Iodine containing porous organosilica nanoparticles trigger tumor spheroids destruction upon monochromatic X-ray irradiation: DNA breaks and K-edge energy X-ray.

X-ray irradiation of high Z elements causes photoelectric effects that include the release of Auger electrons that can induce localized DNA breaks. We have previously established a tumor spheroid-based assay that used gadolinium containing mesoporous silica nanoparticles and synchrotron-generated monochromatic X-rays. In this work, we focused on iodine and synthesized iodine-containing porous organosilica (IPO) nanoparticles. IPO were loaded onto tumor spheroids and the spheroids were irradiated with 33.2 keV monochromatic X-ray. After incubation in CO2 incubator, destruction of tumor spheroids was observed which was accompanied by apoptosis induction, as determined by the TUNEL assay. By employing the γH2AX assay, we detected double strand DNA cleavages immediately after the irradiation. These results suggest that IPO first generate double strand DNA breaks upon X-ray irradiation followed by apoptosis induction of cancer cells. Use of three different monochromatic X-rays having energy levels of 33.0, 33.2 and 33.4 keV as well as X-rays with 0.1 keV energy intervals showed that the optimum effect of all three events (spheroid destruction, apoptosis induction and generation of double strand DNA breaks) occurred with a 33.2 keV monochromatic X-ray. These results uncover the preferential effect of K-edge energy X-ray for tumor spheroid destruction mediated by iodine containing nanoparticles.

Ionizing Radiation-Induced Extracellular Vesicle Release Promotes AKT-Associated Survival Response in SH-SY5Y Neuroblastoma Cells.

Radiation therapy is one of the most effective methods of tumor eradication; however, in some forms of neuroblastoma, radiation can increase the risk of secondary neoplasms, due to the ability of irradiated cells to transmit pro-survival signals to non-irradiated cells through vesicle secretion. The aims of this study were to characterize the vesicles released by the human neuroblastoma cell line SH-SY5Y following X-ray radiations and their ability to increase invasiveness in non-irradiated SH-SY5Y cells. We first purified the extracellular vesicles released by the SH-SY5Y cells following X-rays, and then determined their total amount, dimensions, membrane protein composition, and cellular uptake. We also examined the effects of these extracellular vesicles on viability, migration, and DNA damage in recipient SH-SY5Y cells. We found that exposure to X-rays increased the release of extracellular vesicles and altered their protein composition. These vesicles were readily uptaken by non-irradiated cells, inducing an increase in viability, migration, and radio-resistance. The same results were obtained in an MYCN-amplified SK-N-BE cell line. Our study demonstrates that vesicles released from irradiated neuroblastoma cells stimulate proliferation and invasiveness that correlate with the epithelial to mesenchymal transition in non-irradiated cells. Moreover, our results suggest that, at least in neuroblastomas, targeting the extracellular vesicles may represent a novel therapeutic approach to counteract the side effects associated with radiotherapy.

Dosimetry study on Auger electron-emitting nuclear medicine radioisotopes in micrometer and nanometer scales using Geant4-DNA simulation.

PURPOSE: Dosimetry of Auger electron-emitting nuclear medicine radioisotopes on cellular and DNA scales is essential in order to assess the biological effects and damage of these radioisotopes to the DNA molecule. This study examined the effects of widely used radioisotopes in nuclear medicine and also two therapeutic radioisotopes in the scale of micrometer and nanometer. METHODS: In this paper, on cell scale (micrometer scale), the S-values for widely used Auger electron-emitting diagnostic radioisotopes 123I, 125I, 99mTc, 67Ga, 201Tl and 111In and two therapeutic radioisotopes of 131I and 211At in three different geometrical cell models (spherical, elliptical and cubic) were calculated using Geant4-DNA and the results were compared with the results of other simulation codes as well as the MIRD technique. On DNA scale (nanometer scale), the average number of DNA strand breaks (SSB and DSB) resulting from the direct and indirect effects was calculated for the specified radioisotope. RESULTS: The results showed that in the cell scale, S-values of the diagnostic radioisotopes were mostly greater than the S-values of the therapeutic radioisotope 131I, but they were less those of 211At. On DNA scale, two different geometric models of DNA molecule were simulated and the results of these two models were compared with each other, as well as with the literature. The results showed that the geometric shape of sugar-phosphate groups has a significant effect on the break rates of DNA molecules. CONCLUSIONS: Among the widely used diagnostic radioisotopes, 201Tl and 125I had the greatest effect on the rate of SSBs and DSBs, respectively, while the 131I therapeutic radioisotope almost had no effect and therapeutic radioisotope of 211At had a moderate effect on the number of breaks.

