E. coli aggregation and impaired cell division after terahertz irradiation.

In this study we demonstrated that exposure of Escherichia coli (E. coli) to terahertz (THz) radiation resulted in a change in the activities of the tdcABCDEFGR and matA-F genes (signs of cell aggregation), gene yjjQ (signs of suppression of cell motility), dicABCF, FtsZ, and minCDE genes (signs of suppression of cell division), sfmACDHF genes (signs of adhesin synthesis), yjbEFGH and gfcA genes (signs of cell envelope stabilization). Moreover, THz radiation induced E. coli csg operon genes of amyloid biosynthesis. Electron microscopy revealed that the irradiated bacteria underwent increased aggregation; 20% of them formed bundle-like structures consisting of two to four pili clumped together. This could be the result of changes in the adhesive properties of the pili. We also found aberrations in cell wall structure in the middle part of the bacterial cell; these aberrations impaired the cell at the initial stages of division and resulted in accumulation of long rod-like cells. Overall, THz radiation was shown to have adverse effects on bacterial populations resulting in cells with abnormal morphology.

Light-Regulated Liquid-Liquid Phase Separation for Spatiotemporal Protein Recruitment and Cell Aggregation.

We have previously shown that the upper critical solution temperature-type thermoresponsive ureido polymers such as polyallylurea and poly(2-ureidoethylmethacrylate) derivatives show liquid-liquid phase separation (LLPS), also known as simple coacervation, under physiological conditions below their phase-separation temperatures (Tp). The addition of the polymer-rich coacervate droplets that result from LLPS to a monolayer cell culture induced aggregation of cells into multicellular spheroids. In this study, we prepared a ureido copolymer, poly(vinylamine-co-vinylurea), with azobenzene substituents (Azo-PVU) and demonstrated light-guided assembly and disassembly of LLPS coacervates. Azo-PVUs with Tp values ranging from 10 to 52 °C were prepared by changing the azobenzene content. Ultraviolet light caused a decrease in the Tp of Azo-PVU because of trans-to-cis photoisomerization of the azobenzene and irradiation with visible light increased the Tp. Thus, LLPS of Azo-PVU was reversibly controlled. The coacervate droplets deposited on a dish surface were immediately dissolved by targeted UV irradiation (owing to a decrease in the Tp). Spatially controlled recruitment of proteins on the dish surface was achieved when protein solution was added to the light-patterned surface. Furthermore, the light-guided deposition of coacervates resulted in the spatiotemporal transformation of monolayer cells to aggregates. This light-controlled LLPS will allow the preparation of novel liquid-based materials for biomolecular and cellular engineering.

Human Brain Malignant Glioma (BMG-1) 3D Aggregate Morphology and Screening for Cytotoxicity and Anti-Proliferative Effects.

Two interesting aspects of cell lines grown in 3 Dimensional (3D) conditions are their distinct morphology and production of extracellular matrix (ECM). Also, it is known that 3D aggregates have different susceptibilities to damage-inducing agents compared to their 2D monolayer counterparts. We describe the effect of ECM on 3D aggregate morphology, the effect of cisplatin, bleomycin, and UV on the 3D aggregates and 2 Dimensional (2D) monolayers of the BMG-1 cell line. We also present a rapid method for analyzing cytotoxicity and anti-proliferative effects of 3D aggregates in 96-well plates. We utilized a single-step protocol using the dye resazurin. BMG-1 cells formed floating aggregates on 1% agarose hydrogels. The extent of ECM formed by them was dependent on number of cells seeded irrespective of the seeding density, which in turn directed the 3D aggregate compactness. The 3D aggregates were less susceptible to cisplatin and UV-induced cytotoxicity compared to 2D counterparts. The IC50 value of cisplatin was elevated at 210 µg/ml for the aggregates compared to 170 µg/ml for the monolayers. Exposure to UV for 0, 10, 20, and 30 min gave inhibition values of 2.98%, 8%, 22.99%, and 31.8% for the aggregates as compared to 3.06%, 7.5%, 39.4%, and 46.7% for the monolayers. While bleomycin-induced effects were unapparent when analyzed by vital staining for the doses used, the rapid, single-step method in 96-well plates was able to provide a dose-response for cytotoxicity and anti-proliferative effects. Also, comparative analysis of results obtained from vital staining and the single-step method demonstrates the reliability of the assay described. J. Cell. Physiol. 232: 685-690, 2017. © 2016 Wiley Periodicals, Inc.

