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At least 433 records · Page 24

Altered Gene Expressions and Cytogenetic Repair Efficiency in Cells with Suppressed Expression of XPA after Proton Exposure

Cellular responses to damages from ionizing radiation (IR) exposure are influenced not only by the genes involved in DNA double strand break (DSB) repair, but also by non- DSB repair genes. We demonstrated previously that suppressed expression of several non-DSB repair genes, such as XPA, elevated IR-induced cytogenetic damages. In the present study, we exposed human fibroblasts that were treated with control or XPA targeting siRNA to 250 MeV protons (0 to 4 Gy), and analyzed chromosome aberrations and expressions of genes involved in DNA repair. As expected, after proton irradiation, cells with suppressed expression of XPA showed a significantly elevated frequency of chromosome aberrations compared with control siRNA treated (CS) cells. Protons caused more severe DNA damages in XPA knock-down cells, as 36% cells contained multiple aberrations compared to 25% in CS cells after 4Gy proton irradiation. Comparison of gene expressions using the real-time PCR array technique revealed that expressions of p53 and its regulated genes in irradiated XPA suppressed cells were altered similarly as in CS cells, suggesting that the impairment of IR induced DNA repair in XPA suppressed cells is p53-independent. Except for XPA, which was more than 2 fold down regulated in XPA suppressed cells, several other DNA damage sensing and repair genes (GTSE1, RBBP8, RAD51, UNG and XRCC2) were shown a more than 1.5 fold difference between XPA knock-down cells and CS cells after proton exposure. The possible involvement of these genes in the impairment of DNA repair in XPA suppressed cells will be further investigated.

Zhang, Ye↗

Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems.

Zhang, Ye↗

Broadband Phase Retrieval for Image-Based Wavefront Sensing

A focus-diverse phase-retrieval algorithm has been shown to perform adequately for the purpose of image-based wavefront sensing when (1) broadband light (typically spanning the visible spectrum) is used in forming the images by use of an optical system under test and (2) the assumption of monochromaticity is applied to the broadband image data. Heretofore, it had been assumed that in order to obtain adequate performance, it is necessary to use narrowband or monochromatic light. Some background information, including definitions of terms and a brief description of pertinent aspects of image-based phase retrieval, is prerequisite to a meaningful summary of the present development. Phase retrieval is a general term used in optics to denote estimation of optical imperfections or aberrations of an optical system under test. The term image-based wavefront sensing refers to a general class of algorithms that recover optical phase information, and phase-retrieval algorithms constitute a subset of this class. In phase retrieval, one utilizes the measured response of the optical system under test to produce a phase estimate. The optical response of the system is defined as the image of a point-source object, which could be a star or a laboratory point source. The phase-retrieval problem is characterized as image-based in the sense that a charge-coupled-device camera, preferably of scientific imaging quality, is used to collect image data where the optical system would normally form an image. In a variant of phase retrieval, denoted phase-diverse phase retrieval [which can include focus-diverse phase retrieval (in which various defocus planes are used)], an additional known aberration (or an equivalent diversity function) is superimposed as an aid in estimating unknown aberrations by use of an image-based wavefront-sensing algorithm. Image-based phase-retrieval differs from such other wavefront-sensing methods, such as interferometry, shearing interferometry, curvature wavefront sensing, and Shack-Hartmann sensing, all of which entail disadvantages in comparison with image-based methods. The main disadvantages of these non-image based methods are complexity of test equipment and the need for a wavefront reference.

Dean, Bruce H.↗

Distribution of Chromosome Breakpoints in Human Epithelial Cells Exposed to Low- and High-LET Radiations

The advantage of the multicolor banding in situ hybridization (mBAND) technique is not only its ability to identify simultaneously both inter- and intrachromosome exchanges, but also the ability to measure the breakpoint location along the length of the chromosome in a precision that is unmatched with other traditional banding techniques. Breakpoints on specific regions of a chromosome have been known to associate with specific cancers. The breakpoint distribution in cells after low- and high-LET radiation exposures will also provide the data for biophysical modeling of the chromatin structure, as well as the data for the modeling the formation of radiation-induced chromosome aberrations. In a series of experiments, we studied low- and high-LET radiation-induced chromosome aberrations using the mBAND technique with chromosome 3 painted in 23 different colored bands. Human epithelial cells (CH1 84B5F5/M10) were exposed in vitro to Cs- 137 rays at both low and high dose rates, secondary neutrons with a broad energy spectrum at a low dose rate and 600 MeV/u Fe ions at a high dose rate. The data of both inter- and intrachromosome aberrations involving the painted chromosome have been reported previously. Here we present data of the location of the chromosome breaks along the length of chromosome 3 in the cells after exposures to each of the four radiation scenarios. In comparison to the expected breakpoint distribution based on the length of the bands, the observed distribution appeared to be non-random for both the low- and high-LET radiations. In particular, hot spots towards both ends of the chromosome were found after low-LET irradiations of either low or high dose rates. For both high-LET radiation types (Fe ions and neutrons), the breakpoint distributions were similar, and were much smoother than that for low-LET radiation. The dependence of the breakpoint distribution on the radiation quality requires further investigations.

