Effects of Long-Term and Multigeneration Exposure of Caenorhabditis elegans to 9.4 GHz Microwaves.

A large number of studies on the biological effects of microwaves are carried out using rodents and cells, but the conditions are difficult to control, and the irradiation period is short; the results obtained have always been controversial and difficult to reproduce. In this study, we expose nematodes to an electromagnetic environment for a long-term and multigeneration period to explore the possible biological effects. Wild-type N2 strains of Caenorhabditis elegans are exposed to 9.4 GHz microwaves at a specific adsorption rate of 4 W/kg for 10 h per day from L1 larvae to adults. Then, adult worms are washed off, and the laid eggs are kept to hatch L1 larvae, which are continuously exposed to microwaves until passing through 20 generations. The worms of the 10th, 15th, and 20th generations are collected for index detection. Interestingly, we found that the fecundity of C. elegans decreased significantly in the exposed group from the 15th generation. At the same time, we found that the growth of C. elegans decreased, motility decreased, and oxidative stress occurred in the exposed group from the 10th generation, which may play roles in the decreased spawning in worms. We preliminarily believe that the microwave energy received by worms leads to oxidative stress, which causes a decrease in the spawning rate, and the underlying mechanism needs to be further studied. © 2022 Bioelectromagnetics Society.

Pulsed High-Peak Power Microwaves at 9.4 GHz Do Not Affect Basic Endpoints in Caenorhabditis elegans.

Because of the extensive application of electromagnetic technology, its health impact on humans has attracted widespread attention. Due to the lack of a model organism with a stable response to electromagnetic waves, the research conclusions on the biological effects of electromagnetic waves have been vague. Therefore, the aim of this study was to investigate the effects of irradiation by pulsed 9.4 GHz high-power microwaves with a peak power density of 2126 W/cm2 using Caenorhabditis elegans. The development, movement, egg production, ROS, and lifespan of C. elegans were detected at different times after irradiation with different repetitive frequencies of 10, 20, and 50 Hz for 30 min. The results indicated that no obvious changes in basic life indices were induced compared with the sham radiation group, but the survival rate of positive control was significantly decreased compared with other groups, which is of interest for microwave protection research based on C. elegans and provides data for updating safety standards with respect to pulsed high-peak power microwave. © 2021 Bioelectromagnetics Society.

Functional expression and purification of recombinant full-length human ATG7 protein with HIV-1 Tat peptide in Escherichia coli.

The human autophagy-related protein ATG7 (hATG7), an E1-like ubiquitin enzyme, activates two ubiquitin-like proteins, LC3 (Atg8) and Atg12, and promotes autophagosome formation. While hATG7 plays an essential role for the autophagy conjugation system, the production of full-length functional hATG7 in bacterial systems remains challenging. Previous studies have demonstrated that the HIV-1 virus-encoded Tat peptide ('GRKKRRQRRR') can increase the yield and solubility of heterologous proteins. Here, functional full-length hATG7 was expressed using the pET28b-Tat expression vector in the Escherichia coli BL21 (DE3) strain. Recombinant hATG7 protein aggregated as inclusion bodies while expressed with widely used prokaryotic expression plasmids. In contrast, the solubility of Tat-tagged hATG7 increased significantly with prolonged time compared to Tat-free hATG7. The recombinant proteins were purified to >90% homogeneity under native conditions with a single step of affinity chromatography purification. The results of in vitro pull-down and LC3B-I lipidation assays showed that Tat-tagged hATG7 directly interacted with LC3B-I and promoted LC3B-I lipidation, suggesting that Tat-tagged hATG7 has significant catalytic activity. Overall, this study provides a novel method for improving the functional expression of full-length hATG7 in bacterial systems by fusion with the Tat peptide, a process which may be applied in future studies of hATG7 structure and function.

Effects of 1.5-GHz high-power microwave exposure on the reproductive systems of male mice.

