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Plant leaves contain multiple cell types which achieve distinct characteristics whilst still coordinating development within the leaf. The bundle sheath possesses larger individual cells and lower chloroplast content than the adjacent mesophyll, but how this morphology is achieved remains unknown. To identify regulatory mechanisms determining bundle sheath cell morphology we tested the effects of perturbing environmental (light) and endogenous signals (hormones) during leaf development of Oryza sativa (rice). Total chloroplast area in bundle sheath cells was found to increase with cell size as in the mesophyll but did not maintain a 'set-point' relationship, with the longest bundle sheath cells demonstrating the lowest chloroplast content. Application of exogenous cytokinin and gibberellin significantly altered the relationship between cell size and chloroplast biosynthesis in the bundle sheath, increasing chloroplast content of the longest cells. Delayed exposure to light reduced the mean length of bundle sheath cells but increased corresponding leaf length, whereas premature light reduced final leaf length but did not affect bundle sheath cells. This suggests that the plant hormones cytokinin and gibberellin are regulators of the bundle sheath cell-chloroplast relationship and that final bundle sheath length may potentially be affected by light-mediated control of exit from the cell cycle., (© 2024 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Irreparable DNA damage following ionizing radiation (IR) triggers prolonged DNA damage response and induces premature senescence. Cellular senescence is a permanent state of cell-cycle arrest characterized by chromatin restructuring, altered nuclear morphology and acquisition of secretory phenotype, which contributes to senescence-related inflammation. However, the mechanistic connections for radiation-induced DNA damage that trigger these senescence-associated hallmarks are poorly understood. In our in vitro model of radiation-induced senescence, mass spectrometry-based proteomics was combined with high-resolution imaging techniques to investigate the interrelations between altered chromatin compaction, nuclear envelope destabilization and nucleo-cytoplasmic chromatin blebbing. Our findings confirm the general pathophysiology of the senescence-response, with disruption of nuclear lamin organization leading to extensive chromatin restructuring and destabilization of the nuclear membrane with release of chromatin fragments into the cytosol, thereby activating cGAS-STING-dependent interferon signaling. By serial block-face scanning electron microscopy (SBF-SEM) whole-cell datasets were acquired to investigate the morphological organization of senescent fibroblasts. High-resolution 3-dimensional (3D) reconstruction of the complex nuclear shape allows us to precisely visualize the segregation of nuclear blebs from the main nucleus and their fusion with lysosomes. By multi-view 3D electron microscopy, we identified nanotubular channels formed in lamin-perturbed nuclei of senescent fibroblasts; the potential role of these nucleo-cytoplasmic nanotubes for expulsion of damaged chromatin has to be examined.

Photodynamic therapy (PDT) is based on special light source, photosensitizer (PS), and in the presence of oxygen. Different light sources have been used for PDT applications. Recent studies have focused on LED light sources for PDT applications due to reducing the cost of laser-based PDT and providing easy access for research laboratory or clinic facilities. LED-mediated PDT applications have shown promising results for the treatment of different types of disease. However, few studies have determined the effects of LED-based PDT on cancer cells. For the first time, the aim of this study was to explore the therapeutic effects of 5-aminolevulinic acid (5-ALA)-mediated PDT after LED irradiation on two sub-types (a poorly aggressive MCF-7 and a highly aggressive MDA-MB-231) of breast cancer cell lines. The effectiveness of 5-ALA PDT treatment was evaluated by WST-1, annexin V, and acridine orange staining with different energy levels. The LED system was specially developed with optical power and wavelength stability techniques. The system consists of user interface and embedded LED controller with real-time optic power output calibration by photodiode feedback. Our results demonstrated that the cell viability of breast cancer cells was considerably decreased a LED dose-dependent manner (P < 0.05). Additionally, a significant increase in the percentage of apoptotic cells was detected in breast cancer cells after irradiation with LED at a density of 18 and 30 J/cm 2 energy. Consequently, the LED system could be effectively used for irradiation of 5-ALA in the treatment of breast cancer cells.

Photobiomodulation (PBM) has been shown to improve cell proliferation and cell migration. Many cell types have been investigated, with most studies using deep penetrating red light irradiation. Considering the interest of surface biostimulation of oral mesenchymal cells after surgical wound, the present study aimed to assess green light irradiation effects on Dental Pulp Stem Cells' (DPSC) proliferation and migration. To understand the mechanisms underlying these effects, we investigated cytoskeleton organization and subsequent cell shape and stiffness. A 532-nm wavelength Nd:YAG laser (30 mW) was applied between 30 and 600 s on DPSC in vitro. Cell proliferation was analyzed at 24, 48, and 72 h after irradiation, by cell counting and enzymatic activity quantification (paranitrophenylphosphate phosphatase (pNPP) test). A wound healing assay was used to study cell migration after irradiation. Effects of PBM on cytoskeleton organization and cell shape were assessed by actin filaments staining. Elasticity changes after irradiation were quantified in terms of Young's modulus measured using Atomic Force Microscopy (AFM) force spectroscopy. Green light significantly improved DPSC proliferation with a maximal effect obtained after 300-s irradiation (energy fluence 5 J/cm 2 ). This irradiation had a significant impact on cell migration, improving wound healing after 24 h. These results were concomitant with a decrease of cells' Young's modulus after irradiation. This cell softening was explained by actin cytoskeleton reorganization, with diminution of cell circularity and more abundant pseudopodia. This study highlights the interest of green laser PMB for the proliferation and migration of mesenchymal stem cells, with encouraging results for clinical application, especially for surgical wound healing procedures.

Chemicals and signaling molecules released by injured cells at the beginning of wound healing prompt inflammation. In diabetes, prolonged inflammation is one of the probable causes for delayed wound healing. Increased levels of cyclooxygenase-2 (cox-2), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF- α ) are associated with the inflammatory response and in diabetes, and increased levels of these contribute to chronic wounds that do not heal. Rising levels of cox-2, IL-6, and TNF- α have also been associated with increased oxidative stress. Photobiomodulation (PBM) may impact wound healing processes by affecting the signaling pathways and molecules pertinent to tissue repair. In the present study, the effect of PBM (wavelength: 660 nm; energy density: 5 J/cm 2 ) on levels of cox-2, IL-6, and TNF- α was determined in fibroblast cell culture models. Four WS1 models (normal, normal wounded, diabetic, and diabetic wounded) were irradiated at 660 nm, and the culture media was collected at 0, 24, and 48 h postirradiation. Cells that were not irradiated (0 J/cm 2 ) served as the controls. The following parameters were determined postirradiation: cell morphology using light microscopy, cell viability using the Trypan Blue exclusion assay, and levels of the inflammatory markers cox-2, IL-6, and TNF- α were measured using ELISA. Cell migration increased in the wounded groups over the 48 h interval after PBM; viability improved postirradiation in the diabetic wounded groups at 0 and 24 h ( P ≤ 0.05 and P ≤ 0.01, respectively); levels of cox-2 decreased in normal and diabetic wounded groups at 0 h ( P ≤ 0.001) and increased in the diabetic and diabetic wounded groups at 48 h postirradiation ( P ≤ 0.05 and P ≤ 0.01, respectively), while levels of IL-6 decreased in the normal ( P ≤ 0.01), diabetic ( P ≤ 0.05), and diabetic wounded ( P ≤ 0.001) groups at 24 h and in the diabetic and diabetic wounded groups at 48 h ( P ≤ 0.05) postirradiation. TNF- α was decreased in the normal wounded groups ( P ≤ 0.05) at 48 h. Through its effect on decreased IL-6 levels in diabetic cell models, PBM at 660 nm may be successful at decreasing oxidative stress; however, the present study also found an increase in cox-2 levels at 48 h postirradiation., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Asma Shaikh-Kader et al.)

