Genetically Encoded Photosensitizer for Destruction of Protein or Cell Function.

There are several paths when excited molecules return to the ground state. In the case of fluorescent molecules, the dominant path is fluorescence emission that is greatly contributing to bioimaging. Meanwhile, photosensitizers transfer electron or energy from chromophore to the surrounding molecules, including molecular oxygen. Generated reactive oxygen species has potency to attack other molecules by oxidation. In this chapter, we introduce the chromophore-assisted light inactivation (CALI) method using a photosensitizer to inactivate proteins in a spatiotemporal manner and development of CALI tools, which is useful for investigation of protein functions and dynamics, by inactivation of the target molecules. Moreover, photosensitizers with high efficiency make it possible optogenetic control of cell ablation in living organisms and photodynamic therapy. Further development of photosensitizers with different excitation wavelengths will contribute to the investigation of multiple proteins or cell functions through inactivation in the different positions and timings.

Simvastatin treatment varies the radiation response of human breast cells in 2D or 3D culture.

Background Statins inhibit the cholesterol biosynthesis and are used as cholesterol-lowering agents in fat-metabolism disorders. Furthermore, several studies state that statins have supportive functions in breast cancer treatment. Therefore, simvastatin (SVA) as a potential radiosensitizer should be investigated on the basis of human breast cells. Methods First, an optimal concentration of SVA for normal (MCF10A) and cancer (MCF-7) cells was identified via growth and cytotoxicity assays that, according to the definition of a radiosensitizer in the narrower sense, enhances the effect of radiation therapy but has no cytotoxic effect. Next, in combination with radiation SVA's influence on DNA repair capacity and clonogenic survival in 2D and 3D was determined. Furthermore cell cycle distribution, expression of survivin and connective tissue growth factor (CTGF) as well as ERK1 map kinase were analysed. Results 1 µM SVA was identified as highest concentration without an influence on cell growth and cytotoxicity and was used for further analyses. In terms of early and residual γH2AX-foci, SVA affected the number of foci in both cell lines with or without irradiation. Different radiation responses were detected in 2D and 3D culture conditions. During the 2D cultivation, a radiosensitizing effect within the clonogenic survival was observable, but not in 3D. Conclusion The present study suggests that SVA may have potential for radiosensitization. Therefore, it is important to further investigate the role of SVA in relation to the extent of radiosensitization and how it could be used to positively influence the therapy of breast cancer or other entities.

Precise cell behaviors manipulation through light-responsive nano-regulators: recent advance and perspective.

Nanotechnology-assisted spatiotemporal manipulation of biological events holds great promise in advancing the practice of precision medicine in healthcare systems. The progress in internal and/or external stimuli-responsive nanoplatforms for highly specific cellular regulations and theranostic controls offer potential clinical translations of the revolutionized nanomedicine. To successfully implement this new paradigm, the emerging light-responsive nanoregulators with unparalleled precise cell functions manipulation have gained intensive attention, providing UV-Vis light-triggered photocleavage or photoisomerization studies, as well as near-infrared (NIR) light-mediated deep-tissue applications for stimulating cellular signal cascades and treatment of mortal diseases. This review discusses current developments of light-activatable nanoplatforms for modulations of various cellular events including neuromodulations, stem cell monitoring, immunomanipulation, cancer therapy, and other biological target intervention. In summary, the propagation of light-controlled nanomedicine would place a bright prospect for future medicine.

Enhanced Antitumor Efficacy Achieved Through Combination of nsPEFs and Low-Dosage Paclitaxel.