Hada, Megumi↗

Truly Incomplete and Complex Chromosomal Exchanges in Human Fibroblast Cells Exposed In Situ to Energetic Heavy Ions

Confluent human fibroblast cells (AG 1522) were irradiated with gamma rays, 490 MeV/nucleon Si, or with Fe ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 C for 24 hours after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Incomplete and complex exchanges were analyzed in the irradiated samples. In order to verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after high-LET radiation, and consequently, the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/micron, the highest LET value in the present study. For samples exposed to 200 MeV/nucleon Fe ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique that allow identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy dose of the Fe ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges, values for which were higher than those obtained after a 6 Gy gamma exposure. After 0.7 Gy of Fe ions, most complex aberrations were found to involve three or four chromosomes, which is a likely indication of the maximum number of chromosome domains traversed by a single Fe ion track.

Wu, Honglu↗

Karyotyping of Chromosomes in Human Bronchial Epithelial Cells Transformed by High Energy Fe Ions

Lung cancer induced from exposure to space radiation is believed to be one of the most significant health risks for long-term space travels. In a previous study, normal human bronchial epithelial cells (HBECs), immortalized through the expression of Cdk4 and hTERT, were exposed to gamma rays and high energy Fe ions for the selection of transformed clones induced by low- and high-LET radiation. In this research, we analyzed chromosome aberrations in these selected clones for genomic instability using the multi-color fluorescent in situ hybridization (mFISH), as well as the multi-banding in situ hybridization (mBAND) techniques. In most of the clones, we found chromosomal aberrations involving translocations between different chromosomes, with several of the breaks occurred in the q-arm of chromosome 3. We also identified copy number variations between the transformed clones and the parental HBEC cells regardless of the exposure condition. Our results indicated that the chromosomal aberrations in low- and high radiation-induced transformed clones are inadequately different from spontaneous soft agar growth. Further analysis is underway to reveal the genomic instability in more transformed clones

Yeshitla, Samrawit↗

Simulation of DNA Damage in Human Cells from Space Radiation Using a Physical Model of Stochastic Particle Tracks and Chromosomes

The formation of double-strand breaks (DSBs) and chromosomal aberrations (CAs) is of great importance in radiation research and, specifically, in space applications. We are presenting a recently developed model, in which chromosomes simulated by NASARTI (NASA Radiation Tracks Image) is combined with nanoscopic dose calculations performed with the Monte-Carlo simulation by RITRACKS (Relativistic Ion Tracks) in a voxelized space. The model produces the number of DSBs, as a function of dose for high-energy iron, oxygen, and carbon ions, and He ions. The combined model calculates yields of radiation-induced CAs and unrejoined chromosome breaks in normal and repair deficient cells. The merged computational model is calibrated using the relative frequencies and distributions of chromosomal aberrations reported in the literature. The model considers fractionated deposition of energy to approximate dose rates of the space flight environment. The merged model also predicts of the yields and sizes of translocations, dicentrics, rings, and more complex-type aberrations formed in the G0/G1 cell cycle phase during the first cell division after irradiation.

Ponomarev, Artem↗

Design and Modeling of the Off-Axis Parabolic Deformable (OPD) Mirror Laboratory

Coronagraph-equipped direct imaging missions need an active wavefront control system to cancel out the optical aberrations that degrade the performance of the coronagraphs. A fast steering mirror is used to control Line-of-Sight (LoS) pointing error caused by the telescope jitter. In addition to controlling other low-order aberrations such as astigmatism and coma, high stroke, high actuator density deformable mirrors (DMs) are also used to control the electric field at the required high spatial frequencies. We are designing a testbed to verify a different deformable architecture, where the powered optic in the optical train are controllable and have lower actuator count compared to the existing DMs with flat nominal surfaces. This simplifies the packaging issue for space missions and reduces both cost and risk of having the entire coronagraph instrument's performance depending on one or two high-actuator count DMs. The testbed would also be capable of testing different low-order wavefront sensing algorithms, which focuses in the near-term on a new adaptive Kalman filtering and gradient decent method to estimate the harmonic LoS errors that affect space telescopes. In long run, we would test different machine learning techniques to estimate low-order aberrations and non-linear algorithms for digging the region of high contrast called the dark holes (DH).