Zirconia has been regarded as a promising material for dental implants, and Nd:YAG laser treatment has been proposed as a potential strategy to improve its bioactivity. The main aim of the present study was to evaluate the in vitro behavior of human fetal osteoblasts in contact with laser-textured zirconia implant surfaces assessing the effect of different texture patterns, spacing between laser passes and number of laser passes. Zirconia discs were produced and treated with Nd:YAG laser according to test group variables: texture (microgrooves and micropillar array), distance between surface features (25 μm, 30 μm and 35 μm), and laser passes [1, 2, 4, and 8]. Untextured sandblasted and acid-etched zirconia discs (SBAE) were used as controls. Human osteoblasts (hFOB 1.19) were cultured for 14 days on test and control samples. Morphology and cellular adhesion were observed using scanning electron microscopy (SEM). Cell viability and proliferation were evaluated at 1, 3, 7, and 14 days using a commercial resazurin-based method. Collagen type I was evaluated at 3 days using ELISA. Alkaline phosphatase (ALP) activity was evaluated at 7 days using a colorimetric enzymatic technique. Group comparisons were tested using ANOVA or Mann-Whitney test (Tukey's post hoc) using statistical software, and significance was set at p < 0.05. Cell viability and proliferation increased over time for all groups with statistically higher values for laser-textured groups when compared with control at 7 and 14 days in culture (p < 0.05). Collagen type I levels were higher for study groups (p < 0.05) when compared with control group. No statistically differences were detected for ALP activity levels between texture and control groups (p > 0.05). The results suggest that laser-machined zirconia implant surfaces may benefit biological osteoblast response. However, the type of texture, spacing at the range of 25-35 μm, and number of laser passes did not seem to be relevant variables.

This study aimed to verify the efficacy of low-level laser irradiation (LLLI) on the proliferation of MC3T3-E1 preosteoblasts cultured on poly(lactic acid) (PLA) films. The produced films were characterized by contact angle tests, scanning electron microscopy (SEM), atomic force microscopy, differential scanning calorimetry, and X-ray diffraction. The MC3T3-E1 cells were cultured as three different groups: Control-cultured on polystyrene plastic surfaces; PLA-cultured on PLA films; and PLA + Laser-cultured on PLA films and submitted to laser irradiation (660 nm; 30 mW; 4 J/cm 2 ). Cell proliferation was analyzed by Trypan blue and Alamar blue assays at 24, 48, and 72 h after irradiation. Cell viability was assessed by Live/Dead assay, apoptosis-related events were evaluated by Annexin V/propidium iodide (PI) expression, and cell cycle events were analyzed by flow cytometry. Cell morphology on the surface of films was assessed by SEM. Cell counting and biochemical assay results indicate that the PLA + Laser group exhibited higher proliferation (p < 0.01) when compared with the Control and PLA groups. The Live/Dead and Annexin/PI assays indicate increased cell viability in the PLA + Laser group that also presented a higher percentage of cells in the proliferative cell cycle phases (S and G2/M). These findings were also confirmed by the higher cell density observed in the irradiated group through SEM images. The evidence from this study supports the idea that LLLI increases the proliferation of MC3T3-E1 cells on PLA surfaces, suggesting that it can be potentially applied in bone tissue engineering., (© 2020 John Wiley & Sons, Ltd.)

Ultraviolet (UV) radiation exerts adverse effects on genome stability, alters the normal state of life, and causes several diseases by inducing DNA damage. Although mechanical stimulation such as stretching has significant effects on the prevention and treatment of diseases, its influence on nuclear morphology and/or intranuclear functions involving resistance to DNA damage remains unknown. Here, we investigated the effects of mechanical stimulation by cyclic stretching on nuclear morphology and resistance of DNA to UV damage in NIH3T3 fibroblasts. Adherent cells on silicone elastic membranes were subjected to ~ 10% cyclic uniaxial stretch at a frequency of 0.5 Hz for 12 h. As a result, the intracellular actin cytoskeleton and nucleus were found to be elongated and aligned in the direction of zero normal strain (~ 62° with respect to the stretch direction) in an actomyosin tension-dependent manner. The nuclei of the stretched cells were dramatically compressed by the reorganized actin stress fibers located on their apical and both sides, and a significant increase in the intranuclear DNA density was observed. Intercellular tension, as assessed with live cell atomic force microscopy imaging, also increased following exposure to cyclic stretch. The UV radiation-induced DNA damage, estimated from the fluorescence intensity of the phospho-histone γ-H2AX, significantly decreased in these stretched cells. These results indicate that the cyclic stretch-induced morphological changes in the nucleus may improve the UV radiation resistance of cells, probably owing to the intracellular force-induced condensation of chromatin. To our knowledge, this is the first study to demonstrate the inhibition of the UV radiation-induced DNA damage by mechanical stimulation.

Our previous research implied mouse skin-derived precursors (mSKPs) possessed the capacity of anti-ultraviolet B (UVB) irradiation damage, and the mechanisms might be associated with transforming growth factor-β (TGF-β) signaling pathway activation. In this study, we investigated and compared the response to UVB irradiation between mSKPs and dermal mesenchymal stem cells (dMSCs), and explored the underlying mechanisms. Irradiation damage such as decreased cell viability, cell senescence, and cell death was observed in both mSKPs and dMSCs at 24 h after UVB exposure. In mSKPs, change in cell morphology, viability, cell senescence and death at the following time points implied the recovery of UVB irradiation damage. Additionally, thrombospondin1 (TSP1) and TGF-β1 increased significantly in mSKPs' supernatant after UVB irradiation. The gene expression of TSP1, TGF-β1, metalloproteinase 1 (MMP1), and Collagen I elevated shortly after the UVB exposure. The protein expression of TSP1, TGF-β1, MMP1, Collagen I, smad2/3, and p-smad2/3 at multiple time points after the UVB exposure was consistent with the gene expression results. In dMSCs, no obvious recovery was noticed. Together, these results revealed that in mSKPs, one of the mechanisms to attenuate the UVB irradiation damage might be the early activation of TGF-β/Smad pathway by TSP1. Given that mSKPs could differentiate into fibroblast-like SKP-derived fibroblasts (SFBs) in vivo or with the presence of serum, mSKPs might serve as a therapeutic potential for fibroblasts supplement and UVB irradiation damage treatment. Abbreviations: SKPs: skin-derived precursors; mSKPs: mouse SKPs; UVB: ultraviolet B; TGF-β/Smad: transforming growth factor-β/Smad; TSP1: thrombospondin 1; MMP 13: metalloproteinases 13; TβRII: TGF-β receptor II; SFBs: SKP-derived fibroblasts; KEGG: Kyoto encyclopedia of genes and genomes; DEGs: differentially expressed genes; dMSCs: dermal mesenchymal stem cells; LM: light microscope; CCK-8: cell counting kit 8; ELISA: Enzyme-linked immuno sorbent assay; qRT-PCR: quantitative real-time polymerase chain reaction; TSPs: thrombospondins; ECM: extracellular matrix; R-smads: receptor-regulated smads.

Cranial irradiation is the main therapeutic treatment for primary and metastatic malignancies in the brain. However, cranial radiation therapy produces long-term impairment in memory, information processing, and attention that contribute to a decline in quality of life. The hippocampal neural network is fundamental for proper storage and retrieval of episodic and spatial memories, suggesting that hippocampal signaling dysfunction could be responsible for the progressive memory deficits observed following irradiation. Previous rodent studies demonstrated that irradiation induces significant loss in dendritic spine number, alters spine morphology, and is associated with behavioral task deficits. Additionally, the literature suggests a common mechanism in which synaptic elimination via microglial-mediated phagocytosis is complement dependent and associated with cognitive impairment in aging as well as disease. We demonstrate sexual dimorphisms in irradiation-mediated alterations of microglia activation markers and dendritic spine density. Further, we find that the significant dendritic spine loss observed in male mice following irradiation is microglia complement receptor 3 (CR3)-dependent. By identifying sex-dependent cellular and molecular factors underlying irradiation-mediated spine loss, therapies can be developed to counteract irradiation-induced cognitive decline and improve patient quality of life.

In children, ketamine sedation is often used during radiological procedures. Combined exposure of ketamine and radiation at doses that alone did not affect learning and memory induced permanent cognitive impairment in mice. The aim of this study was to elucidate the mechanism behind this adverse outcome. Neonatal male NMRI mice were administered ketamine (7.5 mg kg -1 ) and irradiated (whole-body, 100 mGy or 200 mGy, 137 Cs) one hour after ketamine exposure on postnatal day 10. The control mice were injected with saline and sham-irradiated. The hippocampi were analyzed using label-free proteomics, immunoblotting, and Golgi staining of CA1 neurons six months after treatment. Mice co-exposed to ketamine and low-dose radiation showed alterations in hippocampal proteins related to neuronal shaping and synaptic plasticity. The expression of brain-derived neurotrophic factor, activity-regulated cytoskeleton-associated protein, and postsynaptic density protein 95 were significantly altered only after the combined treatment (100 mGy or 200 mGy combined with ketamine, respectively). Increased numbers of basal dendrites and branching were observed only after the co-exposure, thereby constituting a possible reason for the displayed alterations in behavior. These data suggest that the risk of radiation-induced neurotoxic effects in the pediatric population may be underestimated if based only on the radiation dose.