Looking for a safe and effective cancer therapy for patients is becoming an important and promising research direction. Nanosecond pulsed electric field (nsPEF) has been found to be a potential non-thermal therapeutic technique with few side effects in pre-clinical studies. On the other hand, paclitaxel (PTX), as a common chemotherapeutic agent, shows full anti-tumor activities and is used to treat a wide variety of cancers. However, the delivery of PTX is challenging due to its poor aqueous solubility. Hence, high dosages of PTX have been used to achieve effective treatment, which creates some side effects. In this study, nsPEF was combined with low-level PTX, in order to validate if this combined treatment could bring about enhanced efficacy and allow reduced doses of PTX in clinical application. Cell proliferation, apoptosis, and cell cycle distribution were examined using MTT and flow cytometry assay, respectively. Results showed that combination treatments of nsPEF and PTX exhibited significant synergistic effects in vitro. The underlying mechanism might be that these two agents acted at different targets and coordinately enhanced MDA-MB-231 cell death.

PROSPECTS FOR METROLOGY RELATED TO BIOLOGICAL RADIATION EFFECTS OF ION BEAMS.

In recent years, several approaches have been proposed to provide an understanding of the enhanced relative biological effectiveness of ion beams based on multi-scale models of their radiation effects. Among these, the BioQuaRT project was the only one which focused on developing metrology for a multi-scale characterization of particle track structure. The progress made within the BioQuaRT project has motivated the formation of a department 'Radiation Effects' at PTB dedicated to metrological research on ionizing radiation effects. This paper gives an overview of the department's present research directions and shortly discusses ideas for the future development of metrology related to biological effects of ion beams that are based on a stakeholder consultation.

Effect of Low-Field High-Frequency nsPEFs on the Biological Behaviors of Human A375 Melanoma Cells.

OBJECTIVE: To evaluate the effect of low-field high-frequency nanosecond pulsed electric fields (nsPEFs) on multiple biological behaviors of human A375 melanoma cells and to optimize suitable parameters for further study and clinical use. METHODS: An nsPEF generator was developed to generate appropriate pulses. Cell apoptosis and the cell cycle were evaluated by flow cytometry. The CCK-8 assay was performed to explore the effect of nsPEFs on the viability of A375 melanoma cells. Cell migration was assessed using a Transwell Boyden Chamber. The proliferation of A375 melanoma cells was determined by the cloning efficacy test. Furthermore, the nude mouse tumorigenicity assay was used to detect the effectiveness of nsPEFs in vivo. RESULTS: The nsPEFs with our tested parameters failed to induce apoptosis of A375 melanoma cells, though nsPEFs with high pulse duration (500 ns) induced necrosis. However, the viability and migration of A375 melanoma cells were significantly inhibited by nsPEFs. nsPEFs also suppressed the proliferation of A375 melanoma cells by restricting cells in G0/G1 phase. Moreover, animal experiments demonstrated that nsPEFs inhibited the growth of melanoma in vivo. CONCLUSION: Low-field high-frequency nsPEFs failed to induce apoptosis but effectively inhibited the growth of melanoma via affecting other biological behaviors of melanoma cells, such as cell viability, proliferation, and migration. SIGNIFICANCE: This study investigated the influence of low-field high-frequency nsPEFs on melanoma through evaluating their effects on multiple biological behaviors and is helpful in the treatment of melanoma and other tumors.

Dose analysis of photobiomodulation therapy on osteoblast, osteoclast, and osteocyte.

The objective of this study was to evaluate the effects of varying light doses on the viability and cellular activity of osteoblasts, osteocytes, and osteoclasts. A light application device was developed to apply 940-nm wavelength light from light-emitting diodes on three cultured cells, MC3T3-E1, MLO-A5, and RANKL-treated RAW264.7 cells. The doses (energy density) on cells were 0, 1, 5, and 7.5 J / cm2. The corresponding light power densities at the cell site were 0, 1.67, 8.33, and 12.5 mW / cm2, respectively, and the duration was 10 min. The results showed that the three cell types respond differently to light and their responses were dose dependent. Low-dose treatment (1 J / cm2) enhanced osteoblast proliferation, osteoclast differentiation, and osteoclastic bone resorption activity. Osteocyte proliferation was not affected by both low- and high-dose (5 J / cm2) treatments. While 1 J / cm2 did not affect viability of all three cell types, 5 J / cm2 significantly decreased viability of osteocytes and osteoclasts. Osteoblast viability was negatively impacted by the higher dose (7.5 J / cm2). The findings suggest that optimal doses exist for osteoblast and osteoclast, which can stimulate cell activities, and there is a safe dose range for each type of cell tested.