High-power microwaves (HPMs) have been reported to have hazardous effects on multiple human and animal organs. However, the biological effects of 1.5-GHz HPMs on the reproductive system are not clear. Here, we studied the effects of 1.5 -GHz HPM whole-body exposure on the pathological structure of the testicles and changes in spermatozoa mobility. C57BL/6 mice of groups L, M, and H were exposed to 1.5-GHz HPM fields for two 15-min intervals at the average specific absorption rates of 3, 6, and 12 W/Kg, respectively. The pathological structure of the testicles and spermatozoa, as well as serum testosterone and sperm motility parameters, were evaluated at 6 h, 1 d, 3 d, and 7 d after exposure. As a result, there were no significant pathological or ultrastructural changes in the testicles or spermatozoa and serum testosterone levels. The number of progressively motile spermatozoa, curvilinear velocity, linear velocity, and average path velocity of the exposure group increased at 6 h, decreased at 1 d, and recovered at 3 d. The opposite results were considered a stress response to the thermal effect of the microwaves. Our results indicated that 1.5-GHz HPM whole-body exposure in mice at SARs of 3, 6, and 12 W/Kg for 30 min did not cause obvious damage to the reproductive system.

Radiofrequency radiation at 2.856 GHz does not affect key cellular endpoints in neuron-like PC12 cells.

To investigate the potential cytotoxicity of radiofrequency (RF) radiation on central nervous system, rat pheochromocytoma (PC12) cells were exposed to 2.856 GHz RF radiation at a specific absorption rate (SAR) of 4 W/kg for 8 h a day for 2 days in 35 mm Petri dishes. During exposure, the real-time variation of the culture medium temperature was monitored in the first hour. Reactive oxygen species (ROS) production, intracellular Ca2+ concentration, and cell apoptosis rate were assessed immediately after exposure by flow cytometry. The results showed that the medium temperature raised about 0.93 °C, but no significant changes were observed in apoptosis, ROS levels or intracellular Ca2+ concentration after treatment. Although several studies suggested that RF radiation does indeed cause neurological effects, this study presented inconsistent results, indicating that 2.856 GHz RF radiation exposure at a SAR of 4 W/kg does not have a dramatic impact on PC12 cells, and suggests the need for further investigation on the key cellular endpoints of other nerve cells after exposure to RF radiation.

Estrogen receptor α enhances the transcriptional activity of ETS-1 and promotes the proliferation, migration and invasion of neuroblastoma cell in a ligand dependent manner.

BACKGROUND: It is well known that estrogen receptor α (ERα) participates in the pathogenic progress of breast cancer, hepatocellular carcinoma and head and neck squamous cell carcinoma. In neuroblastoma cells and related cancer clinical specimens, moreover, the ectopic expression of ERα has been identified. However, the detailed function of ERα in the proliferation of neuroblastoma cell is yet unclear. METHODS: The transcriptional activity of ETS-1 (E26 transformation specific sequence 1) was measured by luciferase analysis. Western blot assays and Real-time RT-PCR were used to examine the expression of ERα, ETS-1 and its targeted genes. The protein-protein interaction between ERα and ETS-1 was determined by co-IP and GST-Pull down assays. The accumulation of ETS-1 in nuclear was detected by western blot assays, and the recruitment of ETS-1 to its targeted gene's promoter was tested by ChIP assays. Moreover, SH-SY5Y cells' proliferation, anchor-independent growth, migration and invasion were quantified using the MTT, soft agar or Trans-well assay, respectively. RESULTS: The transcriptional activity of ETS-1 was significantly increased following estrogen treatment, and this effect was related to ligand-mediated activation of ERα. The interaction between the ERα and ETS-1 was identified, and enhancement of ERα activation would up-regulate the ETS-1 transcription factor activity via modulating its cytoplasm/nucleus translocation and the recruitment of ETS-1 to its target gene's promoter. Furthermore, treatment of estrogen increased proliferation, migration and invasion of neuroblastoma cells, whereas the antagonist of ERα reduced those effects. CONCLUSIONS: In this study, we provided evidences that activation of ERα promoted neuroblastoma cells proliferation and up-regulated the transcriptional activity of ETS-1. By investigating the role of ERα in the ETS-1 activity regulation, we demonstrated that ERα may be a novel ETS-1 co-activator and thus a potential therapeutic target in human neuroblastoma treatment.

Calibration ruler for CW-EPR distance measurement using diradical molecule of rigid structure.