Photobiomodulation therapy (PBMT) can enhance the mesenchymal stem cell (MSC) proliferation, differentiation, and tissue repair and can therefore be used in regenerative medicine. The objective of this study is to investigate the effects of photobiomodulation on the directional neural differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) and provide a theoretical basis for neurogenesis. hUC-MSCs were divided into control, inducer, laser, and lasers combined with inducer groups. A 635-nm laser and an 808-nm laser delivering energy densities from 0 to 10 J/cm 2 were used in the study. Normal cerebrospinal fluid (CSF) and injured cerebrospinal fluid (iCSF) were used as inducers. The groups were continuously induced for 3 days. Cellular proliferation was evaluated using MTT. The marker proteins nestin (marker protein of the neural precursor cells), NeuN (marker protein of neuron), and GFAP (glial fibrillary acidic protein, marker proteins of glial cells) were detected by immunofluorescence and western blot. We found that irradiation with 635-nm laser increased cell proliferation, and that with 808 nm laser by itself and combined with cerebrospinal fluid treatment generated significant neuron-like morphological changes in the cells at 72 h. Nestin showed high positive expression at 24 h in the 808 nm group. The expression of GFAP increased in the 808-nm combined inducer group at 24 h but decreased at 72 h. The expression of neuN protein increased only at 72 h in both the 808-nm combined inducer group and inducer group. We concluded that 808 nm laser irradiation could help CSF to induce neuronal differentiation of hUC-MSCs in early stage and tend to change to neuron rather than glial cells.

Perform a physicochemical and morphological characterization of a Ti-15Mo alloy surface modified by laser beam irradiation and to evaluate in vitro the morphological response and proliferation of osteoblastic cells seeded onto this alloy. Disks were made of two different metals, Ti-15Mo alloy and cpTi, used as control. A total of four groups were evaluated: polished cpTi (cpTi-pol), laser-irradiated cpTi (cpTi-L), polished Ti-15Mo alloy (Ti-15Mo-pol), and laser-irradiated Ti-15Mo alloy (Ti-15Mo-L). Before and after laser irradiation of the surfaces, physicochemical and morphological analyses were performed: scanning electron microscopy (FEG-SEM), energy-dispersive spectroscopy (EDX), and X-ray diffraction (XRD). The wettability of the samples was evaluated by contact angle measurement. Murine preosteoblastic cells MC3T3-E1 were cultured onto the experimental disks for cell proliferation, morphology, and spreading analyses. Laser groups presented irregular-shaped cavities on its surface and a typical microstructured surface with large depressions (FEG-SEM). The contact angle for both laser groups was 0°, whereas for the polished groups was ≈ 77 and ≈ 78 for cpTi-pol and Ti-15Mo-pol, respectively. Cell proliferation analysis demonstrated a higher metabolic activity in the laser groups (p < 0.05). From the fluorescence microscopy, Ti-15Mo-L surface seems to induce greater cellular differentiation compared to the cpTi-L surface. The preliminary biological in vitro analyses suggested possible advantages of laser surface treatment in the Ti-15Mo alloy regarding cell proliferation and maturation.

The feasibility of continuous wave laser-based photoacoustic (CWPA) response technique in detecting the morphological changes in cells during the biological studies, through the features extracted from CWPA signal (i.e., amplitude) is demonstrated here. Various hematological disorders (e.g., sickle cell anemia, thalesemia) produce distinct changes at the cellular level morphologically. In order to explore the photoacoustic response technique to detect these morphological changes, we have applied CWPA technique onto the blood samples. Results of our preliminary study show a distinct change in the signal amplitude of photoacoustic (PA) signal due to a change in the concentration of blood, which signifies the sensitivity of the technique towards red blood cell (RBC) count (related to hematological disease like anemia). Further hypotonic and hypertonic solutions were induced in blood to produce morphological changes in RBCs (i.e., swollen and shrink, respectively) as compared to the normal RBCs. Experiments were performed using continuous wave laser-based photoacoustic response technique to verify the morphological changes in these RBCs. A distinct change in the PA signal amplitude was found for the distinct nature of RBCs (swollen, shrink, and normal). Thus, this can serve as a diagnostic signature for different biological studies based on morphological changes at cellular level. The experiments were also performed using conventional pulsed laser photoacoustic response technique which uses nano-second pulsed laser and the results obtained from both PA techniques were validated to produce identical changes. This demonstrates the utility of continuous wave laser-based photoacoustic technique for different biological studies related to morphological cellular disorders.

High light and high temperature (HLHT) stress may become more frequent and severe as the climate changes, affecting crop growth and resulting in reduced production. However, the mechanism of the response to HLHT stress in rice is not yet fully understood. In the present study, we screened a rice mutant library using HLHT conditions and isolated an HLHT-sensitive mutant, local lesions 1 (ls1), which showed decreased pigment contents, defective stomata and chloroplasts, and a local lesions phenotype under HLHT. We characterized and cloned LS1 by map-based cloning and genetic complementation. LS1 encodes the A subunit of the RNase H2 complex (RNASEH2A). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and comet assays indicated that mutation of LS1 led to severe DNA damage under HLHT stress. Furthermore, we found excessive reactive oxygen species (ROS) accumulation in the ls1 mutant under HLHT stress. Exogenous antioxidants eased the local lesions phenotype of the ls1 mutant under HLHT. DNA damage caused by HLHT stress induces ROS accumulation, which causes the injury and apoptosis of leaf cells in the ls1 mutant. These results enhance our understanding of the regulatory mechanism in the response to HLHT stress in higher plants., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

To investigate the role of Wnt/β-catenin signaling pathway in the restoration of induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) after laser photocoagulation. After differentiation of RPE cells from hiPSCs, laser photocoagulation was performed. Activation of Wnt/β-catenin signaling at days 1 and 5 after laser photocoagulation was evaluated by expression of β-catenin. Cell proliferation and alteration in cell-to-cell contact at day 5 after laser photocoagulation with or without Dickkopf-1 (Dkk-1) treatment were studied using ethynyl-2'-deoxyuridine (EdU) assay and zonula occludens-1 (ZO-1) expression analysis, respectively. The mRNA levels of Wnt genes at day 5 after laser photocoagulation were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Activation of Wnt/β-catenin signaling at days 1 and 5 after laser photocoagulation was confirmed by β-catenin accumulation in the cytoplasm and nucleus of hiPSC-RPE. Many EdU-positive cells also expressed β-catenin, and the number of EdU-positive cells was decreased at day 5 after laser photocoagulation after Dkk-1 treatment, indicating that Wnt/β-catenin signaling mediated hiPSC-RPE proliferation. ZO-1 expression was not decreased with Dkk-1 treatment at day 5 after laser photocoagulation, indicating that Wnt/β-catenin signaling mediated hiPSC-RPE restoration. At day 5, after laser photocoagulation, mRNA levels of Wnt2b, Wnt3, Wnt5a, Wnt7a, and Wnt10b were increased. Wnt/β-catenin signaling has a crucial role in restoration of hiPSC-RPE proliferation after laser photocoagulation. Manipulation of Wnt/β-catenin signaling while elucidating the underlying mechanisms of RPE restoration might have a therapeutic potential in retinal degenerative diseases.

Exposure to natural and artificial light and environmental pollutants are the main factors that challenge skin homeostasis, promoting aging or even different forms of skin cancer through a variety of mechanisms that include accumulation of reactive oxygen species (ROS), engagement of DNA damage responses, and extracellular matrix (ECM) remodeling upon release of metalloproteases (MMPs). Ultraviolet A radiation is the predominant component of sunlight causative of photoaging, while ultraviolet B light is considered a potentiator of photoaging. In addition, different chemicals contribute to skin aging upon penetration through skin barrier disruption or hair follicles, aryl hydrocarbon receptors (AhR) being a major effector mechanism through which toxicity is exerted. Deschampsia antarctica is a polyextremophile Gramineae capable of thriving under extreme environmental conditions. Its aqueous extract (EDA) exhibits anti- photoaging in human skin cells, such as inhibition of MMPs, directly associated with extrinsic aging. EDA prevents cellular damage, attenuating stress responses such as autophagy and reducing cellular death induced by UV. We demonstrate that EDA also protects from dioxin-induced nuclear translocation of AhR and increases the production of loricrin, a marker of homeostasis in differentiated keratinocytes. Thus, our observations suggest a potential use exploiting EDA's protective properties in skin health supplements.

