Use of diamond sensors for a high-flux, high-rate X-ray pass-through diagnostic.

X-ray free-electron lasers (XFELs) deliver pulses of coherent X-rays on the femtosecond time scale, with potentially high repetition rates. While XFELs provide high peak intensities, both the intensity and the centroid of the beam fluctuate strongly on a pulse-to-pulse basis, motivating high-rate beam diagnostics that operate over a large dynamic range. The fast drift velocity, low X-ray absorption and high radiation tolerance properties of chemical vapour deposition diamonds make these crystals a promising candidate material for developing a fast (multi-GHz) pass-through diagnostic for the next generation of XFELs. A new approach to the design of a diamond sensor signal path is presented, along with associated characterization studies performed in the XPP endstation of the LINAC Coherent Light Source (LCLS) at SLAC. Qualitative charge collection profiles (collected charge versus time) are presented and compared with those from a commercially available detector. Quantitative results on the charge collection efficiency and signal collection times are presented over a range of approximately four orders of magnitude in the generated electron-hole plasma density.

Toward an integrated multi-gigahertz ionizing particle diagnostic.

Needs arising at both current and future accelerator facilities call for the development of radiation-hardened position-sensing diagnostics that can operate with multi-GHz repetition rates. Such instruments are likely to also have applications in the diagnosis of rapid plasma behavior. Building on the recent work of our Advanced Accelerator Diagnostics Collaboration, we are exploring the development of integrated multi-GHz ionizing particle detection systems based on chemical-vapor deposition diamond sensors, with the initial goal of producing a quadrant detector that can determine the intensity and centroid position of a particle beam at a repetition rate between 5 and 10 GHz. Results from our initial high-speed characterization work are presented, including those from a single-channel sensor with a GHz response. Approaches to achieving multi-GHz (5-10 GHz) rate capability, including the design of a dedicated Application Specific Integrated Circuit and the use of 3D RF-solver computer aided design software, are presented and discussed in more detail. 3D RF simulations suggest clean pulses of duration less than 250 ps (FWHM less than 125 ps) can be achieved with the approaches developed by this work.

Endogenous late positive component of the evoked potential in cats corresponding to P300 in humans.

A long-latency component of the averaged evoked potential recorded from cats was present only when the evoking stimulus was relevant to the task. The amplitude of this component varied inversely with stimulus probability and was independent of stimulus modality.

Flow cytometric localization within the cell cycle and isolation of viable cells following exposure to cytotoxic agents.

Viable cell sorting, based on flow cytometric analysis of DNA content and cell volume, was used to evaluate the cycle position and survival potential of Adriamycin (AdR)-treated or 1-beta-D-arabinofuranosylcytosine (ara-C)-treated CHO cells. Drug-treated cells initially stained with the vital, DNA-specific fluorochrome, Hoechst 33342, were analyzed for DNA content and volume, and sorting "windows" were established for subsequent sorting of duplicate unstained cell samples based only on cell volume. Another portion of the cell sample was fixed in ethanol, and stained with three fluorochromes for correlated flow cytometric analysis of DNA, RNA, and protein. Similarities in the viable cell volume distributions and the protein content distributions of the ethanol-fixed samples provided a means for indirectly determining the DNA and RNA contents of the sorted cells. Three regions (S, L, and I) were selected in the cell volume distributions corresponding to the range of near normal cell size (S), larger than normal cell size (I), and the extremely large cells (L). Adriamycin-treated or ara-C-treated cells sorted from the S region had survival values, respectively, 46 times and 7 times greater than the abnormally large cells in region L. Cells from the S region also respectively survived 14-fold (AdR-treated) and 7-fold (ara-C-treated) greater than the cells sorted from the I regions. RNA content levels for cells within the L region were three times and two times greater, respectively, than the AdR-treated and Ara-C-treated subpopulations in the S regions. Survival of subpopulations of G2-arrested, AdR-treated cells (I and L regions) was better correlated with relative abnormality in cell size than with position in the cell cycle. In addition to providing further support for the validity of the "balanced growth hypothesis," the results of this study suggest that two-parameter DNA content and cell volume measurements would be extremely useful for providing general guidelines for judging the effectiveness of therapy, especially in clinical diagnoses where cell sorting is impractical or impossible. From these analyses the frequency and cycle position of cells resistant to therapy can be estimated. Such information would be particularly useful for rapidly detecting drug-resistant cells and design of subsequent therapeutic regimens.

