The redox state of the cell can be affected by many cellular conditions. In this study we show that detectable reactive oxygen species (ROS) are also generated in response to DNA damage by the chromatin remodeling factor and monoamine oxidase LSD1/KDM1A. This raised the possibility that the localized generation of hydrogen peroxide produced by LSD1 may affect the function of proximally located DNA repair proteins. The two major pathways for repair of DNA double-strand breaks (DSBs) are homologous recombination (HR) and non-homologous end joining (NHEJ). Cells were exposed to low levels of ectopic H2O2, DNA breaks generated by laser light, and recruitment kinetics of NHEJ protein Ku80 to DNA damage sites determined. Ku80 recruitment to damage sites was significantly decreased in cells pretreated with H2O2 while HR end binding protein Nbs1 was increased. This suggests that the DNA repair pathway choice has the potential to be modulated by the local redox state. This has implications for chemotherapeutic approaches involving generating DNA damage to target actively dividing cancer cells, which may be more or less effective dependent on the redox state of the targeted cells and the predominant repair pathway required to repair the type of DNA damage generated.

Cartilage undergoes characteristic mechanical stress relaxation following laser irradiation below the ablation threshold. Porcine auricular cartilage (1-2 mm thickness) was irradiated with a Nd:YAG laser (λ = 1.32 μm) at two power levels (W/cm2). Surface temperature (S(c)(t) (°C)) (monitored using a single element HgCdTe infrared detector, 10-14 μm spectral range), and integrated back scattered light intensity I(t) were measured during laser irradiation. A HeNe laser beam (λ = 632.8 nm) was incident on the back surface of the cartilage specimen and fractional integrated backscattered light intensity was measured using an integrating sphere and a silicon photodiode. Laser irradiation (5.83 W/cm2, 50 Hz pulse repetition rate (PRR)) continued until surface temperature reached approximately 70°C, during which cartilage mechanical stress relaxation was observed. Integrated back scattered light intensity reached a plateau at about 70°C). At higher laser power (39.45 W/cm2, 50 Hz PRR), a feedback-controlled cryogen spray was used to maintain surface temperature below 5O°C. A similar plateau response was noted in integrated backscattered light intensity. This signal may be used to optimise the process of stress relaxation in laser cartilage reshaping. Several clinical applications involving reconstructive surgery are proposed.

Laser radiation can be used to reshape cartilage tissue into new morphologic configurations. When a critical temperature is attained, mechanically deformed cartilage becomes malleable and may be reshaped into new geometric configurations that harden as the tissue cools. This temperature-dependent process results in mechanical stress relaxation and is characteristic of a phase transformation. The principal advantages of using laser radiation for the generation of thermal energy in tissue are precise control of both the space-time temperature distribution and time-dependent thermal denaturation kinetics. In this study, we illustrate the utility of laser mediated cartilage reshaping in ex vivo porcine model of reconstructive nasal and laryngeal surgery, and attempt to determine the temperature range in which accelerated stress relaxation occurs during laser mediated cartilage reshaping. Optimization of the reshaping process requires identification of the temperature dependence of this phase transformation and its relationship to observed changes in cartilage optical (diffuse scattering), mechanical (internal stress), and thermodynamic properties (heat capacity). Light scattering, infrared radiometry, and modulated differential scanning calorimetry were used to measure temperature-dependent changes in the biophysical properties of cartilage tissue during fast (laser mediated) and show heating (conventional calorimetric heating). Our studies using MDSC and laser probe techniques have identified changes in tissue thermodynamic and optical properties suggestive of a phase transformation occurring near 60°C. Clinically, reshaped cartilage tissue can be used to recreate the underlying cartilagious framework of structures in the head and neck such as the ear, larynx, trachea, and nose.

The redox state of the cell can be affected by many cellular conditions. In this study we show that detectable reactive oxygen species (ROS) are also generated in response to DNA damage by the chromatin remodeling factor and monoamine oxidase LSD1/KDM1A. This raised the possibility that the localized generation of hydrogen peroxide produced by LSD1 may affect the function of proximally located DNA repair proteins. The two major pathways for repair of DNA double-strand breaks (DSBs) are homologous recombination (HR) and non-homologous end joining (NHEJ). Cells were exposed to low levels of ectopic H2O2, DNA breaks generated by laser light, and recruitment kinetics of NHEJ protein Ku80 to DNA damage sites determined. Ku80 recruitment to damage sites was significantly decreased in cells pretreated with H2O2 while HR end binding protein Nbs1 was increased. This suggests that the DNA repair pathway choice has the potential to be modulated by the local redox state. This has implications for chemotherapeutic approaches involving generating DNA damage to target actively dividing cancer cells, which may be more or less effective dependent on the redox state of the targeted cells and the predominant repair pathway required to repair the type of DNA damage generated.

Laser-assisted cartilage reshaping is mediated by thermally induced stress relaxation, and may be used to alter cartilage morphology for reconstructive surgical procedures in the upper airway and face without carving, morselizing, or suturing. Internal stress σ(t), integrated backscattered light intensity I(t) from a He-Ne probe laser (λ=632.8 nm), and radiometric surface temperature Sc(t) were measured during the reshaping of porcine nasal septal cartilage using a pulsed Nd:YAG laser (λ=1.32 μm). Internal stress and integrated backscattered light intensity were observed to increase, plateau, and then decrease in similar ways during laser irradiation. The plateau region occurred when the cartilage front surface temperature approached 65 °C. I(t) was utilized in a feedback control procedure to reshape cartilage specimens from a flat to a curved geometry. Immediately following laser irradiation, the tissues were rehydrated in normal saline for 15 min while wrapped around a small dowel. A stable shape change was retained for 21 days while the specimens were stored in normal saline at 5 °C. The backscattered light intensity signal mirrors underlying changes in internal stress, and further rate of change or slope of I(t) is nearly zero when the surface temperature reaches about 65 °C. Measurements of I(t) (or, equivalently, the fractional change in integrated backscattered light intensity ΔI(t)/I0) may be used to control the process of laser-assisted cartilage reshaping and minimize nonspecific thermal injury due to uncontrolled heating. © 1998 Society of Photo-Optical Instrumentation Engineers.