Tissue engineering aims to take advantage of the ability of undifferentiated stem cells to differentiate into multiple cell types to repair damaged tissue. Photobiomodulation uses either lasers or light-emitting diodes to promote stem cell proliferation and differentiation. The present study aimed to investigate single and dual combinations of laser wavelengths on mesenchymal stem cells (MSCs). MSCs were derived from rabbit iliac bone marrow. One control and eight laser irradiated groups were designated as Infrared (IR, 810 nm), Red (R, 660 nm), Green (G, 532 nm), Blue (B, 485 nm), IR-R, IR-B, R-G, and B-G. Irradiation was repeated daily for 21 days and cell proliferation, osseous, or cartilaginous differentiation was then measured. RT-PCR biomarkers were SOX9, aggrecan, COL 2, and COL 10 expression for cartilage and ALP, COL 1, and osteocalcin expression for bone. Cellular proliferation was increased in all irradiated groups except G. All cartilage markers were significantly increased by IR and IR-B except COL 10 which was suppressed by IR-B combination. ALP expression was highest in R and IR groups during osseous differentiation. ALP was decreased by combinations of IR with B and with R, and also by G alone. R and B-G groups showed stimulated COL 1 expression; however, COL 1 was suppressed in IR-B, IR-R, and G groups. IR significantly increased osteocalcin expression, but in B, B-G, and G groups it was reduced. Cartilage differentiation was stimulated by IR and IR-B laser irradiation. The effects of single or combined laser irradiation were not clear-cut on osseous differentiation. Stimulatory effects on osteogenesis were seen for R and IR lasers, while G laser had inhibitory effects.

Photodynamic therapy (PDT) is considered an effective alternative for the treatment of port-wine stains (PWS) using hemoporfin (hematoporphyrin monomethyl ether, HMME), a novel photosensitizer with better efficacy and lower recurrence. Vascular endothelial growth factor (VEGF) plays an important role in the development of PWS. Therefore, we conducted this study to investigate the effect of HMME-PDT on VEGF expression. Human vascular endothelial cells (HUVECs) were treated with different doses of HMME and irradiated with 410-nm light emitting-diode (LED) light. To assess cell viability, CCK-8 assays were performed. At 48 h after PDT, the expression of VEGF/VEGF receptor (VEGFR) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Measurement of VEGF protein was carried out using western blotting assays. Cell viability was significantly inhibited after HMME-PDT and was dose-dependent within a certain range. HMME-PDT decreased secretion of VEGF 48 h after irradiation in HUVECs as compared to controls. The downregulation of VEGF and VEGFR mRNA as well as VEGF protein expression was more significant in the high HMME concentration group (4 μg/mL) than in the lower concentration group (2 μg/mL). Our outcomes provide evidence, that HMME-PDT can downregulate VEGF expression in cultured HUVECs and may explain the efficacy of hemoporfin PDT for PWS treatment.

To investigate the characteristics of regenerated retinal pigment epithelial (RPE) cells after retinal laser photocoagulation in diabetic mice. C57BL/6J mice were used to induce diabetes using intraperitoneal injection of streptozotocin. The proliferation of RPE cells after laser photocoagulation was determined using the 5-ethynyl-2'-deoxyuridine (EdU) assay in both diabetic and wild-type mice. The morphological changes of RPE cells were evaluated by using Voronoi diagram from immunostaining for ß-catenin. Characteristics of regenerated cells were evaluated by quantifying the mRNA and protein levels of RPE and epithelial-mesenchymal transition (EMT) markers. There were significantly less EdU-positive cells in laser-treated areas in diabetic mice than wild-type mice. Hexagonality was extensively lost in diabetic mice. Many EdU-positive cells were co-localized with Otx2-positive cells in the center of the laser-treated areas in wild-type mice, but only EdU-positive cells were widely distributed in diabetic mice. Quantitative analysis of mRNA and protein levels showed that the expression levels of RPE markers, Pax6, Mitf, and Otx2, were significantly decreased in RPE of diabetic mice compared with that of wild-type mice, whereas the expression levels of EMT markers, vimentin and fibronectin, were significantly increased. The proliferation and hexagonality of regenerating RPE cells were impaired after laser photocoagulation, and the regenerated RPE cells lost their original properties in diabetic mice. Further clinical research is needed to elucidate the RPE response after laser photocoagulation in diabetic patients.

Miniaturization of implantable devices is an important challenge for future brain-computer interface applications, and in particular for achieving precise neuron stimulation. For stimulation that utilizes light, i.e., optogenetics, the light propagation behavior and interaction at the nanoscale with elements within the neuron is an important factor that needs to be considered when designing the device. This paper analyzes the effect of light behavior for a single neuron stimulation and focuses on the impact from different cell shapes. Based on the Mie scattering theory, the paper analyzes how the shape of the soma and the nucleus contributes to the focusing effect resulting in an intensity increase, which ensures that neurons can assist in transferring light through the tissue toward the target cells. At the same time, this intensity increase can in turn also stimulate neighboring cells leading to interference within the neural circuits. This paper also analyzes the ideal placements of the device with respect to the angle and position within the cortex that can enable axonal biophoton communications, which can contain light within the cell to avoid the interference.

One of the major challenges in developing acoustic droplet vaporization (ADV)-associated therapy as an effective and safe strategy is the precise determination of the spatial cellular bioeffects after ADV (cell death or cell membrane permeabilization). In this study, we combined high-speed camera imaging and live-cell microscopic imaging to observe the transient dynamics of droplets during ADV and to evaluate the mechanical force on cells. Methods: C6 glioma cells were co-incubated with DiI-labeled droplets (radius: 1.5, 2.25, and 3.0 μm). We used an acousto-optical system for high-speed bright-field (500 kfps) and fluorescence (40 kfps) microscopic imaging in order to visualize the dynamics of droplets under ultrasound excitation (frequency = 5 MHz, pressure = 5-8 MPa, cycle number = 3, pulse number = 1). Live-cell microscopic imaging was used to monitor the cell morphology, cell membrane permeabilization, and cell viability by membrane-anchored Lyn-yellow fluorescence protein, propidium Iodide staining, and calcein blue AM staining, respectively. Results: We discovered that the spatial distribution of ADV-induced bioeffects could be mapped to the physical dynamics of droplet vaporization. For droplets with a 1.5 μm radius, the distance threshold for ADV-induced cell death (5.5±1.9 μm) and reversible membrane permeabilization (11.3±3.5 μm) was well correlated with the distance of ADV-bubble pressing downward to the floor (5.7±1.3 μm) and maximum distance of droplet expansion (11.5±2.6 μm), respectively. These distances were enlarged by increasing the droplet sizes and insonation acoustic pressures. The live-cell imaging results show that ADV-bubbles can directly disrupt the cell membrane layer and induce intensive intracellular substance leakage. Further, the droplets shed the payload onto nearby cells during ADV, suggesting ADV could directly induce adjacent cell death by physical force and enhancement of chemotherapy to distant cells. Conclusion: This study provide new insights into the ADV-mediated physicochemical synergic effect for medical applications., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background: Ultraviolet (UV) radiation is known to be one of the most important environmental hazards acting on the skin. The most part of UV radiation is absorbed in the epidermis, where keratinocytes are the most abundant and exposed cell type. Lipids have an important role in skin biology, not only for their important contribution to the maintenance of the permeability barrier but also for the production and storage of energy, membrane organization and cell signalling functions. However, the effects on the lipid composition of keratinocytes under UV radiation are little explored., Objective: The present work aims to explore the effects on the phenotype and lipid content of primary human keratinocytes exposed to simulated solar UV radiation., Methods: Keratinocytes were exposed to a single (acute exposure) and repeated simulated solar UV irradiations for 4 weeks (chronic exposure). Cell viability and morphology were explored, as well as the production of reactive oxygen species. Then, lipid extracts were analysed through liquid chromatography coupled to mass spectrometry (LC-MS) and the data generated was processed using the ROIMCR chemometric methodology together with partial least squares discriminant analysis (PLS-DA), to finally reveal the most relevant lipid changes that occurred in keratinocytes upon UV irradiation. Also, the potential induction of keratinocyte differentiation was explored by measuring the increase of involucrin., Results: Under acute irradiation, cell viability and morphology were not altered. However, a general increase of phosphatidylcholines (PC) phosphatidylethanolamines (PE) and phosphatidylglycerol (PG) together with a slight sphingomyelin (SM) decrease were found in UV irradiated cells, among other changes. In addition, keratinocyte cultures did not present any differentiation hallmark. Contrary to acute-irradiated cells, in chronic exposures, cell viability was reduced and keratinocytes presented an altered morphology. Also, hallmarks of differentiation, such as the increase of involucrin protein and the autophagy induction were detected. Among the main lipid changes that accompanied this phenotype, the increase of long-chain ceramides, lysoPC and glycerolipid species were found., Conclusion: Important lipid changes were detected under acute and chronic UV irradiation. The lipid profile under chronic exposure may represent a lipid fingerprint of the keratinocyte differentiation phenotype., (Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Inevitable exposure to ionizing radiation from natural and human-made sources has been increasing over time. After nuclear disasters, such as the Fukushima accident, the public concerns on health risk of radiation exposure because of radioactive contamination of the environment have increased. However, it is very difficult to assess the biological effects of exposure caused by environmental radiation. A reliable and rapid bioassay to monitor the physiological effects of radiation exposure is therefore needed. Here, we quantitatively analyzed the changes in cell shape in Drosophila epidermis after irradiation as a model for biomonitoring of radiation. Interestingly, the number of irregularly shaped epithelial cells was increased by irradiation in a dose-dependent manner. A dose-response curve constructed with the obtained data suggests that the measurement of the number of irregular shaped cell in the epidermis is useful for the assessment of radiation dose. In addition, a comparison of the variation in the different samples and the data scored by different observers showed that our evaluation for cellular morphology was highly reliable and accurate and would, therefore, have immense practical application. Overall, our study suggests that detection of morphological changes in the epithelial cells is one of the efficient ways to quantify the levels of exposure to radioactive radiation from the environment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

