Construction of [89Zr]Zr-Labeled HuL13 for ImmunoPET Imaging of LAG-3 Checkpoint Expression on Tumor-Infiltrating T Cells.

Lymphocyte activation gene 3 (LAG-3) has attracted much attention as a potentially valuable immune checkpoint. Individual identification of LAG-3 expression at screening and during treatment could improve the successful implementation of anti-LAG-3 therapies. HuL13 is a human IgG1 monoclonal antibody that binds to the LAG-3 receptor in T cells. Here, we used [89Zr]Zr-labeled HuL13 to delineate LAG-3+ T-cell infiltration into tumors via positron emission tomography (PET) imaging. A549/LAG-3 cells, which stably express LAG-3, were generated by infection with lentivirus. The uptake of [89Zr]Zr-DFO-HuL13 in A549/LAG-3 cells was greater than that in the negative control (A549/NC) cells at each time point. The equilibrium dissociation constant (Kd) of [89Zr]Zr-DFO-HuL13 for the LAG-3 receptor was 8.22 nM. PET imaging revealed significant uptake in the tumor areas of A549/LAG-3 tumor-bearing mice from 24 h after injection (SUVmax = 2.43 ± 0.06 at 24 h). As a proof of concept, PET imaging of the [89Zr]Zr-DFO-HuL13 tracer was further investigated in an MC38 tumor-bearing humanized LAG-3 mouse model. PET imaging revealed that the [89Zr]Zr-DFO-HuL13 tracer specifically targets human LAG-3 expressed on tumor-infiltrating lymphocytes (TILs). In addition to the tumors, the spleen was also noticeably visible. Tumor uptake of the [89Zr]Zr-DFO-HuL13 tracer was lower than its uptake in the spleen, but high uptake in the spleen could be reduced by coinjection of unlabeled antibodies. Coinjection of unlabeled antibodies increases tracer activity in the blood pool, thereby improving tumor uptake. Dosimetry evaluation of the healthy mouse models revealed that the highest absorbed radiation dose was in the spleen, followed by the liver and heart wall. In summary, these studies demonstrate the feasibility of using the [89Zr]Zr-DFO-HuL13 tracer for the detection of LAG-3 expression on TILs. Further clinical evaluation of the [89Zr]Zr-DFO-HuL13 tracer may be of significant help in the stratification and management of patients suitable for anti-LAG-3 therapy.

Strontium Uptake and Effect in Lettuce and Radish Cultivated Under Hydroponic Conditions.

The accumulation of strontium (Sr) in lettuce and radish under 0 (control), 0.5, 1, 2.5, 5, and 10 mM Sr treatments in hydroponic solution at 16, 23 and 30 days and the effects of Sr stress on six nutrient elements in plants were investigated. The results showed that Sr concentrations in plant aerial and underground parts increased in low-Sr treatments (0.5, 1 and 2.5 mM) and fluctuated in high-Sr treatments (5 and 10 mM) throughout the three sampling periods. Sr concentrations were higher in roots than in leaves, reaching 108.8 ± 14.7 and 134.1 ± 1.2 mg/g in lettuce and radish roots, respectively, after 10 mM Sr treatment. Translocation factor (TF) values (ratio of the Sr concentrations in aerial parts to that in roots) were inversely related to the Sr content in the hydroponic solution, and reached 1.45 ± 0.17 to 0.15 ± 0.03 and 1.06 ± 0.20 to 0.12 ± 0.004 for lettuce and radish. The variation in chlorophyll content was consistent with that in plant biomass.

IER5 is involved in DNA Double-Strand Breaks Repair in Association with PAPR1 in Hela Cells.