Modeling the effect of ion-induced shock waves and DNA breakage with the reactive CHARMM force field.

Ion-induced DNA damage is an important effect underlying ion beam cancer therapy. This article introduces the methodology of modeling DNA damage induced by a shock wave caused by a projectile ion. Specifically it is demonstrated how single- and double strand breaks in a DNA molecule could be described by the reactive CHARMM (rCHARMM) force field implemented in the program MBN Explorer. The entire workflow of performing the shock wave simulations, including obtaining the crucial simulation parameters, is described in seven steps. Two exemplary analyses are provided for a case study simulation serving to: (a) quantify the shock wave propagation and (b) describe the dynamics of formation of DNA breaks. The article concludes by discussing the computational cost of the simulations and revealing the possible maximal computational time for different simulation set-ups.

DNA damage induced during mitosis undergoes DNA repair synthesis.

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.

The effect of hypoxia on the induction of strand breaks in plasmid DNA by alpha-, beta- and Auger electron-emitters 223Ra, 188Re, 99mTc and DNA-binding 99mTc-labeled pyrene.

INTRODUCTION: Radiation-induced DNA damage occurs from direct and indirect effects. The induction is influenced by the physical characteristics of the radionuclide, especially its linear energy transfer. Hypoxia reduces the effect of irradiation treatment in tumor cells and leads to poor patient outcomes. High linear energy transfer emitters can overcome this obstacle. Our aim is to demonstrate the influence of hypoxia on the interaction of different radiation qualities with isolated DNA. METHODS: PuC19 Plasmid DNA was irradiated with 223Ra, 188Re, 99mTc and 99mTc-labeled pyrene with and without DMSO under hypoxia or normoxic conditions. DNA damages in form of single-(SSB) and double-strand breaks (DSB) were analyzed by gel electrophoresis. RESULTS: Radiation doses up to 200 Gy of 223Ra, 188Re and 99mTc led to maximal yields of 80% SSB and 30%, 28% and 32% DSB, respectively. Hypoxia had minor effects on damages from 223Ra, but caused a small enhancement in DSB for 188Re and 99mTc. DMSO prevented DSB completely and reduced SSB from the "free" radionuclides to comparable levels. DNA-binding 99mTc-labeled pyrene induced less SSB and DSB compared to [99mTc]TcO4-. However, the incubation with DMSO could prevent the SSB and DSB induction only to a minor extent. CONCLUSIONS: Hypoxia does not limit DNA damage induced by 223Ra, 188Re, 99mTc and 99mTc-labeled pyrene. Dose-dependent radiation effects were comparable for alpha-emitters and both high- and low-energy electron emitters. The radioprotection by DMSO was not influenced by hypoxia. The results indicate the contribution of mainly indirect radiation effects for 99mTc, 188Re and 223Ra. 99mTc-labeled pyrene caused direct DNA damages and Auger-electrons from 99mTc-labeled pyrene are more effective than high-energy electrons or alpha particles. ADVANCES IN KNOWLEDGE: Without the consideration of DNA repair mechanisms, oxygen has no direct influence in radiation-induced DNA damages by different radiation qualities. IMPLICATIONS FOR PATIENT CARE: The short-time stimulation with oxygen during patient radiation could have minor influence compared to constant oxygen flooding to overcome hypoxic barriers.

Vacuum-UV and Low-Energy Electron-Induced DNA Strand Breaks - Influence of the DNA Sequence and Substrate.