Many experimental factors and uncontrollable factors may introduce errors in the distance measurement by continuous wave electron paramagnetic resonance. To deal with this problem, several C60 nitroxide diradical adducts with rigid structure and definite molecular dimension were used as distance calibration rulers. Based on the improvement of distance calculation program via adding simulation programs of experimental spectra and dipolar broadening function, respectively, the distance calibration method was developed under different conditions such as different solvent, solution concentration, measuring temperature, and microwave power. As a result, stable distance calibration rulers were established within the range of 8-13 Å. The distance calibration effect was evaluated resulting in a corresponding distance measurement precision of 0.84 Å. The results suggested that the influence of non-dipolar spectral broadening factors could be overcome, and the established experimental and calculation methods were suitable to a wide range of situations. The developed method will ensure more accurate and objective distance measurement in biomacromolecular analysis.

MiR-122 Induces Radiosensitization in Non-Small Cell Lung Cancer Cell Line.

MiR-122 is a novel tumor suppresser and its expression induces cell cycle arrest, or apoptosis, and inhibits cell proliferation in multiple cancer cells, including non-small cell lung cancer (NSCLC) cells. Radioresistance of cancer cell leads to the major drawback of radiotherapy for NSCLC and the induction of radiosensitization could be a useful strategy to fix this problem. The present work investigates the function of miR-122 in inducing radiosensitization in A549 cell, a type of NSCLC cells. MiR-122 induces the radiosensitization of A549 cells. MiR-122 also boosts the inhibitory activity of ionizing radiation (IR) on cancer cell anchor-independent growth and invasion. Moreover, miR-122 reduced the expression of its targeted genes related to tumor-survival or cellular stress response. These results indicate that miR-122 would be a novel strategy for NSCLC radiation-therapy.

Emergence of FLASH­radiotherapy across the last 50 years (Review).

A novel radiotherapy (RT) approach termed FLASH-RT, which irradiates areas at ultra-high dose rates, is of current interest to medical researchers. FLASH-RT can maintain equivalent antitumor effects while sparing healthy tissue compared with conventional RT (CONV-RT), which uses low dose rates. The sparing effect on healthy tissue after FLASH-RT is known as the FLASH effect. Owing to the FLASH effect, FLASH-RT can raise the maximum tolerable dose to control tumor growth or eradicate the tumor and provide a new strategy for clinical RT. However, definitive irradiation conditions for reproducing the FLASH effect and the biological mechanism of the FLASH effect have not yet been fully elucidated. The efficacy of FLASH-RT is controversial despite its successful application in clinical RT. The present review recapitulates the progression of FLASH-RT and critically comments on the hypothesis of the FLASH effect. In addition, the review expounds on the current issues with regard to the differential phenomena between in vitro and in vivo studies, and elaborates on the challenges for the application of FLASH-RT that need to be addressed in the future.

A reliable and quick method for screening alternative splicing variants for low-abundance genes.

Alternative splicing (AS) is a universal phenomenon in eukaryotes, and it is still challenging to identify AS events. Several methods have been developed to identify AS events, such as expressed sequence tags (EST), microarrays and RNA-seq. However, EST has limitations in identifying low-abundance genes, while microarray and RNA-seq are high-throughput technologies, and PCR-based technology is needed for validation. To overcome the limitations of EST and shortcomings of high-throughput technologies, we established a method to identify AS events, especially for low-abundance genes, by reverse transcription (RT) PCR with gene-specific primers (GSPs) followed by nested PCR. This process includes two major steps: 1) the use of GSPs to amplify as long as the specific gene segment and 2) multiple rounds of nested PCR to screen the AS and confirm the unknown splicing variants. With this method, we successfully identified three new splicing variants, namely, GenBank Accession No. HM623886 for the bdnf gene (GenBank GeneID: 12064), GenBank Accession No. JF417977 for the trkc gene (GenBank GeneID: 18213) and GenBank Accession No. HM623888 for the glb-18 gene (GenBank GeneID: 172485). In addition to its reliability and simplicity, the method is also cost-effective and labor-intensive. In conclusion, we developed an RT-nested PCR method using gene-specific primers to efficiently identify known and novel AS variants. This approach overcomes the limitations of existing methods for detecting rare transcripts. By enabling the discovery of new isoforms, especially for low-abundance genes, this technique can aid research into aberrant splicing in disease. Future studies can apply this method to uncover AS variants involved in cancer, neurodegeneration, and other splicing-related disorders.