The immediate early response gene 5 (IER5) is a radiation response gene induced in a dose-independent manner, and has been suggested to be a molecular biomarker for biodosimetry purposes upon radiation exposure. Here, we investigated the function of IER5 in DNA damage response and repair. We found that interference on IER5 expression significantly decreased the efficiency of repair of DNA double-strand breaks induced by ionizing radiations in Hela cells. We found that IER5 participates in the non-homologous end-joining pathway of DNA breaks repair. Additionally, we identified a number of potential IER5-interacting proteins through mass spectrometry-based protein assays. The interaction of IER5 protein with poly(ADP-Ribose) polymerase 1 (PARP1) and Ku70 was further confirmed by immunoprecipitation assays. We also found that Olaparib, a PARP1 inhibitor, affected the stability of IER5. These results indicate that targeting of IER5 may be a novel DNA damage response-related strategy to use during cervical cancer radiotherapy or chemotherapy.

Acute hematologic toxicity prediction using dosimetric and radiomics features in patients with cervical cancer: does the treatment regimen matter?

Background: Acute hematologic toxicity (HT) is a prevalent adverse tissue reaction observed in cervical cancer patients undergoing chemoradiotherapy (CRT), which may lead to various negative effects such as compromised therapeutic efficacy and prolonged treatment duration. Accurate prediction of HT occurrence prior to CRT remains challenging. Methods: A discovery dataset comprising 478 continuous cervical cancer patients (140 HT patients) and a validation dataset consisting of 205 patients (52 HT patients) were retrospectively enrolled. Both datasets were categorized into the CRT group and radiotherapy (RT)-alone group based on the treatment regimen, i.e., whether chemotherapy was administered within the focused RT duration. Radiomics features were derived by contouring three regions of interest (ROIs)-bone marrow (BM), femoral head (FH), and clinical target volume (CTV)-on the treatment planning CT images before RT. A comprehensive model combining the radiomics features as well as the demographic, clinical, and dosimetric features was constructed to classify patients exhibiting acute HT symptoms in the CRT group, RT group, and combination group. Furthermore, the time-to-event analysis of the discriminative ROI was performed on all patients with acute HT to understand the HT temporal progression. Results: Among three ROIs, BM exhibited the best performance in classifying acute HT, which was verified across all patient groups in both discovery and validation datasets. Among different patient groups in the discovery dataset, acute HT was more precisely predicted in the CRT group [area under the curve (AUC) = 0.779, 95% CI: 0.657-0.874] than that in the RT-alone (AUC = 0.686, 95% CI: 0.529-0.817) or combination group (AUC = 0.748, 95% CI: 0.655-0.827). The predictive results in the validation dataset similarly coincided with those in the discovery dataset: CRT group (AUC = 0.802, 95% CI: 0.669-0.914), RT-alone group (AUC = 0.737, 95% CI: 0.612-0.862), and combination group (AUC = 0.793, 95% CI: 0.713-0.874). In addition, distinct feature sets were adopted for different patient groups. Moreover, the predicted HT risk of BM was also indicative of the HT temporal progression. Conclusions: HT prediction in cervical patients is dependent on both the treatment regimen and ROI selection, and BM is closely related to the occurrence and progression of HT, especially for CRT patients.

Analysis and Monitoring of Indoor Radon Concentrations of 37 Kindergartens - Beijing Municipality, China, 2023.

Introduction: Radon (222Rn or 222radon) is a radioactive gas emitted from building materials, foundations, and soil. Children are especially susceptible to radon exposure, underscoring the need to assess indoor radon levels in kindergartens. This study monitored radon concentrations in 37 Beijing kindergartens from June to October 2023. Methods: A random sample of 37 kindergartens was selected from 18 administrative districts in Beijing. The indoor radon concentration was measured using the solid track accumulation method, with radon detectors continuously monitored over a 3-month period. Results: The mean indoor radon level in 37 kindergartens, observed at 252 monitoring points, was 84.3 Bq/m3, with values varying from 12.9 to 263.5 Bq/m3. About 20.2% of points showed radon levels between 100.0 and 200.0 Bq/m3, while 2.4% exceeded 200.0 Bq/m3. Notably, radon levels were significantly elevated on the ground floor compared to the upper floors. Conclusion: Indoor radon levels in 37 kindergartens remained below the national standard limit of 300.0 Bq/m3 for buildings (GB/T 16146-2015). Nonetheless, 18.9% of the kindergartens exceeded the 100.0 Bq/m3 limit set for new constructions. It is advised to improve radon monitoring in kindergartens and consider developing a national standard for maximum permissible radon levels in such facilities.