DNA is effectively damaged by radiation, which can on the one hand lead to cancer and is on the other hand directly exploited in the treatment of tumor tissue. DNA strand breaks are already induced by photons having an energy below the ionization energy of DNA. At high photon energies, most of the DNA strand breaks are induced by low-energy secondary electrons. In the present study we quantified photon and electron induced DNA strand breaks in four different 12mer oligonucleotides. They are irradiated directly with 8.44 eV vacuum ultraviolet (VUV) photons and 8.8 eV low energy electrons (LEE). By using Si instead of VUV transparent CaF2 as a substrate the VUV exposure leads to an additional release of LEEs, which have a maximum energy of 3.6 eV and can significantly enhance strand break cross sections. Atomic force microscopy is used to visualize strand breaks on DNA origami platforms and to determine absolute values for the strand break cross sections. Upon irradiation with 8.44 eV photons all the investigated sequences show very similar strand break cross sections in the range of 1.7-2.3×10-16  cm2 . The strand break cross sections for LEE irradiation at 8.8 eV are one to two orders of magnitude larger than the ones for VUV photons, and a slight sequence dependence is observed. The sequence dependence is even more pronounced for LEEs with energies <3.6 eV. The present results help to assess DNA damage by photons and electrons close to the ionization threshold.

Biological effects of non-ionizing electromagnetic fields: Two sides of a coin.

Controversial, sensational and often contradictory scientific reports have triggered active debates over the biological effects of electromagnetic fields (EMFs) in literature and mass media the last few decades. This could lead to confusion and distraction, subsequently hampering the development of a univocal conclusion on the real hazards caused by EMFs on humans. For example, there are lots of publications indicating that EMF can induce apoptosis and DNA strand-breaks in cells. On the other hand, these effects could rather be beneficial, in that they could be effectively harnessed for treatment of various disorders, including cancer. This review discusses and analyzes the results of various in vitro, in vivo and epidemiological studies on the effects of non-ionizing EMFs on cells and organs, including the consequences of exposure to the low and high frequencies EM spectrum. Emphasis is laid on the analysis of recent data on the role of EMF in the induction of oxidative stress and DNA damage. Additionally, the impact of EMF on the reproductive system has been discussed, as well as the relationship between EM radiation and blood cancer. Apart from adverse effects, the therapeutic potential of EMFs for clinical use in different pathologies is also highlighted.

VISUALIZATION OF THE DNA REPAIR PROCESS IN MAMMALIAN CELLS TRANSFECTED WITH EGFP-EXPRESSING PLASMID DNA AFTER EXPOSURE TO X-RAYS IN VITRO.

To investigate the repair process of DNA damage induced by ionizing radiation in isolation from various types of cytoplasmic damage, we transfected X-irradiated enhanced green fluorescent protein (EGFP)-expressing plasmid DNA into non-irradiated mammalian cells using lipofectamine. The repair kinetics of the irradiated plasmids in the cells were visualized under microscopy as the EGFP fluorescence emitted by transfected cells. Using an agarose gel electrophoresis method, the yields of single- and double-strand breaks of the plasmids were also quantified. As positive control experiments, plasmid DNA with single- or double-strand breaks induced by a nicking or restriction enzyme were also transfected into the cells. The DNA repair rates for X-ray-irradiated plasmids were significantly lower than those of the enzymatically digested positive control samples. These results indicate that X-rays could induce less repairable damage than that induced by enzymes.

HOW DETECTION OF PLASMID DNA FRAGMENTATION AFFECTS RADIATION STRAND BREAK YIELDS.

A compromised detection of radiation-induced plasmid DNA fragments results in underestimation of calculated damage yields. Electrophoretic methods are easy and cheap, but they can only detect a part of the fragments, neglecting the shortest ones. These can be detected with atomic force microscopy, but at the expense of time and price. Both methods were used to investigate their capabilities to detect the DNA fragments induced by high-energetic heavy ions. The results were taken into account in calculations of radiation-induced yields of single and double strand breaks. It was estimated that the double strand break yield is twice as high when the fragments are at least partially detected with the agarose electrophoresis, compared to when they were completely omitted. Further increase by 13% was observed when the measured fragments were corrected for the fraction of the shortest fragments up to 300 base pairs, as detected with the atomic force microscopy. The effect of fragment detection on the single strand break yield was diminished.