Subedi, Hari↗

Design and Modeling of the Off-Axis Parabolic Deformable Mirror Laboratory

Coronagraph-equipped direct imaging missions need an active wavefront control system to cancel out the optical aberrations that degrade the performance of the coronagraphs. A fast steering mirror is used to control Line-of-Sight (LoS) pointing error caused by the telescope jitter. In addition to controlling other low-order aberrations such as astigmatism and coma, high stroke, high actuator density deformable mirrors (DMs) are also used to control the electric field at the required high spatial frequencies. We are designing a testbed to verify a different deformable architecture, where the powered optic in the optical train are controllable and have lower actuator count compared to the existing DMs with flat nominal surfaces. This simplifies the packaging issue for space missions and reduces both cost and risk of having the entire coronagraph instrument's performance depending on one or two high-actuator count DMs. The testbed would also be capable of testing different low-order wavefront sensing algorithms, which focuses in the near-term on a new adaptive Kalman filtering and gradient decent method to estimate the harmonic LoS errors that affect space telescopes. In long run, we would test different machine learning techniques to estimate low-order aberrations and non-linear algorithms for digging the region of high contrast called the dark holes (DH).

Communication↗

Star Tracker Accuracy Improvement and Optimization for Attitude Measurement in Three-Axis

High precision attitude measurement systems obviate the need for the beacon from the receiver making it possible for the spacecraft to beam a laser communications signal to a ground station without the ground station advertising its location. The research presented targets new detection and estimation methods to improve the accuracy in locating stars on a focal plane detector, and an understanding of the effects of changes in the optics design parameters and aberration, including defocus, on the navigation solution itself. This understanding can lead to an optimization of the attitude solution with respect to those optics realm parameter changes. The methodology discussed includes the development of a model of a current star tracker system. Using this model, multiple algorithms are implemented, including a multi-hypothesis method (MHT), to detect and estimate the position of the stars on the focal plane detector. It will be shown that using the MHT for detection and estimation, a greater accuracy can be found for each star estimation from more traditional detection and estimation algorithms. The approach then uses the model to develop statistics of the star tracker and the attitude estimation outputs to understand the accuracy, or variance, of the system's attitude solution. This solution is repeated for a range of defocus aberration, and a lower limit to the variance of the attitude solution is shown. A Cramer Rao lower bound solution is derived for the star tracker system and the results are compared to the Monte Carlo analysis from the model and shown to correlate very well. The approach uses a star image not as a Gaussian spot on the focal plane as done in previous work, and use of an image that includes the effects of aberrations of the optic system, and the effects of under-sampling and noise from the focal plane detector as well. Analysis includes exploring a star tracker's accuracy improvement through the combination of focus error and under-sampling effects alone, possibly contradicting conventional wisdom and approaches.

Lichter, Michael J.↗

Science Yield Estimates and Sensitivities With the AstroPIC Integrated Photonic Coronagraph for the Habitable Worlds Observatory

The Habitable Worlds Observatory (HWO) flagship has a goal to survey ~100 of the nearest stellar systems and their habitable zones in order to detector and spectroscopically characterize ~25 potentially “Earth-like planets” (or “Exoearths”). The HWO telescope will feature a >6-m diameter aperture and a coronagraph that will suppress starlight by ten orders of magnitude to enable the detection and characterization of Earth analogues in the habitable zone. Three Exploratory Analytic Cases (EACs) are currently considering different aperture configurations for HWO including on-axis and off-axis apertures and both different hexagonal and keystone segmentation configurations. Photonic Integrated Circuits (PICs) offer a path to miniaturize traditional, bulk optics coronagraph functionality reducing risk margins and increasing mission science exoearth yields for the HWO mission. We have developed a simulation pipeline that enables evaluation of the AstroPIC photonic integrated coronagraph through to estimates mission yields. We present sensitivities to aberrations for different types of segmentation and low-order aberrations for HWO EACs demonstrating that AstroPIC is suitable for both on-axis and off-axis configurations. We perform a parametric sweep of the exoearth science yield sensitivities in terms of key coronagraph parameters including coronagraphic end-to-end throughput, inner working angle, robustness to aberrations, and bandwidth. We consider both a hybrid and a pure photonic architecture; a pure integrated photonic architecture provides the greatest potential in science yield improvements, although a hybrid PIC limited in its numbers of modes can provide complementary science to a traditional, bulk-optic coronagraph and increasing the estimated science yield when operated jointly.