Establishment and Application of a Radiation Dose Rate Model for Nuclear Medicine Examinees.

Introduction: Traditional methods for determining radiation dose in nuclear medicine include the Monte Carlo method, the discrete ordinate method, and the point kernel integration method. This study presents a new mathematical model for predicting the radiation dose rate in the vicinity of nuclear medicine patients. Methods: A new algorithm was created by combining the physical model of "cylinder superposition" of the human body with integral analysis to assess the radiation dose rate in the vicinity of nuclear medicine patients. Results: The model accurately predicted radiation dose rates within distances of 0.1-3.0 m, with a deviation of less than 11% compared to observed rates. The model demonstrated greater accuracy at shorter distances from the radiation source, with a deviation of only 1.55% from observed values at 0.1 m. Discussion: The model proposed in this study effectively represents the spatial and temporal distribution of the radiation field around nuclear medicine patients and demonstrates good agreement with actual measurements. This model has the potential to serve as a radiation dose rate alert system in hospital environments.

Curcumin-induced apoptosis in human hepatocellular carcinoma j5 cells: critical role of ca(+2)-dependent pathway.

The antitumor effects of curcumin, a natural biologically active compound extracted from rhizomes of Curcuma longa, have been studied in many cancer cell types including human hepatocellular carcinoma (HCC). Here, we investigated the effects of Ca(2+) on curcumin-induced apoptosis in human HCC J5 cells. The abrogation of mitochondrial membrane potential (ΔΨ(m)), the increase of reactive oxygen species (ROS) production, and calcium release were demonstrated with flow cytometry as early as 15 minutes after curcumin treatment. In addition, an increase level of cytochrome c in the cytoplasm which led to DNA fragmentation was observed. To verify the role of Ca(2+) in curcumin-induced apoptosis, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an intracellular calcium chelator, was applied. Cell viability was increased, but ΔΨ(m), ROS production, activation of caspase 3, and cell death were decreased in J5 cells pretreated with BAPTA for 2 h followed by the treatment of 25 µM curcumin. These results suggest that the curcumin-induced apoptosis in human HCC J5 cells is via mitochondria-dependent pathway and is closely related to the level of intracellular accumulation of calcium.

Effect of strontium on nutrient uptake, physiological parameters, and strontium localization in lettuce.

Human activities increase the risk of stable and radioactive strontium (Sr) isotopes entering the environment and food chain. In this study, the effects of Sr on the nutrient uptake and physiological responses of lettuce under different "Sr treatment" concentrations (0, control, 1, 2, 3, 4, and 5 mM) and "times" (7, 14, and 21 day) were studied in a hydroponic system. In addition, the distribution of Sr on the surfaces and cross-sections of lettuce leaves was revealed by scanning electron microscopy with energy-dispersive X-ray (SEM-EDX) analysis. A two-way analysis of variance (ANOVA) method was used to analyze the significance of "Sr treatment," "time," and their "interaction." The results showed that an increase in Sr uptake in lettuce could significantly reduce the uptake of calcium (Ca). The contents of sulfur (S), potassium (K), and iron (Fe) in lettuce leaves showed significant differences with the sampling day. Similarly, the fresh weight of lettuce leaves and roots as well as the photosynthetic pigment contents of lettuce leaves was also significantly different with the sampling day. The activities of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD)) showed significant differences with the sampling day. The activities of SOD and CAT decreased significantly with the sampling day, while POD increased significantly. The MDA content increased significantly with increasing hydroponic Sr concentration on the 21st day. SEM-EDX analysis showed that the weight percentage of Sr in the vascular bundle sheath in the cross-section of lettuce leaves was relatively higher than that in the mesophyll. This study aids our understanding of the distribution of Sr in lettuce leaf tissues and the effect of Sr on lettuce physiology.