Quantitative Analysis of Endocytic Recycling of Membrane Proteins by Monoclonal Antibody-Based Recycling Assays.

In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. J. Cell. Physiol. 232: 463-476, 2017. © 2016 Wiley Periodicals, Inc.

Low Concentration of Exogenous Carbon Monoxide Modulates Radiation-Induced Bystander Effect in Mammalian Cell Cluster Model.

During radiotherapy procedures, radiation-induced bystander effect (RIBE) can potentially lead to genetic hazards to normal tissues surrounding the targeted regions. Previous studies showed that RIBE intensities in cell cluster models were much higher than those in monolayer cultured cell models. On the other hand, low-concentration carbon monoxide (CO) was previously shown to exert biological functions via binding to the heme domain of proteins and then modulating various signaling pathways. In relation, our previous studies showed that exogenous CO generated by the CO releasing molecule, tricarbonyldichlororuthenium (CORM-2), at a relatively low concentration (20 µM), effectively attenuated the formation of RIBE-induced DNA double-strand breaks (DSB) and micronucleus (MN). In the present work, we further investigated the capability of a low concentration of exogenous CO (CORM-2) of attenuating or inhibiting RIBE in a mixed-cell cluster model. Our results showed that CO (CORM-2) with a low concentration of 30 µM could effectively suppress RIBE-induced DSB (p53 binding protein 1, p53BP1), MN formation and cell proliferation in bystander cells but not irradiated cells via modulating the inducible nitric oxide synthase (iNOS) andcyclooxygenase-2 (COX-2). The results can help mitigate RIBE-induced hazards during radiotherapy procedures.

8-prenylnaringenin and tamoxifen inhibit the shedding of irradiated epithelial cells and increase the latency period of radiation-induced oral mucositis : cell culture and murine model.

PURPOSE: The major component in the pathogenesis of oral radiation-induced mucositis is progressive epithelial hypoplasia and eventual ulceration. Irradiation inhibits cell proliferation, while cell loss at the surface continues. We conceived to slow down this desquamation by increasing intercellular adhesion, regulated by the E-cadherin/catenin complex. We investigated if 8-prenylnaringenin (8-PN) or tamoxifen (TAM) decrease the shedding of irradiated human buccal epithelial cells in vitro and thus delay the ulcerative phase of radiation-induced mucositis in vivo. MATERIALS AND METHODS: In vitro, aggregates of buccal epithelial cells were irradiated and cultured in suspension for 11 days. 8-PN or TAM were investigated regarding their effect on cell shedding. In vivo, the lower tongue surface of mice was irradiated with graded single doses of 25 kV X-rays. The incidence, latency, and duration of the resulting mucosal ulcerations were analyzed after topical treatment with 8-PN, TAM or solvent. RESULTS: 8-PN or TAM prevented the volume reduction of the irradiated cell aggregates during the incubation period. This was the result of a higher residual cell number in the treated versus the untreated irradiated aggregates. In vivo, topical treatment with 8-PN or TAM significantly increased the latency of mucositis from 10.9 to 12.1 and 12.4 days respectively, while the ulcer incidence was unchanged. CONCLUSION: 8-PN and TAM prevent volume reduction of irradiated cell aggregates in suspension culture. In the tongues of mice, these compounds increase the latency period. This suggests a role for these compounds for the amelioration of radiation-induced mucositis in the treatment of head and neck tumors.

A branching process model for the analysis of abortive colony size distributions in carbon ion-irradiated normal human fibroblasts.