A broadband multi-frequency microwave combined biological exposure setup.

With the rapid popularization of wireless electronic devices, there has been an increasing concern about the impacts of the electromagnetic environment on health. However, most research reports on the biological effects of microwaves have focused on a single frequency point. In reality, people are exposed to complex electromagnetic environments that consist of multiple frequency microwave signals in their daily lives. It is important to investigate whether multi-frequency combined microwave energies have different biological effects compared with single frequency microwave energy. Unfortunately, there are limited reports on this topic due to the lack of suitable platforms for research on multi-frequency microwave energy combined with biological exposure. To address this issue, this study presents a setup that has a very wide working frequency bandwidth and can be compatible with single frequency and multi-frequency microwave combined exposure. Moreover, it can achieve relatively equal exposure to multiple biological samples at any frequency point in the working frequency range, which is crucial for electromagnetic biology research. The experimental results are in good agreement with the simulation results, confirming its capability to facilitate the study of complex electromagnetic environment effects on organisms.

Biological effects of exposure to 2650 MHz electromagnetic radiation on the behavior, learning, and memory of mice.

BACKGROUND: With the development of communication technology, the public is paying increasing attention to whether electromagnetic radiation is harmful to health. Mobile phone communication has entered the 5G era, and there are almost no reports on electromagnetic radiation at 2650 MHz. Therefore, it is necessary to evaluate the risk of adverse effects of 5G mobile phone EMR exposure on the human brain. METHODS: Male animals were continuously exposed to 2650 MHz-EMR for 28 days with a whole-body averaged specific absorption rate (WBSAR) of 2.06 W/kg for 4 h per day. Mouse behavior was assessed using the open-field test (OFT), elevated-plus maze (EPM), and tail suspension test (TST). The Morris water maze (MWM), HE staining, and TUNEL staining were used to evaluate the spatial memory ability and pathological morphology of hippocampal dentate gyrus cells. Additionally, the expression levels of brain-derived neurotrophic factor (BDNF), aminobutyric acid (GABA), and glucocorticoid (GR) in the hippocampus were detected by western blotting and immunohistochemistry, while the corticosterone (CORT) level in serum was detected by ELISA. RESULTS: In the OFT, the total distance traveled, central distance traveled, and residence time significantly decreased in the EMR exposure group (p < .05). In EPM, the percentage of the number of times to open the arm and the percentage of time to open the arm significantly decreased in the EMR exposure group. However, in the TST, the two groups had no significant difference in the 4-min immobility time. In the MWM, the escape latency of the EMR exposure group was shorter than that of the control group, with no significant difference. Furthermore, CORT levels in serum were significantly increased in the EMR exposure group (p < .05), while the expression of BDNF and GR proteins in the hippocampus was reduced (p < .05), but there was no significant difference in GABA expression. CONCLUSIONS: Our results indicate that exposure to 2650 MHz-EMR (WBSAR: 2.06 W/kg, 28 days, 4 h per day) had no significant effect on the spatial memory ability of mice (in comparison to little effect). The exposure may be associated with anxiety-like behavior in mice but not related to depression-like behavior in mice.

X-band TE101 rectangular aperture cavity for in vivo EPR tooth dosimetry after radiation emergency.

The TE101 mode rectangle EPR cavity was newly developed to achieve X-band in vivo EPR tooth dosimetry for the rescue of nuclear emergency. An aperture for sample detection was opened on the cavity's surface. Its characteristics were evaluated by measuring DPPH and intact human incisor samples. Remarkable radiation induced signal from EPR spectrum of 1Gy-8Gy irradiated teeth was observed. In vivo measurements of rat was performed to verify its application for in vivo tooth dosimetry.

The design of X-band EPR cavity with narrow detection aperture for in vivo fingernail dosimetry after accidental exposure to ionizing radiation.