During mobile phone conversations, the temporal lobe neural networks involved in processing auditory information are exposed to electromagnetic fields (EMF) such as pulse-modulated GSM-1800 MHz radiofrequencies that convey wireless communications. The effects of these EMF on the brain affected by a pathological condition remain little investigated. In this study, rats injected with lipopolysaccharide (LPS) to induce neuroinflammation were exposed "head-only" to GSM-1800 MHz signals for two hours at a specific absorption rate (SAR) that reached an average value of 1.55 W/kg in the auditory cortex (ACx). Immunodetection of Iba1, a microglial marker, and electrophysiological recordings in the ACx three to six hours after global system for communication (GSM) exposure, or sham-exposure, showed that exposure to GSM-1800 MHz resulted in a growth of microglial processes and a reduction in spontaneous firing rate. More importantly, there was a significant reduction in evoked responses to artificial and natural stimuli and an increase in response duration. The response latency and the bandwidth of the frequency tuning were unchanged, but the GSM exposure led to a higher proportion of cortical sites exhibiting abnormally high acoustic thresholds. These modifications were not observed in rats exposed to GSM-1800 MHz without pretreatment with LPS. Together our data provide evidence that in neuroinflammatory conditions, acute exposure to GSM-1800 MHz can significantly affect microglia and neuronal activity underling auditory perception., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Hepatitis B virus (HBV) infection is strongly associated with hepatocellular carcinoma due to the main pathogenic X protein of HBV (HBx). Whether HBV infection and the HBx protein could result in macular degeneration (MD) is not known. The aim of this study is to assess the association and underlying mechanisms between HBV infection and MD., Methods: The National Health Research Institutes in Taiwan built a large database, the National Health Insurance Research Database (NHIRD), which includes the claims data from the Taiwan National Health Insurance (NHI) program. The Taiwan NHI is a single-payer, compulsory health insurance program for Taiwan citizens. The data for the present study were derived from the Longitudinal Health Insurance Database, which contains the claims data of 1 million insured people within the NHIRD, including beneficiary registration, inpatient and outpatient files, drug use, and other medical services. In this study, we first investigated the association of HBV infection and the risk of MD by a population-based cohorts study enrolling 39,796 HBV-infected patients and 159,184 non-HBV-infected patients., Results: After adjustment of age, sex, and comorbidities, the risk of MD was significantly higher in the HBV-infected cohort than in the non-HBV-infected cohort (adjusted HR = 1.31; 95% CI = 1.17-1.46). In vitro, we provided evidence to demonstrate that overexpression of HBx in the human retinal pigment epithelial (RPE) cell line, ARPE19, significantly reduced cell viability and clonogenic survival upon UV and blue light irradiation. By gene microarray analysis, we further showed that almost all genes in DNA repair pathways including base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination were significantly down-regulated in the UV-induced cell death of HBx-transfected ARPE19 cells., Conclusions: The HBx protein may sensitize RPE cells to UV and blue light irradiation and increase the risk of HBV-infection-associated MD through down-regulation of multiple DNA repair pathways.

The 1064-nm Q-switched Nd:YAG laser is demonstrated to be effective for non-ablative skin rejuvenation, but the molecular mechanism by which dermis responses to laser-induced damage and initiates skin remodeling is still unclear. HaCaT cells and 3T3 skin fibroblasts were irradiated with the 1064-nm Q-switched Nd:YAG laser at the different doses. Then, cells were collected and lysed for PCR and Western blot analysis. Cell viability was detected by Cell Counting Kit-8 (CCK-8) before and after laser irradiation. The expressions of S100A8, advanced glycosylation end product-specific receptor (RAGE) and inflammatory cytokines in two cell lines were markedly upregulated after laser treatments. The PCR, Western blot, and ELISA analysis showed the significant increase of type I and III procollagen in the 3T3 cells treated with the 1064-nm laser. Interestingly, si S100A8 effectively inhibited the expression of cytokines and collagen, while S100A8 treatments significantly increased them. P-p38 and p-p65 levels were also elevated after the 1064-nm laser irradiation, which is positively related with S100A8. Cell viability and reactive oxygen species (ROS) levels were not changed, while the content of superoxidase dismutase (SOD) in two cells was increased after laser irradiation. Our results demonstrated that the overexpression of S100A8 induced by the 1064-nm laser irradiation triggered inflammatory reactions in skin cells. The inflammatory microenvironment and improvement of skin antioxidant capacity contribute to new collagen synthesis in the skin cells. Thus, S100A8 was required for laser-induced new collagen synthesis in skin cells. p38/MAPK and NF-κB signal pathways were involved in S100A8-mediated inflammatory reactions in response to laser irradiation.

The study of the effects of low-level laser (LLL) radiation on blood is important for elucidating the mechanisms behind the interaction of LLL radiation and biologic tissues. Different therapy methods that involve blood irradiation have been developed and used for clinical purposes with beneficial effects. The aim of this study was to compare the effects of different irradiation protocols using a diode-pumped solid-state LLL (λ = 405 nm) on samples of human blood by measuring the erythrocyte sedimentation rate (ESR). Human blood samples were obtained through venipuncture into tubes containing EDTA as an anticoagulant. Every sample was divided into two equal aliquots to be used as an irradiated sample and a non-irradiated control sample. The irradiated aliquot was subjected to a laser beam with a wavelength of 405 nm and an energy density of 72 J/cm 2 . The radiation source had a fixed irradiance of 30 mW/cm 2 . The ESR change was observed for three different experimental protocols: irradiated whole blood, irradiated red blood cells (RBCs) samples re-suspended in non-irradiated blood plasma, and non-irradiated RBCs re-suspended in irradiated blood plasma. The ESR values were measured after laser irradiation and compared with the non-irradiated control samples. Irradiated blood plasma in which non-radiated RBCs were re-suspended was found to result in the largest ESR decrease for healthy human RBCs, 51%, when compared with RBCs re-suspended in non-irradiated blood plasma. The decrease in ESR induced by LLL irradiation of the plasma alone was likely related to changes in the plasma composition and an increase in the erythrocyte zeta potential upon re-suspension of the RBCs in the irradiated blood plasma.

Photobiomodulation (PBM) therapy contributes to pain relief, wound healing, and tissue regeneration. The pulsed wave (PW) mode has been reported to be more effective than the continuous wave (CW) mode when applying PBM to many biological systems. However, the reason for the higher effectiveness of PW-PBM is poorly understood. Herein, we suggest using delayed luminescence (DL) as a reporter of mitochondrial activity after PBM treatment. DL originates mainly from mitochondrial electron transport chain systems, which produce reactive oxygen species (ROS) and adenosine triphosphate (ATP). The decay time of DL depends on the pulse frequencies of applied light, which correlate with the biological responses of human dental pulp stem cells (hDPSCs). Using a low-power light whose wavelength is 810 nm and energy density is 38 mJ/cm 2 , we find that a 300-Hz pulse frequency prolonged the DL pattern and enhanced alkaline phosphatase activity. In addition, we analyze mitochondrial morphological changes and their volume density and find evidence supporting mitochondrial physiological changes from PBM treatment. Our data suggest a new methodology for determining the effectiveness of PBM and the specific pulse frequency dependency of PBM in the differentiation of hDPSCs.