Cdc25B is transcriptionally inhibited by IER5 through the NF-YB transcription factor in irradiation-treated HeLa cells.

Cervical cancer (CC) is a type of pelvic malignant tumor that severely threatens women's health. Current evidence suggests that IER5, as a potential radiosensitizer, promotes irradiation-induced apoptosis in CC tissues in patients undergoing chemoradiotherapy. IER5 has been shown to be involved in the G2/M-phase transition. In the present study, we used Cdc25B as the breakthrough point to explore the underlying mechanism of IER5 in the cell cycle regulation of radiation-damaged HeLa cells. IER5 was evidently upregulated after irradiation, but Cdc25B was significantly downregulated. In monoclonal IER5-silenced HeLa cells, irradiation-induced downregulation of Cdc25B was attenuated. The effect of irradiation on Cdc25B promoter activity was determined by dual-luciferase reporter assays. The response elements on the Cdc25B promoter related to irradiation were predicted by JASPAR. These conserved sequences were mutated individually or in combination by splicing-by-overlap extension PCR, and their function was confirmed by dual-luciferase reporter assays. The enrichment efficiency of transcription factors after irradiation was determined by chromatin immunoprecipitation (ChIP) assay. Both Sp1/Sp3 and NF-YB binding sites were involved in irradiation-mediated regulation of Cdc25B. IER5 was involved in irradiation-mediated regulation of Cdc25B through the NF-YB binding site. Furthermore, ChIP assays showed that IER5 bound to the Cdc25B promoter, and the binding of IER5 to the Cdc25B promoter region in irradiation-induced HeLa cells induced the release of the coactivator p300 through interaction with NF-YB. Taken together, these findings indicate that IER5 is the transcriptional repressor that accelerates the downregulation of Cdc25B expression after irradiation.

Gamma-ray up-regulated holocarboxylase synthetase gene.

PURPOSE: To investigate the effects of holocarboxylase synthetase (HCS) gene to irradiation in a time- and dose-dependent manner. MATERIALS AND METHODS: AHH-1 cells, Hela cells, and the nude mice inoculated with tumor cells were exposed to gamma-ray of cobalt 60. The mRNA level of HCS was detected using real-time PCR. The profiles of mRNA of HCS after radiation were analyzed and described. RESULTS: The expression of HCS gene was higher in AHH-1 cells than that in Hela cells. Furthermore, both AHH-1 and Hela displayed similar time-dependent transcriptional levels of HCS gene to radiations at the dose of 2 and 10 Gy. We set the parameters of D, V, R, F, N to quantitatively analyze HCS gene regulation in response to irradiation. We also observed that irradiation resulted in higher levels of HCS in human hepatocyte xenografts, compared with three other types of human tumor xenografts. 2 and 4 Gy radiation had little influence on the HCS gene of human lung cancer and brain cancer, mammary gland cancer was more sensitive to the 4 Gy gamma-ray dose compared with the 2 Gy gamma-ray dose. CONCLUSION: HCS is a good radiation-responsive gene. It may be used as a candidate for developing novel biomarkers of radiation damage and it has a great potential to be used in radiation-therapy of liver tumors.

Prediction of IER5 structure and function using a bioinformatics approach.