A single cell can form a colony, and ionizing irradiation has long been known to reduce such a cellular clonogenic potential. Analysis of abortive colonies unable to continue to grow should provide important information on the reproductive cell death (RCD) following irradiation. Our previous analysis with a branching process model showed that the RCD in normal human fibroblasts can persist over 16 generations following irradiation with low linear energy transfer (LET) γ-rays. Here we further set out to evaluate the RCD persistency in abortive colonies arising from normal human fibroblasts exposed to high-LET carbon ions (18.3 MeV/u, 108 keV/µm). We found that the abortive colony size distribution determined by biological experiments follows a linear relationship on the log-log plot, and that the Monte Carlo simulation using the RCD probability estimated from such a linear relationship well simulates the experimentally determined surviving fraction and the relative biological effectiveness (RBE). We identified the short-term phase and long-term phase for the persistent RCD following carbon-ion irradiation, which were similar to those previously identified following γ-irradiation. Taken together, our results suggest that subsequent secondary or tertiary colony formation would be invaluable for understanding the long-lasting RCD. All together, our framework for analysis with a branching process model and a colony formation assay is applicable to determination of cellular responses to low- and high-LET radiation, and suggests that the long-lasting RCD is a pivotal determinant of the surviving fraction and the RBE.

Magnetic manipulation and spatial patterning of multi-cellular stem cell aggregates.

The controlled assembly and organization of multi-cellular systems to mimic complex tissue structures is critical to the engineering of tissues for therapeutic and diagnostic applications. Recent advances in micro-scale technologies to control multi-cellular aggregate formation typically require chemical modification of the interface between cells and materials and lack multi-scale flexibility. Here we demonstrate that simple physical entrapment of magnetic microparticles within the extracellular space of stem cells spheroids during initial formation enables scaffold-free immobilization, translocation and directed assembly of multi-cellular aggregates across multiple length and time scales, even under dynamic suspension culture conditions. The response of aggregates to externally applied magnetic fields was a direct function of microparticle incorporation, allowing for rapid and transient control of the extracellular environment as well as separation of heterogeneous populations. In addition, spatial patterning of heterogeneous spheroid populations as well as individual multi-cellular aggregates was readily achieved by imposing temporary magnetic fields. Overall, this approach provides novel routes to examine stem cell differentiation and tissue morphogenesis with applications that encompass the creation of new model systems for developmental biology, scaffold-free tissue engineering strategies and scalable bioprocessing technologies.

Ophthalmic pterygium: a stem cell disorder with premalignant features.

Pterygia are common ocular surface lesions thought to originate from limbal stem cells altered by chronic UV exposure. Traditionally regarded as a degenerative condition, pterygia also display tumor-like features, such as a propensity to invade normal tissue and high recurrence rates following resection, and may coexist with secondary premalignant lesions. This study was initiated to determine the rate of concurrent ocular surface diseases in patients with pterygia recruited from the practice of a single surgeon operating in a Sydney metropolitan hospital. One hundred pterygium specimens were histopathologically reviewed and selected cases were immunohistochemically assessed to confirm diagnosis. Along with previously documented typical features including epithelial proliferation, goblet cell hyperplasia, angiogenesis, inflammation, elastosis, stromal plaques, and Bowman's membrane dissolution, we identified five cases of ocular surface squamous neoplasia, six cases of primary acquired melanosis, two compound nevi (one suspect invasive melanoma), and one dermoid-like lesion. In 18 specimens, clusters of basal epithelial cells that coexpressed cytokeratin-15/-19 and p63-α were identified at the head of the pterygium, coinciding with clinical observation of Fuchs' flecks. Our data show that significant preneoplastic lesions may be associated with pterygium and that all excised pterygia should undergo histological examination. The presence of p63-α-positive epithelial cell clusters supports the hypothesis that pterygia develop from limbal epithelial progenitors.

Methods for photocrosslinking alginate hydrogel scaffolds with high cell viability.