This study aimed to evaluate the effects of an ultrashort pulsed laser (USPL) (1064 nm, 20 ps, 100 kHz) with different laser fluences (F, 4, 6, 8 J/cm 2 ) and pulse overlaps (PO, 0, 50%) on human periodontal ligament cells (hPDLs) behavior. Dentin samples were ablated with USPL with different combinations of fluences and pulse overlaps; some samples were ablated with an Er:YAG laser (2940 nm, 150 µs, 100 mJ/pulse, 5 J/cm 2 ) and some samples were ground with a carbide bur. Then hPDLs were grown on the samples after different treatments. Dentin morphology and cell adhesion were observed with SEM and gene expressions were measured by RT-PCR. The results showed dentin surfaces ablated with USPL when F = 4 J/cm 2 , PO = 0, and F = 6 J/cm 2 , PO = 0 were partially intact with obvious ridges and valleys and cells on these surfaces grew mostly along the valleys. USPL ablated surfaces in other groups were entirely ablated and cell cluster formation was observed. The RT-PCR results showed an upregulation of osteocalcin of cells grown on the dentin after some laser treatment. It can be concluded that USPL could improve the attachment and differentiation of hPDLs and thus potentially promote periodontal tissue regeneration.

Migration of stem cells underpins the physiology of metazoan animals. For tissues to be maintained, stem cells and their progeny must migrate and differentiate in the correct positions. This need is even more acute after tissue damage by wounding or pathogenic infection. Inappropriate migration also underpins metastasis. Despite this, few mechanistic studies address stem cell migration during repair or homeostasis in adult tissues. Here, we present a shielded X-ray irradiation assay that allows us to follow stem cell migration in planarians. We demonstrate the use of this system to study the molecular control of stem cell migration and show that snail-1 , snail-2 and zeb-1 EMT transcription factor homologs are necessary for cell migration to wound sites and for the establishment of migratory cell morphology. We also observed that stem cells undergo homeostatic migration to anterior regions that lack local stem cells, in the absence of injury, maintaining tissue homeostasis. This requires the polarity determinant notum Our work establishes planarians as a suitable model for further in-depth study of the processes controlling stem cell migration in vivo ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

This study aimed to compare the effects of photobiomodulation (PBM) in different energy densities and irradiances on maintaining cell viability, and proliferation of pulp fibroblasts from human primary teeth (HPF) were cultured in DMEM and used between the fourth and eighth passages. Then, HPF were irradiated with the following different energy densities: 1.25 J/cm 2 (a), 2.50 J/cm 2 (b), 3.75 J/cm 2 (c), 5.00 J/cm 2 (d), and 6.25 J/cm 2 (e); but varying either the time of irradiation (groups 1a-1e) or the output power (groups 2a-2e). Positive (groups 1f and 2f) and negative controls (groups 1g and 2g), respectively, comprised non-irradiated cells grown in regular nutritional conditions (10% fetal bovine serum [FBS]) and under nutritional deficit (1% FBS). Cell viability and proliferation were respectively assessed through MTT and crystal violet (CV) assays at 24, 48, and 72 h after irradiation. Statistical analysis was performed by two-way ANOVA, followed by Tukey test (P < 0.05). The negative controls showed significantly lower viability in relation to most of the corresponding subgroups, both for MTT and CV assays. For both assays, the intragroup comparison showed that the periods of 24 h exhibited lower viability than the periods of 48 and 72 h for most of the subgroups, except the negative controls with lower viability. The different irradiation protocols (equal energy densities applied with different irradiances) showed no statistically significant differences on cell viability and proliferation at the evaluated periods. The proposed PBM in different energy densities and irradiance did not affect the viability and proliferation of pulp fibroblasts from human primary teeth.

Moderate to intense light is reported to damage the chick retina, which is cone dominated. Light damage alters neurotransmitter pools, such as those of glutamate. Glutamate level in the retina is regulated by glutamate-aspartate transporter (GLAST) and glutamine synthetase (GS). We examined immunolocalization patterns and the expression levels of both markers and of glial fibrillary acidic protein (GFAP, a marker of neuronal stress) in chick retina exposed to 2000 lux under 12-h light:12-h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod), and 24L:0D (constant light) at post-hatch day 30. Retinal damage (increased death of photoreceptors and inner retinal neurons and Müller cell hypertrophy) and GFAP expression in Müller cells were maximal in 24L:0D condition compared to that seen in 12L:12D and 18L:6D conditions. GS was present in Müller cells and GLAST expressed in Müller cell processes and photoreceptor inner segments. GLAST expression was decreased in 24L:0D condition, and the expression levels between 12L:12D and 18L:6D, though increased marginally, were statistically insignificant. Similar was the case with GS expression that significantly decreased in 24L:0D condition. Our previous study with chicks exposed to 2000 lux reported increased retinal glutamate level in 24L:0D condition. The present results indicate that constant light induces decreased expressions of GLAST and GS, a condition that might aggravate glutamate-mediated neurotoxicity and delay neuroprotection in a cone-dominated retina.

Even with the advances of conventional treatment techniques, the nervous system cancer prognosis is still not favorable to the patient which makes alternative therapies needed to be studied. Photodynamic therapy (PDT) is presented as a promising therapy, which employs a photosensitive (PS) agent, light wavelength suitable for the PS agent, and molecular oxygen, producing reactive oxygen species in order to induce cell death. The aim of this study is to observe the PDT action in gliosarcoma cell using a chlorin (Photodithazine, PDZ). The experiments were done with 9L/lacZ lineage cells, grown in a DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution and put in a culture chamber at 37 °C with an atmosphere of 5% CO 2 . The PS agent used was the PDZ to an LED light source device (Biopdi/IRRAD-LED 660) in the 660-nm region. The location of the PS agent was analyzed by fluorescence microscopy, and cell viability was analyzed by MTT assay (mitochondrial activity), exclusion by trypan blue (cell viability), and morphological examination through an optical microscope (Leica MD 2500). In the analysis of the experiments with PDZ, there was 100% cell death at different concentrations and clear morphological differences in groups with and without treatment. Furthermore, it was observed that the photodithazine has been focused on all nuclear and cytoplasmic extension; however, it cannot be said for sure whether the location is in the inside core region or on the plasma membrane. In general, the PDZ showed a promising photosensitive agent in PDT for the use of gliosarcoma.

Thrombocytopenia is an important cause of hemorrhage and death after radiation injury, but the pathogenesis of radiation-induced thrombocytopenia has not been fully characterized. Here, we investigated the influence of radiation-induced endothelial cell injury on platelet regeneration. We found that human umbilical vein endothelial cells (HUVECs) underwent a high rate of apoptosis, accompanied by a significant reduction in the expression of vascular endothelial growth factor (VEGF) at 96 h after radiation. Subsequent investigations revealed that radiation injury lowered the ability of HUVECs to attract migrating megakaryocytes (MKs). Moreover, the adhesion of MKs to HUVECs was markedly reduced when HUVECs were exposed to radiation, accompanied by a decreased production of platelets by MKs. In vivo study showed that VEGF treatment significantly promoted the migration of MKs into the vascular niche and accelerated platelet recovery in irradiated mice. Our studies demonstrate that endothelial cell injury contributes to the slow recovery of platelets after radiation, which provides a deeper insight into the pathogenesis of thrombocytopenia induced by radiation., (© The Author 2017. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

Epidemiological studies have indicated a possible association between extremely low-frequency magnetic field (ELF-MF) exposure and the risk of nervous system diseases. However, laboratory studies have not provided consistent results for clarifying this association, despite many years of studies. In this study, we have systematically investigated the effects of 50 Hz MF exposure on DNA damage and cellular functions in both neurogenic tumor cell lines (U251, A172, SH-SY5Y) and primary cultured neurogenic cells from rats (astrocytes, microglia, cortical neurons). The results showed that exposure to a 50 Hz MF at 2.0 mT for up to 24 h did not influence γH2AX foci formation (an early marker of DNA double-strand breaks) in any of six different neurogenic cells. Exposure to a 50 Hz MF did not affect cell cycle progression, cell proliferation or cell viability in neurogenic tumor U251, A172 or SH-SY5Y cells. Furthermore, the MF exposure for 24 h did not significantly affect the secretion of cytokines (TNF-α, IL-6 or IL-1β) in astrocytes or microglia, or the phagocytic activity of microglia. In addition, MF exposure for 1 h per day did not significantly influence expression levels of microtubule-associated protein tau, microtubule-associated protein 2, postsynaptic density 95 or gephyrin in cortical neurons, indicating an absence of effects of MF exposure on the development of cortical neurons. In conclusion, our data suggest that exposure to a 50 Hz MF at 2.0 mT did not elicit DNA damage effects or abnormal cellular functions in the neurogenic cells studied., (© The Author 2017. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

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., (© 2016 Wiley Periodicals, Inc.)