MODELING DNA DAMAGE BY PHOTONS AND LIGHT IONS OVER ENERGY RANGES USED IN MEDICAL APPLICATIONS.

Comprehensive track structure-based simulations of DNA damage induced in human cells by photons (5 keV-1.3 MeV) and light ions (0.25-512 MeV/u) were performed with PARTRAC. DNA strand breaks, double-strand breaks and their clustering were scored. Effective LET values were established for photons that provide LET-dependent damage yields in agreement with the data for ions. The resulting database captures the variations of biological effectiveness with radiation quality. In particular, it can help compare the effectiveness of conventional radiotherapy using photon beams with techniques relying on proton or ion beams.

Dose-Rate Effects in Breaking DNA Strands by Short Pulses of Extreme Ultraviolet Radiation.

In this study, we examined dose-rate effects on strand break formation in plasmid DNA induced by pulsed extreme ultraviolet (XUV) radiation. Dose delivered to the target molecule was controlled by attenuating the incident photon flux using aluminum filters as well as by changing the DNA/buffer-salt ratio in the irradiated sample. Irradiated samples were examined using agarose gel electrophoresis. Yields of single- and double-strand breaks (SSBs and DSBs) were determined as a function of the incident photon fluence. In addition, electrophoresis also revealed DNA cross-linking. Damaged DNA was inspected by means of atomic force microscopy (AFM). Both SSB and DSB yields decreased with dose rate increase. Quantum yields of SSBs at the highest photon fluence were comparable to yields of DSBs found after synchrotron irradiation. The average SSB/DSB ratio decreased only slightly at elevated dose rates. In conclusion, complex and/or clustered damages other than cross-links do not appear to be induced under the radiation conditions applied in this study.

The degree of radiation-induced DNA strand breaks is altered by acute sleep deprivation and psychological stress and is associated with cognitive performance in humans.

Study Objectives: Sleep deprivation is associated with impaired immune responses, cancer, and morbidity and mortality, and can degrade cognitive performance, although individual differences exist in such responses. Sleep deprivation induces DNA strand breaks and DNA base oxidation in animals, and psychological stress is associated with increased DNA damage in humans. It remains unknown whether sleep deprivation or psychological stress in humans affects DNA damage response from environmental stressors, and whether these responses predict cognitive performance during sleep deprivation. Methods: Sixteen healthy adults (ages 29-52 years; mean age ± SD, 36.4 ± 7.1 years; seven women) participated in a 5-day experiment involving two 8 hr time-in-bed (TIB) baseline nights, followed by 39 hr total sleep deprivation (TSD), and two 8-10 hr TIB recovery nights. A modified Trier Social Stress Test was conducted on the day after TSD. The Psychomotor Vigilance Test measured behavioral attention. DNA damage was assessed in blood cells collected at 5 time points, and blood cells were irradiated ex vivo. Results: TSD, alone or in combination with psychological stress, did not induce significant increases in DNA damage. By contrast, radiation-induced DNA damage decreased significantly in response to TSD, but increased back to baseline when combined with psychological stress. Cognitively vulnerable individuals had more radiation-induced DNA strand breaks before TSD, indicating their greater sensitivity to DNA damage from environmental stressors. Conclusions: Our results provide novel insights into the molecular consequences of sleep deprivation, psychological stress, and performance vulnerability. They are important for fields involving sleep loss, radiation exposure, and cognitive deficits, including cancer therapy, environmental toxicology, and space medicine.

Apoptosis and cell proliferation in short-term and long-term effects of radioiodine-131-induced kidney damage: an experimental and immunohistochemical study.