Photonics↗

A (S)TEM Gas Cell Holder with Localized Laser Heating for In Situ Experiments

The advent of aberration correction for transmission electron microscopy has transformed atomic resolution imaging into a nearly routine technique for structural analysis. Now an emerging frontier in electron microscopy is the development of in situ capabilities to observe reactions at atomic resolution in real time and within realistic environments. Here we present a new in situ gas cell holder that is designed for compatibility with a wide variety of sample type (i.e., dimpled 3-mm discs, standard mesh grids, various types of focused ion beam lamellae attached to half grids). Its capabilities include localized heating and precise control of the gas pressure and composition while simultaneously allowing atomic resolution imaging at ambient pressure. The results show that 0.25-nm lattice fringes are directly visible for nanoparticles imaged at ambient pressure with gas path lengths up to 20 μm. Additionally, we quantitatively demonstrate that while the attainable contrast and resolution decrease with increasing pressure and gas path length, resolutions better than 0.2 nm should be accessible at ambient pressure with gas path lengths less than the 15 μm utilized for these experiments.

aberration correction↗

Performance predictions of a focused ion beam from a laser cooled and compressed atomic beam

Focused ion beams are indispensable tools in the semiconductor industry because of their ability to image and modify structures at the nanometer length scale. Here, we report on performance predictions of a new type of focused ion beam based on photo-ionization of a laser cooled and compressed atomic beam. Particle tracing simulations are performed to investigate the effects of disorder-induced heating after ionization in a large electric field. They lead to a constraint on this electric field strength which is used as input for an analytical model which predicts the minimum attainable spot size as a function of, amongst others, the flux density of the atomic beam, the temperature of this beam, and the total current. At low currents (I < 10 pA), the spot size will be limited by a combination of spherical aberration and brightness, while at higher currents, this is a combination of chromatic aberration and brightness. It is expected that a nanometer size spot is possible at a current of 1 pA. The analytical model was verified with particle tracing simulations of a complete focused ion beam setup. A genetic algorithm was used to find the optimum acceleration electric field as a function of the current. At low currents, the result agrees well with the analytical model, while at higher currents, the spot sizes found are even lower due to effects that are not taken into account in the analytical model.

43 PARTICLE ACCELERATORS↗

Examination of clonal evolution in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is one of the most frequent lymphoproliferative diseases. CLL is characterized by unusual heterogeneity, which probably reflects its biological and genetic lack of homogeneity. Clonal chromosome aberrations belong to the most important prognostic and predictive factors in CLL. This research was aimed at observing clonal evolution in CLL at the chromosomal level, and assessing its clinical significance in relation to selected prognostic factors. The study involved 72 untreated patients with CLL. The preliminary investigations using cytogenetic banding analysis (CBA) and FISH were performed at the time of diagnosis, and again after about 24 months to observe clonal changes (clonal evolution). In addition, other parameters were evaluated, i.e., the expression of ZAP-70 kinase, CD38 antigen, and the mutation statuses of IGVH and NOTCH1 genes. Classic prognostic factors, i.e., categorized ZAP70 and CD38 expressions as well as mutations in IGVH and NOTCH1 genes did not influence the course of clonal evolution in the examined group of patients. Clonal evolution was detected in 45.8% of patients by means of CBA, and in 19.4% patients with FISH. Analysis of chromosomal aberrations in the examined group of patients showed that the incidence of 17p deletions and translocations in karyotypes has a negative impact on overall survival. CE was found to be a risk factor for the occurrence of disease progression (OR = 2.22). Our observations indicate that combined CBA and FISH are the most optimal techniques for monitoring clonal evolution in the course of CLL.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Cytogenetic Low-Dose Hyperradiosensitivity Is Observed in Human Peripheral Blood Lymphocytes