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., (© 2016 Wiley Periodicals, Inc.)

Exposure of the embryo to ionizing radiation (IR) is detrimental as it can cause genotoxic stress leading to immediate and latent consequences such as functional defects, malformations, or cancer. Human embryonic stem (hES) cells can mimic the preimplantation embryo and help to assess the biological effects of IR during early development. In this study, we describe the alterations H9 hES cells exhibit after X-ray irradiation in respect to cell cycle progression, apoptosis, genomic stability, stem cell signaling, and their capacity to differentiate into definitive endoderm. Early postirradiation, hES cells responded with an arrest in G2/M phase, elevated apoptosis, and increased chromosomal aberrations. Significant downregulation of stem cell signaling markers of the TGF beta-, Wnt-, and Hedgehog pathways was observed. Most prominent were alterations in the expression of activin receptors. However, hES cells responded differently depending on the culture conditions chosen for maintenance. Enzymatically passaged cells were less sensitive to IR than mechanically passaged ones showing fewer apoptotic cells and fewer changes in the stem cell signaling 24 h after irradiation, but displayed higher levels of chromosomal aberrations. Even though many of the observed changes were transient, surviving hES cells, which were differentiated 4 days postirradiation, showed a lower efficiency to form definitive endoderm than their mock-irradiated counterparts. This was demonstrated by lower expression levels of SOX17 and microRNA miR-375. In conclusion, hES cells are a suitable tool for the IR risk assessment during early human development. However, careful choice of the culture methods and a vigorous monitoring of the stem cell quality are mandatory for the use of these cells. Exposure to IR influences the stem cell properties of hES cells even when immediate radiation effects are overcome. This warrants consideration in the risk assessment of radiation effects during the earliest stages of human development.

In recent years, electromagnetic field (EMF) and low-level laser (LLL) have been found to affect various biological processes, the growth and proliferation of cells, and especially that of stem cells. The aim of this study was to investigate the effects of EMF and LLL on proliferation of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and thus to examine the impact of these therapeutic physical modalities on stem cell engraftment. hAT-MSCs were isolated from subcutaneous adipose tissue of six persons ranging in age from 21 to 56 years. EMF was applied for a period of 7 days, once a day for 30 min, via a magnetic cushion surface at a frequency of 50 Hz and an intensity of 3 mT. LLL was applied also for 7 days, once a day for 5 min, at radiation energies of 3 J/cm 2 , with a wavelength of 808 nm, power output of 200 mW, and power density of 0.2 W/cm 2 . Nonexposed cells (control) were cultivated under the same culture conditions. Seven days after treatment, the cells were examined for cell viability, proliferation, and morphology. We found that after 7 days, the number of EMF-treated hAT-MSCs was significantly higher than the number of the untreated cells, LLL-treated hAT-MSCs were more numerous than EMF-treated cells, and hAT-MSCs that were treated with the combination of EMF and LLL were the most numerous. EMF and/or LLL treatment did not significantly affect hAT-MSC viability by itself. Changes in cell morphology were also observed, in terms of an increase in cell surface area and fractal dimension in hAT-MSCs treated with EMF and the combination of EMF and LLL. In conclusion, EMF and/or LLL treatment accelerated the proliferation of hAT-MSCs without compromising their viability, and therefore, they may be used in stem cell tissue engineering.

Photosensitive semiconducting polymer (SP) combined with light stimulation has shown the capability in promoting the proliferation of human dermal fibroblasts (HDFs). However, the high cytotoxicity of the used SP hindered its further application in bioactive scaffolds. In this contribution, we designed and synthesized a SP, poly (N,N-bis(2-octyldodecyl)-3,6-di(thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione-alt-thieno[3,2-b]thiophene) (PDBTT) with low cytotoxicity and strong absorbance in red and near-infrared region (600-1200nm). The photosensitive SP was then applied in electrospun poly(ε-caprolactone) (PCL) nanofibrous scaffold and evaluated its proliferative effect on HDFs under the illumination from red light-emitting diode (LED) with high tissue penetration. After 9days of continuous stimulation, the hybrid electrospun PCL/PDBTT nanofibers with low cytotoxicity showed excellent support for HDFs adhesion, proliferation and collagen secretion than neat PCL nanofibers and HDFs on the stimulated PCL/PDBTT nanofibers gained typical spindle morphology, indicating the well cell spreading on the stimulated PCL/PDBTT nanofibers. The incorporation of functional materials within synthetic biomaterials could be a novel way in improving the performance of engineered tissue constructs by providing multiple cues (e.g. electrical stimulation) to the attached cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

CO 2 laser has a beneficial effect on stem cells by mechanisms that are not clearly elucidated. We hypothesize that the effect of fractional CO 2 laser on human adipose-derived stem cells (ADSC) could be due to changes in redox homeostasis and secretion of factors contributing to cellular proliferation and angiogenic potential. ADSC incubated in medium containing 0.5 or 10 % FBS were exposed to a single irradiation of a 10,600-nm fractional CO 2 laser; non-irradiated ADSC were used as control. Viability/proliferation of ADSC was assessed by MTT assay; the intracellular reactive oxygen species (ROS) levels and the mitochondrial membrane potential (∆Ψ m ) were determined with DCFH-DA and JC-1 fluorescent probes, respectively. Molecules secreted by ADSC in the medium were determined by ELISA assay, and their capacity to support endothelial tube-like formation by the Matrigel assay. The results showed that compared to controls, ADSC kept in low FBS medium and irradiated with CO 2 laser at 9 W exhibited: (a) increased proliferation (∼20 %), (b) transient increase of mitochondrial ROS and the capacity to restore Δψ m after rotenone induced depolarization, and (c) augmented secretion in the conditioned medium of MMP-2 (twofold), MMP-9 (eightfold), VEGF (twofold), and adiponectin (∼50 %) that have the capacity to support angiogenesis of endothelial progenitor cells. In conclusion, the mechanisms underlying the benefic effect of CO 2 laser on ADSC are the activation of the redox pathways which increases cell proliferation and enhances secretion of angiogenic molecules. These results explain, in part, the mechanisms involved in the increased regenerative potential of CO 2 laser-exposed ADSC that could be exploited for clinical applications.