OBJECTIVE: Radioiodine-131 is a radionuclide that is used for therapeutic purposes in hyperthyroidism and thyroid cancer. The aim of this study was to evaluate apoptotosis and proliferative changes in radioiodine-related kidney damage. MATERIALS AND METHODS: Three groups (n=10/group) of rats were used as follows: the rats were in group 1 untreated, and the rats in groups 2 and 3 were treated once with oral radioiodine (111 MBq). The animals in group 2 were killed at the end of the seventh day and the rats in group 3 were killed at the end of the 10th week. The kidneys were removed and evaluated immunohistochemically. The presence of radioiodine in the kidneys was shown by the Na+/I-symporter antibody and proliferating cell nuclear antigen, Ki-67, caspase-3, caspase-8, caspase-9, and terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay were used to detect cell proliferation and apoptosis. RESULTS: Na+/I-symporter protein accumulation in the kidneys was observed to be significantly greater in group 2 than in group 3 (P<0.05). All the immunohistochemical analyses showed that cell proliferation and apoptosis began on the seventh day and peaked in the 10th week. The proliferating cell nuclear antigen, Ki-67, and caspase expressions and terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling values were all found to be statistically significantly increased in group 3 compared with the other groups (P<0.05). CONCLUSION: Radioiodine caused cell proliferation and apoptosis as shown by immunohistochemistry.

Enhanced radiation damage caused by iodinated contrast agents during CT examination.

OBJECTIVE: To access the effect of iodinate contrast agent (ICA) on DNA double-stand breaks (DSBs) in human peripheral blood lymphocytes during computed tomography (CT) examinations. MATERIALS AND METHODS: This present study was approved by the institutional ethics committee; written informed patient consent was obtained from 70 patients. A total of 48 patients underwent computed tomography urography (CTU), in which only one time CT scanning was examined after injecting ICA, and 22 patients received unenhanced whole abdominal CT, among them 10 patients were selected to get ICA injection immediately after irradiation. Blood samples were taken from all patients prior to and immediately after CT scan, as well as 8min after the injection of ICA. The lymphocytes in these blood samples were separated by using density-gradient centrifugation, fixed and immunostained with γH2AX antibody. The average number of phosphorylated histone H2AX (γH2AX) foci per lymphocyte was counted under a fluorescence microscopy. Differences in the number of γH2AX-foci were statistically analyzed using independent sample t test and one way ANOVA. RESULT: The three patient groups had no significant differences in the baseline foci numbers(P>0.05). The γH2AX-focus levels increased in both groups after CT scan. Patients who underwent CTU examinations had a greater DSBs level (mean±standard error of mean, 0.945±0.184 foci per cell) than those who received unenhanced whole abdominal CT scan (mean±standard error of mean, 0.700±0.112 foci per cell), increasing by about 37.9%; The ICA injected before CT scan itself had an effect on the DSBs, which increased DSBs level by approximately 90.3% (0.059±0.018vs 0.031±0.025, P<0.05), but no significant difference was found if added after irradiation, increasing DSBs level only by 3.2% approximately (0.711±0.091vs 0.689±0.108, P=0.499). CONCLUSION: The iodinated contrast agent itself can lead to an increase in the level of DSBs as assessed with γH2AX foci formation, and the application of ICA can amplify DNA damage induced by diagnostic x-ray procedures such as whole abdominal CT.

Cell damage caused by ultraviolet B radiation in the desert cyanobacterium Phormidium tenue and its recovery process.

Phormidium tenue, a cyanobacterium that grows in the topsoil of biological soil crusts (BSCs), has the highest recovery rate among desert crust cyanobacteria after exposure to ultraviolet B (UV-B) radiation. However, the mechanism underlying its recovery process is unclear. To address this issue, we measured chlorophyll a fluorescence, generation of reactive oxygen species (ROS), lipid peroxidation, and repair of DNA breakage in P. tenue following exposure to UV-B. We found that UV-B radiation at all doses tested reduced photosynthesis and induced cell damage in P. tenue. However, P. tenue responded to UV-B radiation by rapidly reducing photosynthetic activity, which protects the cell by leaking less ROS. Antioxidant enzymes, DNA damage repair systems, and UV absorbing pigments were then induced to mitigate the damage caused by UV-B radiation. The addition of exogenous antioxidant chemicals ascorbate and N-acetylcysteine also mitigated the harmful effects caused by UV-B radiation and enhanced the recovery process. These chemicals could aid in the resistance of P. tenue to the exposure of intense UV-B radiation in desertified areas when inoculated onto the sand surface to form artificial algal crusts.

Radiation-Induced Deletions in Mouse Spermatogonia are Usually Large (over 200 kb) and Contain Little Sequence Similarity at the Junctions.