For the purpose of assessing the radiation dose of the victims involved in the nuclear emergency or radiation accident, a new type of X-band EPR resonant cavity for in vivo fingernail EPR dosimetry was designed and a homemade EPR spectrometer for in vivo fingernail detection was constructed. The microwave resonant mode of the cavity was rectangular TE101, and there was a narrow aperture for fingernail detection opened on the cavity's wall at the position of high detection sensitivity. The DPPH dot sample and the fingernail samples were measured based on the in vivo fingernail EPR spectrometer. The measurements of the DPPH dot sample verified the preliminary functional applicable of the EPR spectrometer and illustrated the microwave power and modulation response features. The fingernails after irradiation by gamma-ray were measured and the radiation-induced signal was acquired. The results indicated that the cavity and the in vivo EPR dosimeter instrument was able to detect the radiation-induced signal in irradiated fingernail, and preliminarily verified the basic function of the instrument and its potential for emergency dose estimate after a radiation accident.

A normalization method of the volume and geometry of tooth for X-band in vivo EPR dosimetry.

The accuracy of in vivo EPR tooth dosimetry may be influenced by the volume and geometry variations in teeth, especially when there is considerable non-uniform sensitivity distribution in the active detection area of the cavity. To solve this problem, the present research proposed a normalization method specifically for X-band EPR in vivo tooth dosimetry. The volume and geometry of the measured tooth were reconstructed by digital image processing with images of the tooth impression slices, which were obtained by a custom-made impression module. The sensitivity distribution in the active detection area was established based on experiments with a point sample. Consequently, a composite normalization process that could calibrate the evaluated dose effectively was carried out by taking into account the influences not only from tooth volume and geometry but also from the non-uniform distribution of sensitivity. The effect and practicability of the method were evaluated by incisor samples. Results showed that the standard deviation could be reduced a maximum of 54.8% approximately after the composite normalization, an improvement compared to results from solely tooth volume. The correlation coefficient of the dose-response curve could be improved from 0.731 to 0.986. The preliminary method provides an approach potentially useful on site after radiation accidents when dealing with the influence of variations in the tooth volume and geometry for X-band EPR in vivo dose estimations.

Effect of the tooth surface water on the accuracy of dose reconstructions in the X-band in vivo EPR dosimetry.

The X-band in vivo EPR tooth dosimetry is promising as a tool for the initial triage after a large-scale radiation accident. The dielectric losses caused by water on the tooth surface (WTS) are one of the major sources of inaccuracies in this method. The effect was studied by theoretical simulation calculations and experiments with water films of various thicknesses on teeth. The results demonstrate the possibility of sufficiently accurate measurements of the radiation-induced signal of the tooth enamel provided that the thickness of the water film on the tooth is below 60 µm. The sensitivity of the cavity decreases with increasing thickness of the water layer. The interference of WTS can be diminished by normalization of the radiation-induced signal to the signal of a reference sample permanently present in the cavity.

Study of new practical ESR dosimeter based on carbonated hydroxyapatite and its dosimetric properties.

The development of new dosimeters with good dosimetric properties is important for quality control in radiation applications. A new practical electron spin resonance (ESR) dosimeter based on carbonated hydroxyapatite that simulated the composition and structure of tooth enamel was specially synthesized. The synthesized material was investigated by transmission electron microscope, X-ray diffraction, fourier transform infrared spectroscopy and X-ray photo electron spectroscopy to confirm to the main composition of carbonated hydroxyapatite with CO32- successfully doped into the crystal lattice through optimizing the synthesis process of C/P molar ratio, pH value dynamical adjustment, annealing temperature and time. The dosimetric properties were systematically investigated by ESR spectroscopy. The results indicated that the radiation induced signal had a good dose response within a relatively wide dose range. The dose response was linear in the dose range of 0-400 Gy with a correlation coefficient of 0.9999 and had dose accumulative effect in the experimental dose range of 0-100 Gy. In a wider dose range up to 30 kGy, the dose response also presented linear feature in double-logarithmic coordinate system with a correlation coefficient of 0.9970. The dose detection limit was about 0.34Gy with a given probability of 95% confidence level depending upon a rigid calculation algorithm. The signal was extremely stable in the observation time of 360 days with a variation coefficient of 3.8%. The radiation sensitivity of the material showed no remarkable variation against photon energy from 662 KeV to 1.25 MeV and dose rate from 0.86 Gy/min to 12.17 Gy/min. The material showed more sensitive in lower photon energy range below 662 keV, which hint additional calibration may need when using in special photon energy condition. The preliminary results suggested that this newly developed dosimeter was potential to become a practical dosimeter that would expand the application fields of ESR dosimetry.