Unlabelled

BACKGROUND AND OBJECTIVE To examine the biophysical effects of photothermal heating on herniated intervertebral discs during laser decompression surgery.

Study design/materials and methods

Ex vivo bovine nucleus pulposus specimens were irradiated with a Nd: YAG laser (lambda = 1.32 microm, 100 seconds exposure time, 9-31 W/cm(2), 4.8 mm spot diameter), whereas changes in tissue thermal, mechanical, and optical properties were monitored by using, respectively, infrared radiometry, tissue tension measurements, and diffuse reflectance from a HeNe probe laser. Morphologic changes and mass reduction were monitored by recording shape changes on video and weighing specimens before and after laser exposure.

Results

At power densities below 20 W/cm(2), evaporation of water and specimen volume reduction (shrinking) were consistently observed on video during irradiation. In contrast, above 20 W/cm(2), vapor bubbles formed within the specimen matrix and subsequently ruptured (releasing heated vapors). When radiometric surface temperature approaches approximately 60 to 70 degrees C (denaturation threshold for tissue), tissue tension begins to increase, which is consistent with observations of specimen length reduction. The onset of this change in tissue tension is also reflected in characteristic alterations in diffuse reflectance. With cessation of laser irradiation, a sustained increase in tissue tension is observed, which is consistent with changes in specimen length and volume. Higher laser power results in a faster heating rate and subsequently an accelerated tension change. Specimen mass reduction increased with irradiance from 19 to 72% of the initial mass for 9--31 W/cm(2), respectively. Irradiated specimens did not return to their original shape after immersion in saline (48 hours) in contrast to air-dried specimens (24 hours), which returned to their original shape and size.

Conclusion

These observations suggest that photothermal heating results in irreversible matrix alteration causing shape change and volume reduction (observed on video and evidenced by the increase in tissue tension) taking place at approximately 65 degrees C. Inasmuch as high laser power results in vapor bubble formation and specimen tearing, the heating process must be controlled. Diffuse reflectance measurements provide a noncontact, highly sensitive means to monitor dynamically changes in tension of nucleus purposus.

Pyrrole-imidazole (Py-Im) polyamides are synthetic non-genotoxic minor groove-binding small molecules. We hypothesized that Py-Im polyamides can modulate the cellular response to ionizing radiation. Pre-treatment of cells with a Py-Im polyamide prior to exposure to ionizing radiation resulted in a delay in resolution of phosphorylated γ-H2AX foci, increase in XRCC1 foci, and reduced cellular replication potential. RNA-sequencing of cell lines exposed to the polyamide showed induction of genes related to the ultraviolet radiation response. We observed that the polyamide is almost 10-fold more toxic to a cell line deficient in DNA ligase 3 as compared to the parental cell line. Alkaline single cell gel electrophoresis reveals that the polyamide induces genomic fragmentation in the ligase 3 deficient cell line but not the corresponding parental line. The polyamide interferes directly with DNA ligation in vitro. We conclude that Py-Im polyamides may be further explored as sensitizers to genotoxic therapies.

Cartilage undergoes characteristic deformation following laser irradiation below the ablation threshold. Measurements of surface temperature and integrated scattered light intensity were performed during laser irradiation. Porcine auricular cartilage (1 - 2 mm thickness) was irradiated with an Nd:YAG laser (lambda equals 1.32 micrometer) with varying dose (J/cm2). Surface temperature was monitored using a single element HgCdTe infrared detector, responsive between 10 - 14 micrometer. A HeNe laser beam (lambda equals 632.8 nm) was incident on the back surface of the cartilage specimen and fractional integrated back scattered light intensity was measured using an integrating sphere and a silicon photodiode. Laser irradiation (2 W, 5.83 W/cm2, 50 Hz PRR) was allowed to proceed until surface temperature reached 70 degrees Celsius. Cartilage deformation was observed in each instance. Integrated scattered light intensity reached a plateau before the peak temperature (70 degrees) was reached. At increased laser power (10 W, 39.45 W/cm2, 50 Hz PRR), a feedback controlled cryogen spray was used to maintain surface temperature below 50 degrees Celsius. A similar plateau response was also noted in integrated scattered light intensity. This signal may be used to optimize the process of stress relaxation in laser cartilage reshaping. Several clinical applications are discussed. ©2005 Copyright SPIE - The International Society for Optical Engineering.

Glioblastoma multiforme (GBM) is a life-threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem-like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator of mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the mitotic kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase-dependent manner. This MELK-dependent activation of FOXM1 results in a subsequent increase in mitotic regulatory genes in GSCs. MELK-driven FOXM1 activation is regulated by the binding and subsequent trans-phosphorylation of FOXM1 by another kinase PLK1. Using mouse neural progenitor cells (NPCs), we found that transgenic expression of FOXM1 enhances, while siRNA-mediated gene silencing diminishes neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases as cells progress from NPCs, to pretumorigenic progenitors and GSCs. The antibiotic Siomycin A disrupts MELK-mediated FOXM1 signaling with a greater sensitivity in GSC compared to neural stem cell. Treatment with the first-line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cells, and addition of Siomycin A to Temozolomide treatment in mice harboring GSC-derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1-dependent manner and thus, this protein complex is a potential therapeutic target for GBM.