Immediate­early response gene 5 (IER5) is a gene involved in the regulation of the cell cycle, and its structure and function have been investigated by bioinformatics analyses. The present study determined the sites of promoter methylation and gene ontology (GO) annotations associated with IER5. In addition, we conducted a prediction analysis to determine the physical and chemical properties, hydrophobicity/hydrophilicity, posttranslational modification, subcellular localization, transmembrane structure, signal peptide and secondary and tertiary structures of IER5. One CpG island and several methylated sites were identified close to the promoter of IER5. The GO analysis suggested that IER5 could bind ions and proteins that were mainly associated with metabolic processes. IER5 comprised 327 amino acids and was reported to be an unstable hydrophilic protein with an isoelectric point of 4.91. A total of 18 O­glycosylation sites and 22 phosphorylation sites were identified within this protein. The subcellular localization of IER5 was mainly in the nucleus, and its main secondary structural element was the α­helix. Bioinformatic analyses of the features of IER5 may improve understanding of its structure and function; however, experimental verification is required.

Overexpression of the immediate early response 5 gene increases the radiosensitivity of HeLa cells.

The effects of the immediate early response 5 (IER5) gene on the sensitivity of HeLa cells to radiation remain unclear. In the present study, stably transfected HeLa cells resulting in the knockdown or overexpression of IER5 were investigated. In addition, xenografts of normal, IER5-silenced and -overexpressed HeLa cells were injected into nude mice and examined. The results demonstrated that the radiosensitivity of the IER5-overexpressed HeLa cells was significantly increased compared with that of the normal and IER5-silenced cells. The upregulation of IER5 effectively decreased cell proliferation and IER5 silencing promoted cell proliferation compared with that in the normal HeLa cells. Following irradiation of the cells with IER5 knockdown, cell cycle was arrested at the G2/M phase and an increase in the proportion of S phase cells was observed. By contrast, the overexpression of IER5 led to an increase in the proportion of G1 phase cells. Furthermore, the upregulation of IER5 inhibited tumor growth in vivo. The present findings demonstrate that the IER5 gene affects the radiosensitivity of HeLa cells and serves an important role in cell proliferation, suggesting that this gene may be a potential radiotherapeutic target in cervical cancer.

IER5 as a promising predictive marker promotes irradiation-induced apoptosis in cervical cancer tissues from patients undergoing chemoradiotherapy.

PURPOSE: To define the role of immediate-early 5 (IER5) gene as a promising biomarker in predicting the radiosensitivity and prognosis of cervical cancer patients receiving cisplatin-based concurrent chemoradiotherapy (DDP-CCRT). RESULTS: Our investigations found that IER5 level was markedly elevated in cervical cancer patients after being treated with irradiation, which indicated IER5 was closely dose induced. By contrast, the correlation between IER5 and radiosensitivity cannot be confirmed by the present study. The up-regulation of IER5 expression effectively increased cell apoptosis after administration of irradiation (P < 0.05). Using an ANOVA model for repeated-measures, we found significant association between the IER5 level and tumor size (P < 0.05). MATERIALS AND METHODS: Forty-three cervical cancer patients stage IIb-IIIb received DDP-CCRT were registered. Biopsy tissues were obtained after administration of irradiation dose of 0 Gy, 2~6 Gy, 10 Gy, 20 Gy, 30 Gy, respectively. The IER5 protein and mRNA levels were measured by immunohistochemistry, western blot and quantitative polymerase chain reaction, respectively; besides, the apoptosis rate was assessed by transferase-mediated dUTP nick end labeling. CONCLUSIONS: Mechanistically, we confirmed that IER5 induced by radiation dose enhanced apoptosis of cervical cancer, was inversely associated with tumor size. In conclusion, our studies indicate target IER5 is improved to be a potential radiosensitizer for developing effective therapeutic strategies against cervical cancer to radiotherapy and a predictive biomarker for radiosensitivity.

Radiation-induced expression of IER5 is dose-dependent and not associated with the clinical outcomes of radiotherapy in cervical cancer.

