Previous research indicates that the cytoskeleton microtubules (MT) have resonance frequencies in the radiofrequency (RF) range. In this study, cells were labeled with cytoskeletal markers and were exposed to a range of GHz frequencies. Confocal microscopy analysis was performed following exposures to examine effects due to RF exposures to evaluate changes in cell morphology, and on the organization of MT and overall cytoskeletal network. MT are involved in various cellular processes; therefore, it is important to understand RF energy coupling with MT and associated cytoskeletal organization to enable possibilities for RF energy in future development of non-contact MT-based applications.
Human keratinocytes were exposed to continuous wave 900 MHz RF field for 60 minutes. High-throughput RNA sequencing was used to examine global changes in gene expression and differentially expressed genes (DEGs) were defined as having adjusted p value ≤ 0.05 and absolute fold change ≥ 1.3. We used gene enrichment analysis and functional annotation clustering via DAVID Bioinformatics Resources to analyze pathways and gene sets enriched in DEGs. Numerous gene sets related to keratinocyte differentiation and development were enriched in the RF exposed cells.
Recent studies suggest that microtubules (MTs) and tubulin proteins exhibit resonant frequencies in the radiofrequency (RF) range. We hypothesize that exposing neurons to externally applied RF waves tuned to an intrinsic resonant frequency of MTs or tubulin could disrupt the natural signaling occurring in and around them, leading to neurophysiological changes. To test this hypothesis, we assembled custom exposure systems that allow stable RF exposures of cell cultures in a controlled environment (37°C, 5% CO2, 95% humidity). We then exposed differentiated NG108-15 neuronal cells to RF waves tuned to selected resonance peaks for tubulin (91 MHz and 281 MHz) and for MTs (3.0 GHz) for 1 hr at a power density of 0.24 mW/cm2 (SAR = 0.012, 0.087, and 0.53 mW/kg, respectively). We used fluorescence imaging of endogenous MTs and current-clamp electrophysiology to investigate changes following RF exposures compared to sham. The results from the imaging data show a clear difference in the localization of fluorescent MTs between the sham and the RF exposed neuronal cells. The sham cells exhibited more fluorescence in the neurite projections, whereas the RF exposed cells showed a more diffuse pattern, with a stronger fluorescence in the cell body. The electrophysiological results showed that resting membrane potentials of the RF exposed neuronal cells were more depolarized than those of the sham cells. Consequently, we observed spontaneous action potentials in the RF exposed cells, which were not present in the sham cells. Overall, our results suggest that exposing neurons to MTs or tubulin resonant frequencies might affect MTs normal behavior, leading to neurophysiological changes. However, to confirm the specificity of resonant frequency effect and validate this idea, studies investigating exposures to nonresonant frequencies and additional tubulin and MTs resonant frequencies are warranted.
Infrared laser (IRL) exposure can induce a rapid temperature change (fast thermal gradient or FTG) that is able to stimulate or inhibit neurons and, thereby, modify neurological functions. Despite extensive research into this effect, the fundamental mechanism(s) underlying how FTG causes neurological stimulation or inhibition remains unclear. While it is hypothesized that IRL-induced FTG acts directly on the neuronal plasma membrane (PM), it is uncertain if the neurological effects observed in previous studies are mostly derived from presynaptic effects (i.e., modifications in action potential (AP) firing) or also from postsynaptic effects (i.e., alteration of the synaptic responses of the excitatory and inhibitory neuronal receptors). In the present study, we present an analysis of FTG-mediated changes in neuronal PM, AP firing rate, and miniature postsynaptic excitatory and inhibitory currents (mEPSCs and mIPSCs). Our results suggest FTG induces changes in both presynaptic and postsynaptic neurophysiological mechanisms. Specifically, we found that, after IRL pulse (IRLP)-induced FTG exposure, the amplitudes of APs are reduced, but the rate of APs are increased. In contrast, the quantities of both mEPSCs and mIPSCs are reduced, but the peak-to-peak frequency and peak amplitudes are increased. The results outlined in this study demonstrate the impact of FTG on neurons and neuronal network. This information is critical for understanding the complexity of the effects of FTG on neurological functions and for demonstrating how post-synaptic mechanisms might play a crucial role in neurological excitation or inhibition seen following IRL pulse exposure.