Purpose: The shape of the ionizing radiation response curve at very low doses has been the subject of considerable debate. Linear-no-threshold (LNT) models are widely used to estimate risks associated with low-dose exposures. However, the low-dose hyperradiosensitivity (HRS) phenomenon, in which cells are especially sensitive at low doses but then show increased radioresistance at higher doses, provides evidence of nonlinearity in the low-dose region. HRS is more prominent in the G2 phase of the cell cycle than in the G0/G1 or S phases. Here we provide the first cytogenetic mechanistic evidence of low-dose HRS in human peripheral blood lymphocytes using structural chromosomal aberrations. Methods and Materials: Human peripheral blood lymphocytes from 2 normal healthy female donors were acutely exposed to cobalt 60 γ rays in either G0 or G2 using closely spaced doses ranging from 0 to 1.5 Gy. Structural chromosomal aberrations were enumerated, and the slopes of the regression lines at low doses (0-0.4 Gy) were compared with doses of 0.5 Gy and above. Results: HRS was clearly evident in both donors for cells irradiated in G2. No HRS was observed in cells irradiated in G0. The radiation effect per unit dose was 2.5- to 3.5-fold higher for doses ≤0.4 Gy than for doses >0.5 Gy. Conclusions: These data provide the first cytogenetic evidence for the existence of HRS in human cells irradiated in G2 and suggest that LNT models may not always be optimal for making radiation risk assessments at low doses.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Detection and Automated Scoring of Dicentric Chromosomes in Nonstimulated Lymphocyte Prematurely Condensed Chromosomes After Telomere and Centromere Staining

Purpose: To combine telomere and centromere (TC) staining of premature chromosome condensation (PCC) fusions to identify dicentrics, centric rings, and acentric chromosomes, making possible the realization of a dose–response curve and automation of the process. Methods and Materials: Blood samples from healthy donors were exposed to {sup 60}Co irradiation at varying doses up to 8 Gy, followed by a repair period of 8 hours. Premature chromosome condensation fusions were carried out, and TC staining using peptide nucleic acid probes was performed. Chromosomal aberration (CA) scoring was carried out manually and automatically using PCC-TCScore software, developed in our laboratory. Results: We successfully optimized the hybridization conditions and image capture parameters, to increase the sensitivity and effectiveness of CA scoring. Dicentrics, centric rings, and acentric chromosomes were rapidly and accurately detected, leading to a linear-quadratic dose–response curve by manual scoring at up to 8 Gy. Using PCC-TCScore software for automatic scoring, we were able to detect 95% of dicentrics and centric rings. Conclusion: The introduction of TC staining to the PCC fusion technique has made possible the rapid scoring of unstable CAs, including dicentrics, with a level of accuracy and ease not previously possible. This new approach can be used for biological dosimetry in radiation emergency medicine, where the rapid and accurate detection of dicentrics is a high priority using automated scoring. Because there is no culture time, this new approach can also be used for the follow-up of patients treated by genotoxic therapy, creating the possibility to perform the estimation of induced chromosomal aberrations immediately after the blood draw.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Breaking the Crowther limit: Combining depth-sectioning and tilt tomography for high-resolution, wide-field 3D reconstructions

To date, high-resolution (<1 nm) imaging of extended objects in three-dimensions (3D) has not been possible. A restriction known as the Crowther criterion forces a tradeoff between object size and resolution for 3D reconstructions by tomography. Further, the sub-Angstrom resolution of aberration-corrected electron microscopes is accompanied by a greatly diminished depth of field, causing regions of larger specimens (>6 nm) to appear blurred or missing. We demonstrate a three-dimensional imaging method that overcomes both these limits by combining through-focal depth sectioning and traditional tilt-series tomography to reconstruct extended objects, with high-resolution, in all three dimensions. The large convergence angle in aberration corrected instruments now becomes a benefit and not a hindrance to higher quality reconstructions. A through-focal reconstruction over a 390 nm 3D carbon support containing over 100 dealloyed and nanoporous PtCu catalyst particles revealed with sub-nanometer detail the extensive and connected interior pore structure that is created by the dealloying instability.

3D imaging↗

A short story of imaging and spectroscopy of two-dimensional materials by scanning transmission electron microscopy

Here we present a short historical account of when single adatom impurities where first identified in two-dimensional materials by scanning transmission electron microscopy (STEM). We also present a study of the graphene low-loss (below 50 eV) response as a function of number of layers using electron energy-loss spectroscopy (EELS). The study shows that as few as three layers of graphene behave as bulk graphite for losses above 10 eV We also show examples of how point and extended defects can easily be resolved and structural dynamics can be readily capture by using aberration-corrected STEM imaging. Lastly, we show that the new generation of monochromators has opened up possibilities to explore new physics with an electron microscope. All these capabilities were enabled by the development of spherical aberration correctors and monochromators, where Ondrej Krivanek has played a key role.

2D materials↗