Ionizing radiation can induce mutations, and the majority of radiation-induced mutations in mammalian cells are deletions. The most critical types of radiation-induced DNA damage are DNA double-strand breaks, and these breaks are repaired by either the homologous recombination (HR) pathway or the non-homologous end joining (NHEJ) pathway. The HR pathway is not as mutagenic as the NHEJ pathway, and it is expected that radiation-induced deletions would usually have little sequence similarity around the deletion junction points. Here we report sequence data from the regions around the rejoined junctions of 33 de novo copy-number mutations (27 deletions and 6 duplications) obtained from offspring sired by male mice that were irradiated at the spermatogonia stage and from nonirradiated controls. The results indicate that deletions can be classified into three major groups. In group 1, nine deletions were found to share long blocks of similar sequences (200-6,000 bp) at the junctions and the deletion size varied extensively (1 kb to 2 Mb) (e.g., illegitimate recombination). In group 2, five deletions shared short identical sequences (0-7 bp) at the junctions, and the deletion sizes were shorter than 200 kb (e.g., micro-homology-mediated repair). Additional three-deletion candidates of this group were also found but turned out to be inherited from mosaic parents. They are therefore not included in germline mutations. In group 3, twelve deletions shared little sequence similarity (only 0-2 bp) at the junctions (likely due to NHEJ repair) and deletion sizes were longer than 200 kb. Group 1 consisted of deletions found in both spontaneous and irradiated genomes and thus, were probably caused by spontaneous events during meiosis or DNA replication. Group 2 consisted mainly of deletions found in nonexposed genomes. Group 3 consisted primarily of deletions that occurred in the irradiated genomes. Among the duplications, we found no indication of any association with radiation exposures. These results indicate that large size (>200 kb) and little sequence similarity around the rejoined sites are likely to be a hallmark of radiation-induced deletions in mice.

Protective Effect of Juglans regia L. Walnut Extract Against Oxidative DNA Damage.

Walnuts (Juglans regia L.) are relevant components of the Mediterranean diet providing important macronutrients, micronutrients and other bioactive constituents including unsaturated fatty acids, proteins, fiber, vitamins, minerals, phytosterols and polyphenols. Although the walnut beneficial effects in human health are widely recognized by a lot of epidemiologic studies very little is known regarding its effect on damaged DNA. The aim of the present study was to investigate the effect of Juglans regia L. ethanolic extract from kernel on the induction of DNA strand breaks by thiol/Fe3+/O2 mixed function oxidase, tert-butyl hydroperoxide or UVC radiations in acellular and cellular models. Plasmid DNA cleavage and fast Halo assay were used to monitor oxidative damage to DNA. Both approaches showed protection of oxidatively injured DNA. These results agree with a lot of scientific proofs which recommend walnut as dietary adjunct in health promotion and prevention as well as in treatment of lifestyle-related oxidative diseases.

Histone H3 Lysine 9 Acetylation Obstructs ATM Activation and Promotes Ionizing Radiation Sensitivity in Normal Stem Cells.

Dynamic spatiotemporal modification of chromatin around DNA damage is vital for efficient DNA repair. Normal stem cells exhibit an attenuated DNA damage response (DDR), inefficient DNA repair, and high radiosensitivity. The impact of unique chromatin characteristics of stem cells in DDR regulation is not yet recognized. We demonstrate that murine embryonic stem cells (ES) display constitutively elevated acetylation of histone H3 lysine 9 (H3K9ac) and low H3K9 tri-methylation (H3K9me3). DNA damage-induced local deacetylation of H3K9 was abrogated in ES along with the subsequent H3K9me3. Depletion of H3K9ac in ES by suppression of monocytic leukemia zinc finger protein (MOZ) acetyltransferase improved ATM activation, DNA repair, diminished irradiation-induced apoptosis, and enhanced clonogenic survival. Simultaneous suppression of the H3K9 methyltransferase Suv39h1 abrogated the radioprotective effect of MOZ inhibition, suggesting that high H3K9ac promoted by MOZ in ES cells obstructs local upregulation of H3K9me3 and contributes to muted DDR and increased radiosensitivity.