Low frequency Raman spectroscopy is a highly sensitive and non-destructive technique used to investigate the vibrational and rotational modes of biological and non-biological materials. The Raman spectra measured provide information about the chemical structure and nature of these materials. In this study, we present the design and construction of a low frequency Raman spectroscopy system that is able to measure signals <10 cm-1 to <400 cm-1. The system consisted of a 514.5nm monochromatic laser directed through a polarizing beam cube and half waveplate to adjust the intensity of the beam. The beam was expanded and reflected off a 514.5 nm high pass filter before passing through a 50x Mitutoyo objective, which focuses it onto the sample. The back scattered light was recollimated through the objective. The high pass filter and three 514.5 nm Bragg filters were used to reduce the Rayleigh signal. The remaining Raman signal was focused into a Shamrock 303i spectrometer with a cooled ANDOR CCD camera. Using high dynamic range data acquisition with background subtraction, this system allowed low frequency Raman spectroscopy of reduced cytochrome C, bovine serum albumin, microtubules and collagen in solution. The system has the advantage of enabling the measurement of the low frequency Raman signal without sacrificing the ability to perform traditional Raman spectroscopy.
Exposures to non-ionizing electromagnetic (EM) waves in the radiofrequency (RF) range have been shown to influence gene expression in various cell and tissue types. However, the specific mechanism(s) by which exposure to these waves alter gene expression is not completely clear. Recent studies have suggested changes in epigenetics as a plausible mechanism for the gene expression alterations observed in response to exposures to RF waves. In this study, we investigated if exposures to RF fields can influence epigenetics. Specifically, we examined modifications in DNA methylation patterns in response to exposures to 900 MHz RF fields in primary human keratinocytes. We assembled a custom system to allow the stable exposure of cell cultures to 900 MHz RF fields at a range of applied powers and resultant E fields. We used methylation sensitive restriction enzyme digestion and Global DNA Methylation ELISA assay to quantify the status of global DNA methylation in cells exposed to 900 MHz RF fields for different time durations and power densities. Results show significant changes in global DNA methylation in the RF exposed cells compared to the sham (unexposed) counterparts. Importantly, these changes occur in the absence of cell death and without a concomitant increase in temperature during exposures, suggesting that alterations in DNA methylation are not associated with toxic or thermal effects of the RF fields. This suggests that RF exposure changes DNA methylation patterns and can potentially alter gene expression.
Short infrared laser pulses induce a variety of effects in cells and tissues, including neural stimulation and inhibition. However, the mechanism behind these physiological effects is poorly understood. It is known that the fast thermal gradient induced by the infrared light is necessary for these biological effects. Therefore, this study tests the hypothesis that the fast thermal gradient induced in a cell by infrared light exposure causes a change in the membrane fluidity. To test this hypothesis, we used the membrane fluidity dye, di-4-ANEPPDHQ, to investigate membrane fluidity changes following infrared light exposure. Di-4-ANEPPDHQ fluorescence was imaged on a wide-field fluorescence imaging system with dual channel emission detection. The dual channel imaging allowed imaging of emitted fluorescence at wavelengths longer and shorter than 647 nm for ratiometric assessment and computation of a membrane generalized polarization (GP) value. Results in CHO cells show increased membrane fluidity with infrared light pulse exposure and this increased fluidity scales with infrared irradiance. Full recovery of pre-infrared exposure membrane fluidity was observed. Altogether, these results demonstrate that infrared light induces a thermal gradient in cells that changes membrane fluidity.
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