Methods for seeding high-viability (>85%) three-dimensional (3D) alginate-chondrocyte hydrogel scaffolds are presented that employ photocrosslinking of methacrylate-modified alginate with the photoinitiator VA-086. Comparison with results from several other photoinitiators, including Irgacure 2959, highlights the role of solvent, ultraviolet exposure, and photoinitiator cytotoxicity on process viability of bovine chondrocytes in two-dimensional culture. The radicals generated from VA-086 photodissociation are shown to be noncytotoxic at w/v concentrations up to 1.5%, enabling photocrosslinking without significant cell death. The applicability of these photoinitiators for generating 3D tissue-engineered constructs is evaluated by measuring cell viability in 3D constructs with aggregate moduli in the 10-20 kPa range. Hydrogels with encapsulated bovine chondrocytes were constructed with >85% viability using VA-086. While the commonly used Irgacure 2959 is noncytotoxic in its native state and crosslinks the alginate at weight fractions much lower than VA-086, the cytotoxicity of IRG2959's photogenerated radical leads to viabilities below 70% in the conditions tested.

Ionizing radiation induces macrophage foam cell formation and aggregation through JNK-dependent activation of CD36 scavenger receptors.

PURPOSE: Irradiated arteries of cancer patients can be associated with atherosclerosis-like lesions containing cholesterol-laden macrophages (foam cells). Endothelial cell damage by irradiation does not completely explain the foam cell formation. We investigated the possible underlying mechanisms for ionizing radiation (IR)-induced foam cell formation. METHODS AND MATERIALS: Human peripheral blood monocytes were activated by macrophage colony-stimulating factor and then treated with varying doses of IR in vitro in the absence of endothelial cells. Scavenger receptor expression and foam cell formation of IR-treated macrophages were investigated in the presence or absence of oxidized low-density lipoprotein. We also assessed the importance of mitogen-activated protein kinase activity in the macrophage colony-stimulating factor-activated human monocytes (macrophages) for the foam cell formation. RESULTS: We found that IR treatment of macrophage colony-stimulating factor-activated human peripheral blood monocytes resulted in the enhanced expression of CD36 scavenger receptors and that cholesterol accumulated in the irradiated macrophages with resultant foam cell formation in the presence of oxidized low-density lipoprotein. Furthermore, when cultured on collagen gels, human macrophages formed large foam cell aggregates in response to IR. Antibodies against CD36 inhibited the IR-induced foam cell formation and aggregation, indicating that the IR-induced foam cell formation and the subsequent aggregation are dependent on functional CD36. In addition, we found that IR of human macrophages resulted in c-Jun N-terminal kinase activation and that c-Jun N-terminal kinase inhibition suppressed IR-induced CD36 expression and the subsequent foam cell formation and aggregation. CONCLUSION: Taken together, these results suggest that IR-induced foam cell formation is mediated by c-Jun N-terminal kinase-dependent CD36 activation.

Color of illumination during growth affects LHCII chiral macroaggregates in pea plant leaves.

To determine whether the color of illumination under which plants are grown, affects the structure of photosynthetic antennae, pea plants were grown under either blue-enriched, red-enriched, or white light. Carotenoid content of isolated chloroplasts was found to be insensitive to the color of illumination during growth, while chlorophyll a/b ratio in chloroplasts isolated from young illuminated leaves showed susceptibility to color. Color of illumination affects the LHCII chiral macroaggregates in intact leaves and isolated chloroplasts, providing light-induced alteration of the handedness of the LHCII chiral macroaggregate, as measured with circular dichroism and circularly polarized luminescence. The susceptibility of handedness to current illumination (red light excitation of chlorophyll fluorescence) is dependent on the color under which the plants were grown, and was maximal for the red-enriched illumination. We propose the existence of a long-term (growth period) color memory, which influences the susceptibility of the handedness of LHCII chiral macroaggregates to current light.

Magnetic optimization in a multicellular magnetotactic organism.