Optochemical Control of Biological Processes in Cells and Animals.

Biological processes are naturally regulated with high spatial and temporal control, as is perhaps most evident in metazoan embryogenesis. Chemical tools have been extensively utilized in cell and developmental biology to investigate cellular processes, and conditional control methods have expanded applications of these technologies toward resolving complex biological questions. Light represents an excellent external trigger since it can be controlled with very high spatial and temporal precision. To this end, several optically regulated tools have been developed and applied to living systems. In this review we discuss recent developments of optochemical tools, including small molecules, peptides, proteins, and nucleic acids that can be irreversibly or reversibly controlled through light irradiation, with a focus on applications in cells and animals.

DNA double strand break (DSB) induction and cell survival in iodine-enhanced computed tomography (CT).

A multi-scale Monte Carlo model is proposed to assess the dosimetric and biological impact of iodine-based contrast agents commonly used in computed tomography. As presented, the model integrates the general purpose MCNP6 code system for larger-scale radiation transport and dose assessment with the Monte Carlo damage simulation to determine the sub-cellular characteristics and spatial distribution of initial DNA damage. The repair-misrepair-fixation model is then used to relate DNA double strand break (DSB) induction to reproductive cell death. Comparisons of measured and modeled changes in reproductive cell survival for ultrasoft characteristic k-shell x-rays (0.25-4.55 keV) up to orthovoltage (200-500 kVp) x-rays indicate that the relative biological effectiveness (RBE) for DSB induction is within a few percent of the RBE for cell survival. Because of the very short range of secondary electrons produced by low energy x-ray interactions with contrast agents, the concentration and subcellular distribution of iodine within and near cellular targets have a significant impact on the estimated absorbed dose and number of DSB produced in the cell nucleus. For some plausible models of the cell-level distribution of contrast agent, the model predicts an increase in RBE-weighted dose (RWD) for the endpoint of DSB induction of 1.22-1.40 for a 5-10 mg ml-1 iodine concentration in blood compared to an RWD increase of 1.07 ± 0.19 from a recent clinical trial. The modeled RWD of 2.58 ± 0.03 is also in good agreement with the measured RWD of 2.3 ± 0.5 for an iodine concentration of 50 mg ml-1 relative to no iodine. The good agreement between modeled and measured DSB and cell survival estimates provides some confidence that the presented model can be used to accurately assess biological dose for other concentrations of the same or different contrast agents.

Development and performance evaluation of a three-dimensional clinostat synchronized heavy-ion irradiation system.

Outer space is an environment characterized by microgravity and space radiation, including high-energy charged particles. Astronauts are constantly exposed to both microgravity and radiation during long-term stays in space. However, many aspects of the biological effects of combined microgravity and space radiation remain unclear. We developed a new three-dimensional (3D) clinostat synchronized heavy-ion irradiation system for use in ground-based studies of the combined exposures. Our new system uses a particle accelerator and a respiratory gating system from heavy-ion radiotherapy to irradiate samples being rotated in the 3D clinostat with carbon-ion beams only when the samples are in the horizontal position. A Peltier module and special sample holder were loaded on a static stage (standing condition) and the 3D clinostat (rotation condition) to maintain a suitable temperature under atmospheric conditions. The performance of the new device was investigated with normal human fibroblasts 1BR-hTERT in a disposable closed cell culture chamber. Live imaging revealed that cellular adhesion and growth were almost the same for the standing control sample and rotation sample over 48h. Dose flatness and symmetry were judged according to the relative density of Gafchromic films along the X-axis and Y-axis of the positions of the irradiated sample to confirm irradiation accuracy. Doses calculated using the carbon-ion calibration curve were almost the same for standing and rotation conditions, with the difference being less than 5% at 1Gy carbon-ion irradiation. Our new device can accurately synchronize carbon-ion irradiation and simulated microgravity while maintaining the temperature under atmospheric conditions at ground level.