This study aimed to investigate the expression of the immediate-early response 5 (IER5) gene in cervical cancer tissues and explore the association between the expression of IER5 and the clinical outcomes of radiotherapy. We collected specimens by surgery or biopsy and obtained 53 specimens from tissues after radiotherapy and 16 specimens from tissues before radiotherapy. Immunohistochemistry and western blotting were used to assess the protein expression levels of IER5. Quantitative polymerase chain reaction (qPCR) was performed to assess the mRNA expression levels of IER5. The protein and mRNA expression levels of IER5 in cervical cancer patients treated with radiation doses ≥20 Gy were significantly higher than in those treated with radiation doses <20 Gy (P<0.05) and before treatment with radiotherapy. Moreover, the expression of IER5 was significantly positively correlated with the radiation dose (immunohistochemistry: r=0.548, P=0.019; qPCR: r=0.671, P=0.002; western blotting: r=0.573, P<0.0001). Radiotherapy induced the upregulated expression of IER5 and this was dependent on the radiation dose. However, the radiation-induced expression of IER5 was not associated with the clinical outcomes of radiotherapy in cervical cancer.

IER5 promotes irradiation- and cisplatin-induced apoptosis in human hepatocellular carcinoma cells.

PURPOSE: To elucidate the mechanisms of the immediate-early response gene 5 (IER5) effect on the apoptosis induced by irradiation and cisplatin (CDDP) in human hepatocellular carcinoma (HepG2) cells. METHODS: We generated IER5 overexpression stable cells (HepG2/IER5) using Lipofectamine 2000 transfection HepG2 cells. Cell apoptosis was induced by irradiation and cisplatin treatments, and cell proliferation (viability) and apoptosis were evaluated by MTT and flow cytometry assays. Protein expression was determined by Western blot. RESULTS: The growth of the IER5 overexpression cells was significantly inhibited after six days of (60)Co γ-irradiation exposure (p<0.01) compared with the cell growth of vector control cells. Furthermore, the HepG2/IER5 cells were arrested at the G2/M phases. We also found that the expression of phospho-Akt was reduced, and the levels of cleaved caspase-3 and PARP were increased after the treatment of HepG2/IER5 cells with γ-irradiation and cisplatin. CONCLUSION: Our results suggest that the overexpression of IER5 can inhibit cell growth and enhance the cell apoptosis induced by exposure to radiation or cisplatin. The overexpression of IER5 can be utilized as a targeting strategy to improve the outcomes of radiotherapy used for the treatment of patients with liver cancer.

Induced expression of the IER5 gene by gamma-ray irradiation and its involvement in cell cycle checkpoint control and survival.

The immediate-early response gene 5 (IER5) was previously shown, using microarray analysis, to be upregulated by ionizing radiation. Here we further characterized the dose- and time-dependency of radiation-induced expression of IER5 at doses from 0.5 to 15 Gy by quantitative real-time PCR analyses in HeLa cells and human lymphoblastoid AHH-1 cells. A radiation-induced increase in the IER5 mRNA level was evident 2 h after irradiation with 2 Gy in both cell lines. In AHH-1 cells the expression reached a peak at 4 h and then quickly returned to the control level, while in HeLa cells the expression only remained increased for a short period of time at around 2 h after irradiation before returning to the control. After high-dose irradiation (10 Gy), the induction of the IER5 expression was lower and delayed in AHH-1 cells as compared with 2-Gy irradiated cells. In HeLa cells, at this dose, two peaks of increased expression were observed 2 h and 12-24 h post-irradiation, respectively. RNA interference technology was employed to silence the IER5 gene in HeLa cells. siRNA-mediated suppression of IER5 resulted in an increased proliferation of HeLa cells. Cell growth and survival analyses demonstrated that suppression of IER5 significantly increased the radioresistance of HeLa cells to radiation doses of up to 6 Gy, but barely affected the sensitivity of cells at 8 Gy. Moreover, suppression of IER5 potentiated radiation-induced arrest at the G2-M transition and led to an increase in the fraction of S phase cells. Taken together, we propose that the early radiation-induced expression of IER5 affects the radiosensitivity via disturbing radiation-induced cell cycle checkpoints.