eggs to a MF increased the conductivity of the Hb molecules. The pronounced increase in the conductivity of the exposed eggs might be attributed to an increase in the surface charge of the Hb macromolecules, resulted from the formation of highly active molecular species. This speculation can be supported by the increase in the relaxation time of the exposed groups. The solubilization process of the RBC membrane indicates a loss in the mobility of RBCs in the blood of hatching chicks.

Evaluation of UV radiation-induced toxicity and biophysical changes in various skin cells with photo-shielding molecules.

Ultraviolet radiation (UVR) triggers many complex events in different types of skin cells, including benign, malignant and normal cells. Chromophores present in these cells play a crucial role in various cellular processes. Unprecedented methods are required for the real-time monitoring of changes in an in vitro model exposed to intermittent mild and intense UVR to determine the mechanisms underlying cell degeneration and the effects of unexpected toxic, agonist and antagonist agents. This study reports the analytical application of a whole cell-based sensor platform for examining the biophysical effects of UVR. We used human keratinocyte, melanocyte and fibroblast cell lines to determine the normal, pathological and protective roles of UVR. In addition, we examined the real-time morphological, biophysical and biomechanical changes associated with cell degeneration induced by UVR at 254 and 365 nm. Information on UVR-induced changes in the cytoskeleton ultrastructure, cellular integrity, cell spreading area, actin microfilament distribution inflammation, microtubule damage, membrane damage, rupture and death was characterized by examining the loss or increase in biophysical and biomechanical properties of these cells. All cells exposed to UVR at 254 and 365 nm showed a significant increase in surface roughness and stiffness in a time-dependent manner. UVR-induced toxicity in differently pigmented skin cells was compared with that in cells pretreated with melanin, keratin and basic fibroblast growth factor to analyze the shielding efficiency of these agents. Melanin exerted a significant shielding effect compared to the other two agents. The biophysical and biomechanical information obtained in this study could advance our understanding of the UVR-induced degeneration process, and help in developing new interventions strategies.

The spatial pattern of cochlear amplification.

Sensorineural hearing loss, which stems primarily from the failure of mechanosensory hair cells, changes the traveling waves that transmit acoustic signals along the cochlea. However, the connection between cochlear mechanics and the amplificatory function of hair cells remains unclear. Using an optical technique that permits the targeted inactivation of prestin, a protein of outer hair cells that generates forces on the basilar membrane, we demonstrate that these forces interact locally with cochlear traveling waves to achieve enormous mechanical amplification. By perturbing amplification in narrow segments of the basilar membrane, we further show that a cochlear traveling wave accumulates gain as it approaches its peak. Analysis of these results indicates that cochlear amplification produces negative damping that counters the viscous drag impeding traveling waves; targeted photoinactivation locally interrupts this compensation. These results reveal the locus of amplification in cochlear traveling waves and connect the characteristics of normal hearing to molecular forces.

PEG hydrogels formed by thiol-ene photo-click chemistry and their effect on the formation and recovery of insulin-secreting cell spheroids.

Hydrogels provide three-dimensional frameworks with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. While recent research efforts have created diverse macromer chemistry to form hydrogels, the mechanisms of hydrogel polymerization for in situ cell encapsulation remain limited. Hydrogels prepared from chain-growth photopolymerization of poly(ethylene glycol) diacrylate (PEGDA) are commonly used to encapsulate cells. However, free radical associated cell damage poses significant limitation for this gel platform. More recently, PEG hydrogels formed by thiol-ene photo-click chemistry have been developed for cell encapsulation. While both chain-growth and step-growth photopolymerizations offer spatial-temporal control over polymerization kinetics, step-growth thiol-ene hydrogels offer more diverse and preferential properties. Here, we report the superior properties of step-growth thiol-ene click hydrogels, including cytocompatibility of the reactions, improved hydrogel physical properties, and the ability for 3D culture of pancreatic ß-cells. Cells encapsulated in thiol-ene hydrogels formed spherical clusters naturally and were retrieved via rapid chymotrypsin-mediated gel erosion. The recovered cell spheroids released insulin in response to glucose treatment, demonstrating the cytocompatibility of thiol-ene hydrogels and the enzymatic mechanism of cell spheroids recovery. Thiol-ene click reactions provide an attractive means to fabricate PEG hydrogels with superior gel properties for in situ cell encapsulation, as well as to generate and recover 3D cellular structures for regenerative medicine applications.

RBE of low energy electrons and photons.

Relative biological effectiveness (RBE) compares the severity of damage induced by a radiation under test at a dose D relative to the reference radiation D(x) for the same biological endpoint. RBE is an important parameter in estimation of risk from exposure to ionizing radiation (IR). The present work provides a review of the recently published data and the knowledge of the RBE of low energy electrons and photons. The review presents RBE values derived from experimental data and model calculations including cell inactivation, chromosome aberration, cell transformation, micronuclei formation and induction of double-strand breaks. Biophysical models, including physical features of radiation track, and microdosimetry parameters are presented, analysed and compared with experimental data. The biological effects of low energy electrons and photons are of particular interest in radiation biology as these are strongly absorbed in micrometer and sub-micrometer layers of tissue. RBE values not only depend on the electron and photon energies but also on the irradiation condition, cell type and experimental conditions.

The impact of blue light on leaf mesophyll conductance.

Blue light has many direct and indirect effects on photosynthesis. The impact of blue light on mesophyll conductance (g(m)), one of the main diffusive limitation to photosynthesis, was investigated in leaves of Nicotiana tabacum and Platanus orientalis, characterized by high and low g(m), respectively. Leaves were exposed to blue light fractions between 0% and 80% of incident light intensity (300 micromol photons m(-2) s(-1)), the other fraction being supplied as red light. Leaves exposed to blue light showed reduced photosynthesis and unaltered stomatal conductance. The g(m), measured using the chlorophyll fluorescence-based method, was strongly reduced in both plant species. Such a reduction of g(m) may not be real, as several assumptions used for the calculation of g(m) by fluorescence may not hold under blue light. To assess possible artefacts, the electron transport rate measured by fluorescence (J(f)) and by gas-exchange (J(c)) were compared in leaves exposed to different fractions of blue light under non-photorespiratory conditions. The two values were only equal, a prerequisite for correct g(m) measurements, when the illumination was totally provided as red light. Under increasing blue light levels an increasing discrepancy was observed, which suggests that J(f) was not correctly calculated, and that such an error could also upset g(m) measurements. Blue light was not found to change the absorbance of light by leaves, whereas it slightly decreased the distribution of light to PSII. To equate J(f) and J(c) under blue light, a further factor must be added to the J(f) equation, which possibly accounted for the reduced efficiency of energy transfer between the pigments predominantly absorbing blue light (the carotenoids) and the chlorophylls. This correction reduced by about 50% the effect of blue light on g(m). However, the residual reduction of g(m) under blue light was real and significant, although it did not appear to limit the chloroplast CO(2) concentration and, consequently, photosynthesis. Reduction of g(m) might be caused by chloroplast movement to avoid photodamage, in turn affecting the chloroplast surface exposed to intercellular spaces. However, g(m) reduction occurred immediately after exposure to blue light and was complete after less than 3 min, whereas chloroplast relocation was expected to occur more slowly. In addition, fast g(m) reduction was also observed after inhibiting chloroplast movement by cytochalasin. It is therefore concluded that g(m) reduction under blue light is unlikely to be caused by chloroplast movement only, and must be elicited by other, as yet unknown, factors.