Monte Carlo N-Particle (MCNP) Modeling of the Cellular Dosimetry of 64Cu: Comparison with MIRDcell S Values and Implications for Studies of Its Cytotoxic Effects.

64Cu emits positrons as well as ß- particles and Auger and internal conversion electrons useful for radiotherapy. Our objective was to model the cellular dosimetry of 64Cu under different geometries commonly used to study the cytotoxic effects of 64Cu. METHODS: Monte Carlo N-Particle (MCNP) was used to simulate the transport of all particles emitted by 64Cu from the cell surface (CS), cytoplasm (Cy), or nucleus (N) of a single cell; monolayer in a well (radius = 0.32-1.74 cm); or a sphere (radius = 50-6,000 µm) of cells to calculate S values. The radius of the cell and N ranged from 5 to 12 µm and 2 to 11 µm, respectively. S values were obtained by MIRDcell for comparison. MCF7/HER2-18 cells were exposed in vitro to 64Cu-labeled trastuzumab. The subcellular distribution of 64Cu was measured by cell fractionation. The surviving fraction was determined in a clonogenic assay. RESULTS: The relative differences of MCNP versus MIRDcell self-dose S values (Sself) for 64Cu ranged from -0.2% to 3.6% for N to N (SN←N), 2.3% to 8.6% for Cy to N (SN←Cy), and -12.0% to 7.3% for CS to N (SN←CS). The relative differences of MCNP versus MIRDcell cross-dose S values were 25.8%-30.6% for a monolayer and 30%-34% for a sphere, respectively. The ratios of SN←N versus SN←Cy and SN←Cy versus SN←CS decreased with increasing ratio of the N of the cell versus radius of the cell and the size of the monolayer or sphere. The surviving fraction of MCF7 /: HER2-18 cells treated with 64Cu-labeled trastuzumab (0.016-0.368 MBq/µg, 67 nM) for 18 h versus the absorbed dose followed a linear survival curve with α = 0.51 ± 0.05 Gy-1 and R2 = 0.8838. This is significantly different from the linear quadratic survival curve of MCF7 /: HER2-18 cells exposed to γ-rays. CONCLUSION: MCNP- and MIRDcell-calculated S values agreed well. 64Cu in the N increases the dose to the N in isolated single cells but has less effect in a cell monolayer or small cluster of cells simulating a micrometastasis, and little effect in a sphere analogous to a tumor xenograft compared with 64Cu in the Cy or on the CS. The dose deposited by 64Cu is less effective for cell killing than γ-rays.

Manipulation of cells with laser microbeam scissors and optical tweezers: a review.

The use of laser microbeams and optical tweezers in a wide field of biological applications from genomic to immunology is discussed. Microperforation is used to introduce a well-defined amount of molecules into cells for genetic engineering and optical imaging. The microwelding of two cells induced by a laser microbeam combines their genetic outfit. Microdissection allows specific regions of genomes to be isolated from a whole set of chromosomes. Handling the cells with optical tweezers supports investigation on the attack of immune systems against diseased or cancerous cells. With the help of laser microbeams, heart infarction can be simulated, and optical tweezers support studies on the heartbeat. Finally, laser microbeams are used to induce DNA damage in living cells for studies on cancer and ageing.

Functionalized Stress Component onto Bio-template as a Pathway of Cytocompatibility.