Cell phones, an indispensable element of daily life, are today used at almost addictive levels by adolescents. Adolescents are therefore becoming increasingly exposed to the effect of the electromagnetic field (EMF) emitted by cell phones. The purpose of this study was to investigate the effect of exposure to a 900-MHz EMF throughout adolescence on the lumbar spinal cord using histopathological, immunohistochemical and biochemical techniques. Twenty-four Sprague Dawley (28.3-43.9g) aged 21days were included in the study. These were divided equally into three groups - control (CG), sham (SG) and electromagnetic (ELMAG). No procedure was performed on the CG rats until the end of the study. SG and ELMAG rats were kept inside an EMF cage (EMFC) for 1h a day every day at the same time between postnatal days 22 and 60. During this time, ELMAG rats were exposed to the effect of a 900-MHz EMF, while the SG rats were kept in the EMFC without being exposed to EMF. At the end of the study, the lumbar regions of the spinal cords of all rats in all groups were extracted. Half of each extracted tissue was stored at -80°C for biochemical analysis, while the other half was used for histopathological and immunohistochemical analyses. In terms of histopathology, a lumbar spinal cord with normal morphology was observed in the other groups, while morphological irregularity in gray matter, increased vacuolization and infiltration of white matter into gray matter were pronounced in the ELMAG rats. The cytoplasm of some neurons in the gray matter was shrunken and stained dark, and vacuoles were observed in the cytoplasms. The apoptotic index of glia cells and neurons were significantly higher in ELMAG compared to the other groups. Biochemical analysis revealed a significantly increased MDA value in ELMAG compared to CG, while SOD and GSH levels decreased significantly. In conclusion, our study results suggest that continuous exposure to a 900-MHz EMF for 1h a day through all stages of adolescence can result in impairments at both morphological and biochemical levels in the lumbar region spinal cords of Sprague Dawley rats., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Photodynamic therapies (PDT) have become increasingly popular in the adjuvant treatment of different tumour entities. Chemotherapeutic agents, such as cisplatin may be used in combination with low-level laser therapy (LLLT) as laser photochemotherapy. The aim of this study was to investigate the effect of LLLT on cell bioviability of normal and malignant bone cells under chemotherapeutic conditions with either cisplatin or zolendronic acid in vitro., Methods: Primary human osteoblasts (HOB) and an osteosarcoma cell line (Saos-2) were treated with different concentrations of zolendronic acid or cisplatin and irradiated twice with a diode laser (wavelength 670 nm, 120 s, energy outputs of 100mW/cm 2 , continuous wave mode). Cell viability was tested by XTT-assay and via histomorphological analysis., Results: LLLT alone increased bioviability for both cell lines. LLLT lowered HOB viability at the three highest concentrations of cisplatin and zolendronic acid. For Saos-2, LLLT reduced cell viability at every concentration of cisplatin. In cases of incubation with zolendronic acid, similar to osteoblasts, LLLT lowered cell viability at the highest concentration only., Conclusions: Based on the conditions of this study, laser photochemotherapy may be able to raise the cytotoxicity of cisplatin and zolendronic acid in benign and malignant bone cells. This could be of interest in the development of new therapeutic treatment modalities against neoplastic bone diseases like osteosarcoma., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Most previous studies on Haematococcus pluvialis have been focused on growth and astaxanthin accumulation. However, the relationships between photorespiration and astaxanthin accumulation have not been clarified. The purpose of this study was to examine the role of photorespiration during the process of astaxanthin accumulation in H. pluvialis. During astaxanthin accumulation, the astaxanthin content was reduced significantly when photorespiration was inhibited by its specific inhibitor, carboxymethoxylamine. The inhibition of photorespiration did not change the dry weight, chlorophyll content and OJIP transients during the incubation; however, the inhibition of photorespiration significantly decreased the photochemistry of photosystem II and total photosynthetic O2 evolution capacity. Moreover, the restriction in photorespiration was synchronized with a decrease of astaxanthin accumulation. These results suggest that the photorespiratory pathway in H. pluvialis can accelerate astaxanthin accumulation. We speculate that photorespiration can enhance astaxanthin accumulation in the following ways: (i) photorespiration directly affects the glycerate-3-phosphate (PGA) level, which is intrinsically related to the accumulation of astaxanthin in H. pluvialis; (ii) the photorespiratory pathway indirectly affects the PGA level by effecting the dark reactions of photosynthesis, which then results in the enhancement of astaxanthin accumulation in H. pluvialis., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The effect of delayed colony appearance by irradiated cells exemplifying genetic instability is confirmed to be more expressed for diploid wild-type yeast cells characterized by a sigmoidal shape of the survival curve and capable of recovery from radiation damage in contrast to isogenic haploid strain incapable of diploid-specific recovery and characterized by the exponential form of the survival curve. The dependence of the delayed appearance of colonies by diploid yeast cells on the dose of ionizing radiation shows more pronounced manifestation after the action of α-particles (RBE = 4.2 ± 0.3) than after irradiation with y-rays. This effect may be associated with the greater efficiency of densely ionizing radiation to produce lethal radiation damage and accompanying sublesions responsible for the delay in the formation of colonies by the cells surviving after irradiation. It is shown that the dependence of the delayed colony appearance by diploid yeast cells on their survival was substantially the same after exposure to sparsely and densely ionizing radiation. Since exposure to ionizing radiation of different quality induced equally effective number of lethal damage and the same survival rates, these data indicate that the identical number of the accompanying sublesions responsible for the delayed colony appearance are produced by irradiated cells.

The specific properties of gold nanoparticles (AuNPs) make them a novel class of photothermal agents that can induce cancer cell damage and even death through the conversion of optical energy to thermal energy. Most relevant studies have focused on increasing the precision of cell targeting, improving the efficacy of energy transfer, and exploring additional functions. Nevertheless, most cells can uptake nanosized particles through nonspecific endocytosis; therefore, before hyperthermia via AuNPs can be applied for clinical use, it is important to understand the adverse optical-thermal effects of AuNPs on nontargeted cells. However, few studies have investigated the thermal effects induced by pulsed laser-activated AuNPs on nearby healthy cells due to nonspecific treatment. The aim of this study is to evaluate the photothermal effects induced by AuNPs plus a pulsed laser on MG63, an osteoblast-like cell line, specifically examining the effects on cell morphology, viability, death program, and differentiation. The cells were treated with media containing 50 nm AuNPs at a concentration of 5 ppm for 1 hour. Cultured cells were then exposed to irradiation at 60 mW/cm(2) and 80 mW/cm(2) by a Nd:YAG laser (532 nm wavelength). We observed that the cytoskeletons of MG63 cells treated with bare AuNPs followed by pulsed laser irradiation were damaged, and these cells had few bubbles on the cell membrane compared with those that were not treated (control) or were treated with AuNPs or the laser alone. There were no significant differences between the AuNPs plus laser treatment group and the other groups in terms of cell viability, death program analysis results, or alkaline phosphatase and calcium accumulation during culture for up to 21 days. However, the calcium deposit areas in the cells treated with AuNPs plus laser were larger than those in other groups during the early culture period.

The stomatal apparatus consists of a pair of guard cells and regulates gas exchange between the leaf and atmosphere. In guard cells, blue light (BL) activates H(+)-ATPase in the plasma membrane through the phosphorylation of its penultimate threonine, mediating stomatal opening. Although this regulation is thought to be widely adopted among kidney-shaped guard cells in dicots, the molecular basis underlying that of dumbbell-shaped guard cells in monocots remains unclear. Here, we show that H(+)-ATPases are involved in the regulation of dumbbell-shaped guard cells. Stomatal opening of rice was promoted by the H(+)-ATPase activator fusicoccin and by BL, and the latter was suppressed by the H(+)-ATPase inhibitor vanadate. Using H(+)-ATPase antibodies, we showed the presence of phosphoregulation of the penultimate threonine in Oryza sativa H(+)-ATPases (OSAs) and localization of OSAs in the plasma membrane of guard cells. Interestingly, we identified one H(+)-ATPase isoform, OSA7, that is preferentially expressed among the OSA genes in guard cells, and found that loss of function of OSA7 resulted in partial insensitivity to BL. We conclude that H(+)-ATPase is involved in BL-induced stomatal opening of dumbbell-shaped guard cells in monocotyledon species., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Published gene expression studies for radiation-induced thyroid carcinogenesis have used various methodologies. In this study, we identified differential gene expression in a human thyroid epithelial cell line after exposure to high-dose γ-radiation. HTori-3 cells were exposed to 5 or 10 Gy of ionizing radiation using two dose rates (high-dose rate: 4.68 Gy/min, and low-dose rate: 40 mGy/h) and then implanted into the backs of BALB/c nude mice after 4 (10 Gy) or 5 weeks (5 Gy). Decreases in cell viability, increases in giant cell frequency, anchorage-independent growth in vitro, and tumorigenicity in vivo were observed. Particularly, the cells irradiated with 5 Gy at the high-dose rate or 10 Gy at the low-dose rate demonstrated more prominent tumorigenicity. Gene expression profiling was analyzed via microarray. Numerous genes that were significantly altered by a fold-change of >50% following irradiation were identified in each group. Gene expression analysis identified six commonly misregulated genes, including CRYAB, IL-18, ZNF845, CYP24A1, OR4N4 and VN1R4, at all doses. These genes involve apoptosis, the immune response, regulation of transcription, and receptor signaling pathways. Overall, the altered genes in high-dose rate (HDR) 5 Gy and low-dose rate (LDR) 10 Gy were more than those of LDR 5 Gy and HDR 10 Gy. Thus, we investigated genes associated with aggressive tumor development using the two dosage treatments. In this study, the identified gene expression profiles reflect the molecular response following high doses of external radiation exposure and may provide helpful information about radiation-induced thyroid tumors in the high-dose range., (© The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)