The Application and Distribution of Magnetic Field Modulation in the Detection Apertures of X-band EPR Cavities for In Vivo Tooth Dosimetry.

In vivo electron paramagnetic resonance tooth dosimetry could be a practical and ideal tool for quick mass triage of victims in the rescue following a disaster event involving irradiation radiation. Magnetic field modulation is an important issue to improve the sensitivity of X-band in vivo tooth dosimetry. We designed a couple of trapezoidal modulation coil sets fixed on the magnet poles that could be used to apply sufficient magnet field modulation into the detection aperture of the resonant cavity. Measurements of irradiated teeth with such coil sets demonstrated significant radiation-induced signals. The modulation generation efficiencies and magnetic field distributions in apertures with different cavity geometries were analytically calculated, simulated by a finite element method and evaluated by measurements of a free radical point sample to study the influences caused by the geometries of the apertures and other factors.

Effects of pulsed 2.856 GHz microwave exposure on BM-MSCs isolated from C57BL/6 mice.

The increasing use of microwave devices over recent years has meant the bioeffects of microwave exposure have been widely investigated and reported. However the exact biological fate of bone marrow MSCs (BM-MSCs) after microwave radiation remains unknown. In this study, the potential cytotoxicity on MSC proliferation, apoptosis, cell cycle, and in vitro differentiation were assayed following 2.856 GHz microwave exposure at a specific absorption rate (SAR) of 4 W/kg. Importantly, our findings indicated no significant changes in cell viability, cell division and apoptosis after microwave treatment. Furthermore, we detected no significant effects on the differentiation ability of these cells in vitro, with the exception of reduction in mRNA expression levels of osteopontin (OPN) and osteocalcin (OCN). These findings suggest that microwave treatment at a SAR of 4 W/kg has undefined adverse effects on BM-MSCs. However, the reduced-expression of proteins related to osteogenic differentiation suggests that microwave can the influence at the mRNA expression genetic level.

Tat peptide-mediated soluble expression of the membrane protein LSECtin-CRD in Escherichia coli.

The human liver and lymph node sinusoidal endothelial cell C-type lectin (hLSECtin), a type II integral membrane protein, containing a Ca(2+)-dependent carbohydrate recognition domain (CRD), has a well-established biological activity, yet its three-dimensional structure is unknown due to low expression yields and aggregation into inclusion bodies. Previous study has demonstrated that the HIV-1 virus-encoded Tat peptide ('YGRKKRRQRRR') can increase the yields and the solubility of heterologous proteins. However, whether the Tat peptide could promote the high-yield and soluble expression of membrane proteins in Escherichia coli is not known. Therefore, the prokaryotic expression vector pET28b-Tat-hLSECtin-CRD (using pET28b and pET28b-hLSECtin-CRD as controls) was constructed, and transformed into E. coli BL21 (DE3) cells and induced with isopropyl-ß-d-thiogalactoside (IPTG) followed with identifying by SDS-PAGE and Western blot. Subsequently, the bacterial subcellular structure, in which overexpressed the heterologous proteins Tat-hLSECtin-CRD and Tat-free hLSECtin-CRD, was analyzed by transmission electron microscope (TEM) respectively, and the mannose-binding activity of Tat-hLSECtin-CRD was also determined. Expectedly, the solubility of Tat-LSECtin-CRD significantly increased compared to Tat-free LSECtin-CRD (**p < 0.01) with prolonged time, and the Tat-LSECtin-CRD had a significant mannose-binding activity. The subcellular structure analysis indicated that the bacterial cells overexpressed Tat-hLSECtin-CRD exhibited denser region compared with controls, while dot denser region aggregated in the two ends of bacterial cells overexpressed Tat-free hLSECtin-CRD. This study provided a novel method for improving the soluble expression of membrane proteins in prokaryotic systems by fusion with the Tat peptide, which may be potentially expanded to the expression of other membrane proteins.