This in-vitro study introduces residual stress as a third dimension of cell stimulus to modulate the interaction between cells and bio-template, without the addition of either chemical or physical stimuli onto the bio-template surface. Ultrashort Pulsed Laser (USPL) irradiation of silicon-based bio-template causes recrystallization of silicon, which mismatches the original crystal orientation of the virgin silicon. Consequently, subsurface Induced Residual Stress (IRS) is generated. The IRS components demonstrated a strong cytocompatibility, whereas the peripheral of IRS, which is the interface between the IRS component and the virgin silicon surface, a significant directional cell alignment was observed. Fibroblast cells shown to be more sensitive to the stress component than Hela cancer cells. It revealed that cytocompatibility in terms of cell migration and directional cell alignment is directly proportional to the level of the IRS component. Higher stress level results in more cell alignment and border migration width. There is a stress threshold below which the stress component completely loses the functionality. These results pointed to a functionalized bio-template with tunable cytocompatibility. This study may lead to a new tool for the designing and engineering of bio-template.

Effects of the pulsed electromagnetic field PST® on human tendon stem cells: a controlled laboratory study.

BACKGROUND: Current clinical procedures for rotator cuff tears need to be improved, as a high rate of failure is still observed. Therefore, new approaches have been attempted to stimulate self-regeneration, including biophysical stimulation modalities, such as low-frequency pulsed electromagnetic fields, which are alternative and non-invasive methods that seem to produce satisfying therapeutic effects. While little is known about their mechanism of action, it has been speculated that they may act on resident stem cells. Thus, the purpose of this study was to evaluate the effects of a pulsed electromagnetic field (PST®) on human tendon stem cells (hTSCs) in order to elucidate the possible mechanism of the observed therapeutic effects. METHODS: hTSCs from the rotator cuff were isolated from tendon biopsies and cultured in vitro. Then, cells were exposed to a 1-h PST® treatment and compared to control untreated cells in terms of cell morphology, proliferation, viability, migration, and stem cell marker expression. RESULTS: Exposure of hTSCs to PST® did not cause any significant changes in proliferation, viability, migration, and morphology. Instead, while stem cell marker expression significantly decreased in control cells during cell culturing, PST®-treated cells did not have a significant reduction of the same markers. CONCLUSIONS: While PST® did not have significant effects on hTSCs proliferation, the treatment had beneficial effects on stem cell marker expression, as treated cells maintained a higher expression of these markers during culturing. These results support the notion that PST® treatment may increase the patient stem cell regenerative potential.

The role of dose rate in radiation cancer risk: evaluating the effect of dose rate at the molecular, cellular and tissue levels using key events in critical pathways following exposure to low LET radiation.

PURPOSE: This review evaluates the role of dose rate on cell and molecular responses. It focuses on the influence of dose rate on key events in critical pathways in the development of cancer. This approach is similar to that used by the U.S. EPA and others to evaluate risk from chemicals. It provides a mechanistic method to account for the influence of the dose rate from low-LET radiation, especially in the low-dose region on cancer risk assessment. Molecular, cellular, and tissues changes are observed in many key events and change as a function of dose rate. The magnitude and direction of change can be used to help establish an appropriate dose rate effectiveness factor (DREF). CONCLUSIONS: Extensive data on key events suggest that exposure to low dose-rates are less effective in producing changes than high dose rates. Most of these data at the molecular and cellular level support a large (2-30) DREF. In addition, some evidence suggests that doses delivered at a low dose rate decrease damage to levels below that observed in the controls. However, there are some data human and mechanistic data that support a dose-rate effectiveness factor of 1. In summary, a review of the available molecular, cellular and tissue data indicates that not only is dose rate an important variable in understanding radiation risk but it also supports the selection of a DREF greater than one as currently recommended by ICRP ( 2007 ) and BEIR VII (NRC/NAS 2006 ).

Characterization of in vitro radiosensitization in mammalian cells using biomathematical modelling: implications for hypofractionated radiotherapy with a combined modality approach.