Unicellular magnetotactic prokaryotes, which typically carry a natural remanent magnetic moment equal to the saturation magnetic moment, are the prime example of magnetically optimized organisms. We here report magnetic measurements on a multicellular magnetotactic prokaryote (MMP) consisting of 17 undifferentiated cells (mean from 148 MMPs) with chains of ferrimagnetic particles in each cell. To test if the chain polarities of each cell contribute coherently to the total magnetic moment of the MMP, we used a highly sensitive magnetization measurement technique (1 fAm(2)) that enabled us to determine the degree of magnetic optimization (DMO) of individual MMPs in vivo. We obtained DMO values consistently above 80%. Numerical modeling shows that the probability of reaching a DMO > 80% would be as low as 0.017 for 17 randomly oriented magnetic dipoles. We simulated different scenarios to test whether high DMOs are attainable by aggregation or self-organization of individual magnetic cells. None of the scenarios investigated is likely to yield consistently high DMOs in each generation of MMPs. The observed high DMO values require strong Darwinian selection and a sophisticated reproduction mechanism. We suggest a multicellular life cycle as the most plausible scenario for transmitting the high DMO from one generation to the next.

Effects of a 50 Hz magnetic field on Dictyostelium discoideum (Protista).

Some studies have demonstrated that a few biological systems are affected by weak, extremely low frequency (ELF) electromagnetic fields (EMFs), lower than 10 mT. However, to date there is scanty evidence of this effect on Protists in the literature. Due to their peculiarity as single-cell eukaryotic organisms, Protists respond directly to environmental stimuli, thus appearing as very suitable experimental systems. Recently, we showed the presence of propionylcholinesterase (PrChE) activity in single-cell amoebae of Dictyostelium discoideum. This enzyme activity was assumed to be involved in cell-cell and cell-environment interactions, as its inhibition affects cell aggregation and differentiation. In this work, we have exposed single-cell amoebae of D. discoideum to an ELF-EMF of about 200 microT, 50 Hz, for 3 h or 24 h at 21 degrees C. A delay in the early phase of the differentiation was observed in 3 h exposed cells, and a significant decrease in the fission rate appeared in 24 h exposed cells. The PrChE activity was significantly lower in 3 h exposed cells than in the controls, whereas 24 h exposed cells exhibited an increase in this enzyme activity. However, such effects appeared to be transient, as the fission rate and PrChE activity values returned to the respective control values after a 24 h stay under standard conditions.

Rhodopsin-mediated photoreception in cryptophyte flagellates.

We show that phototaxis in cryptophytes is likely mediated by a two-rhodopsin-based photosensory mechanism similar to that recently demonstrated in the green alga Chlamydomonas reinhardtii, and for the first time, to our knowledge, report spectroscopic and charge movement properties of cryptophyte algal rhodopsins. The marine cryptophyte Guillardia theta exhibits positive phototaxis with maximum sensitivity at 450 nm and a secondary band above 500 nm. Variability of the relative sensitivities at these wavelengths and light-dependent inhibition of phototaxis in both bands by hydroxylamine suggest the involvement of two rhodopsin photoreceptors. In the related freshwater cryptophyte Cryptomonas sp. two photoreceptor currents similar to those mediated by the two sensory rhodopsins in green algae were recorded. Two cDNA sequences from G. theta and one from Cryptomonas encoding proteins homologous to type 1 opsins were identified. The photochemical reaction cycle of one Escherichia-coli-expressed rhodopsin from G. theta (GtR1) involves K-, M-, and O-like intermediates with relatively slow (approximately 80 ms) turnover time. GtR1 shows lack of light-driven proton pumping activity in E. coli cells, although carboxylated residues are at the positions of the Schiff base proton acceptor and donor as in proton pumping rhodopsins. The absorption spectrum, corresponding to the long-wavelength band of phototaxis sensitivity, makes this pigment a candidate for one of the G. theta sensory rhodopsins. A second rhodopsin from G. theta (GtR2) and the one from Cryptomonas have noncarboxylated residues at the donor position as in known sensory rhodopsins.

A long-range attraction between aggregating 3T3 cells mediated by near-infrared light scattering.