OBJECTIVE: It is unclear whether radiosensitization is beneficial when radiotherapy is administered at a high dose per fraction. The aim of this study was to assess the impact of radiation dose on the effectiveness of a broad range of radiosensitizers. METHODS: We analyzed 653 pairs of clonogenic survival curves in 285 published articles, in which modifications of radiosensitivity were studied using the colony-forming assay. The modifications of radiosensitivity were arbitrarily classified into 20 classes. The survival curves were fitted to two biomathematical models: the linear-quadratic model and the repair-misrepair (RMR) model. RESULTS: We found that radiosensitization was predominantly characterized by an increase of the α value (α-sensitization) without an increase of the ß value (ß-sensitization). A subset analysis revealed that all 20 classes showed significant α-sensitization. In contrast, only oxygen/hypoxic sensitizers (oxygen) and poly(adenosine diphosphate-ribose) polymerase inhibition (PARPi) exhibited ß-sensitization. An analysis using the RMR model revealed two major sources of radiosensitization: an increased residual DNA lesion through repair inhibition and a shift from linear repairs to quadratic misrepairs, leading to enhanced lethal chromosomal aberrations. CONCLUSION: Oxygen and PARPi were found to show ß-sensitization, which was favourable for eliciting a comparable degree of sensitization in the higher dose range. Reduced fidelity of the repair was suggested to be a possible mechanism of ß-sensitization. Further study targeting ß-sensitization is needed to develop a novel combined modality therapy with high-dose-per-fraction radiotherapy. ADVANCES IN KNOWLEDGE: Radiosensitization can be classified into two groups, α- and ß-sensitizations. These two phenomena may stem from distinct underlying mechanisms.

Quantification of Adaptive Protection Following Low-dose Irradiation.

The question whether low doses and low dose-rates of ionizing radiation pose a health risk to people is of public, scientific and regulatory concern. It is a subject of intense debate and causes much fear. The controversy is to what extent low-dose effects, if any, cause or protect against damage such as cancer. Even if immediate molecular damage in exposed biological systems rises linearly with the number of energy deposition events (i.e., with absorbed dose), the response of the whole biological system to that damage is not linear. To understand how initial molecular damage affects a complex living system is the current challenge.

Response of Biological Systems to Low Doses of Ionizing Radiation.

Radiation is ubiquitous in the environment. Biological effects of exposure to low doses of ionizing radiation are subjected to several modulating factors. Two of these, bystander response and adaptive protections, are discussed briefly.

Compelling Issues Compounding the Understanding of Low Dose Radiation Effects: But Do They Matter?

Recent advances in low dose radiation research have raised a number of compelling issues that have compounded the understanding of low dose radiation effects. Here some of them are outlined: the linear no-threshold model for predicting effects at low radiation doses, dose rate effectiveness factor, attributability, and public perception of low dose radiation effects. The impact of changes in any of these hotly debated issues on radiation protection is considered.

Magnification of Inter-individual Variation in Biological Responses After Low Doses and Dose-Rates of Ionizing Radiation.

Biological responses of human cells and tissues to ionizing radiation (IR) are strongly influenced by dose and dose-rate. Unlike the robust activation of cellular DNA damage responses (DDR) seen after high IR doses, the efficiency of activation of DNA damage repair and signaling pathways after much lower doses and dose-rates varies appreciably among different individuals. Genomic and functional assays measuring low dose and dose-rate IR responses repeatedly show increased inter-individual variability when cells and tissues experience DNA damage levels comparable to those experienced endogenously (due to aerobic metabolism, diet, lifestyle, etc). Complicating matters for risk assessment are recent observations of dose-response non-linearity (hyper-linearity) in the low dose range. With both physical and biological factors strongly influencing individual responses to IR at low doses and dose-rates, further radiobiological research is required to assist regulatory agencies in determining appropriate radiological protection standards for such exposures.