At what range can a mammalian cell sense the presence of another cell and through what medium? To approach these questions, the formation of aggregates of a 3T3 cell variant (3T3x cells) grown on solid substrates was studied. Each of the aggregates consisted of cells that, at the time of their seeding, were single and located randomly. Yet somehow they seemed to detect each other within a certain range (R(a)) and move together to form aggregates. The article describes a simple assay to measure the value of R(a). When applied to 3T3x cells with altered intensities of near-infrared light scattering (I(sc)) the assay showed that (i) R(a) was much larger than one cell diameter, and (ii) R(a) was directly related to I(sc). The results suggest that near-infrared light scattering by the cells mediate a long-range attraction between them, which does not require physical contact and enables them to detect each other's presence.

Electroendocytosis: exposure of cells to pulsed low electric fields enhances adsorption and uptake of macromolecules.

This study demonstrates alteration of cell surface, leading to enhanced adsorption of macromolecules (bovine serum albumin (BSA), dextran, and DNA), after the exposure of cells to unipolar pulsed low electric fields (LEF). Modification of the adsorptive properties of the cell membrane also stems from the observation of LEF-induced cell-cell aggregation. Analysis of the adsorption isotherms of BSA-fluorescein isothiocyanate (FITC) to the surface of COS 5-7 cells reveals that the stimulated adsorption can be attributed to LEF-induced increase in the capacity of both specific and nonspecific binding. The enhanced adsorption was consequently followed by increased uptake. At 20 V/cm the maximal binding and subsequent uptake of BSA-FITC attached to specific sites are 6.5- and 7.4-fold higher than in controls, respectively. The nonspecific LEF-induced binding and uptake of BSA are 34- and 5.2-fold higher than in controls. LEF-enhanced adsorption is a temperature-independent process, whereas LEF-induced uptake is a temperature-dependent one that is abolished at 4 degrees C. The stimulation of adsorption and uptake is reversible, revealing similar decay kinetics at room temperature. It is suggested that electrophoretic segregation of charged components in the outer leaflet of the cell membrane is responsible for both enhanced adsorption and stimulated uptake via changes of the membrane elastic properties that enhance budding and fission processes.

Numerical study of induced current perturbations in the vicinity of excitable cells exposed to extremely low frequency magnetic fields.

Realistic three-dimensional cell morphologies were modelled to determine the current density induced in excitable cell culture preparations exposed to 60 Hz magnetic fields and to identify important factors that can influence the responses of cells to these fields. Cell morphologies representing single spherical adrenal chromaffin cells, single elongated smooth muscle cells and chromaffin cell aggregates in a Petri dish containing culture medium were modelled using the finite element method. The computations for a spherical cell revealed alterations in the magnitude and spatial distribution of the induced current density in the immediate vicinity of the cell. Maxima occurred at the equatorial sides and minima at the poles. Proximity of cells to each other as well as cell aggregate shape, size and orientation with respect to the induced current influenced the magnitude and spatial distribution of the induced current density. For an elongated cell, effects on the induced current density were highly dependent on cell orientation with respect to the direction of the induced current. These results provide novel insights into the perturbations in induced current that occur in excitable cell culture preparations and lay a foundation for understanding the mechanisms of interaction with extremely low frequency magnetic fields at the tissue level.

Beyond input-output computings: error-driven emergence with parallel non-distributed slime mold computer.

The emergence derived from errors is the key importance for both novel computing and novel usage of the computer. In this paper, we propose an implementable experimental plan for the biological computing so as to elicit the emergent property of complex systems. An individual plasmodium of the true slime mold Physarum polycephalum acts in the slime mold computer. Modifying the Elementary Cellular Automaton as it entails the global synchronization problem upon the parallel computing provides the NP-complete problem solved by the slime mold computer. The possibility to solve the problem by giving neither all possible results nor explicit prescription of solution-seeking is discussed. In slime mold computing, the distributivity in the local computing logic can change dynamically, and its parallel non-distributed computing cannot be reduced into the spatial addition of multiple serial computings. The computing system based on exhaustive absence of the super-system may produce, something more than filling the vacancy.