Structural requirements for intracellular transport of pulmonary surfactant protein B (SP-B).

Human SP-B is synthesized by the alveolar Type II epithelial cell as a 381 amino acid preproprotein. The 79 residue mature SP-B peptide is extremely hydrophobic and flanked by propeptides of 200 and 102 amino acids at its NH2- and COOH-termini, respectively. The purpose of this study was to identify peptide domains of the SP-B proprotein necessary for trafficking of the mature peptide in the secretory pathway. To this end several constructs were generated, by subcloning the full length human SP-B (SP-B), COOH-terminally truncated SP-B (SP-B delta C, in which residues 201-381 were deleted), NH2-terminally deleted SP-B (SP-B delta N, in which residues 28-200 were deleted), NH2-terminal propeptide (SP-BN), mature SP-B (SP-BM) and COOH-terminal propeptide (SP-BC), into the mammalian expression vector pcDNA3. The resulting expression constructs were characterized by DNA sequencing and in vitro transcription/translation and subsequently transfected into Chinese hamster ovary cells. 48 h after transfection, cells were labeled with [35S]-met/cys and analyzed by immunoprecipitation, SDS-PAGE and autoradiography. Proteins encoded by SP-B, SP-B delta C, SP-BN and SP-BC constructs were secreted into media; in contrast, SP-B constructs lacking the NH2-terminal propeptide (SP-B delta N) remained in the endoplasmic reticulum (as assessed by endoglycosidase H sensitivity) and were rapidly degraded. We conclude that (1) 27 amino acids at the NH2-terminus of SP-B contain a functional signal peptide and (2) the NH2-terminal propeptide of the SP-B precursor is necessary and sufficient for intracellular trafficking of the mature peptide.

Relationships between oxidative metabolism, macrophage activation, and antilisterial activity.

Previous reports from this laboratory have revealed that macrophages obtained from 7-day Listeria-immune mice elicited 15 h before harvest with heat-killed homologous microorganisms were able to kill Listeria monocytogenes while resident or elicited cells were not [14, 16]. In the present study, experiments were conducted to determine if phagocytosis-associated oxidative metabolic activity participates in the enhanced destruction of Listeria by activated macrophages. Investigations into production of oxygen radicals by zymosan-stimulated macrophages revealed that Listeria-immune antigen-elicited (LIAE) cells produced significantly more superoxide and hydrogen peroxide than did resident, thioglycolate, or Listeria antigen-elicited macrophages. Additionally, the percentage of nitroblue tetrazolium (NBT) dye positive cells following exposure to zymosan was maximal in the immune-elicited population. Utilizing a luminol-dependent assay, a short-term chemiluminescent (CL) burst was noted in phagocytizing macrophages. This response was greatest in the LIAE population that exhibited a tenfold increase in peak chemiluminescence over other cell types. Prolonged in vitro culture of these cells diminishes their capacity to generate oxygen radicals. Additionally, LIAE macrophages cultured in excess of 38 h exhibited a significant decrease in zymosan-stimulated hydrogen peroxide release while the decline in superoxide generation was minimal. A substantial diminution in the Listeria-stimulated CL response was also noted during this time period. However, phagocytosis of Listeria by LIAE cells failed to induce the level of oxygen metabolites seen when zymosan was used as the particulate stimulant. In addition, scavengers of oxygen radicals were found to be relatively ineffective in reducing the killing of L monocytogenes by immunologically activated macrophages in culture. It therefore appears that toxic oxygen species do not play a major role in the heightened killing of Listeria by activated macrophages.

Differential fluorochromasia of human lymphocytes as measured by flow cytometry.

Peripheral human lymphocytes reacted with fluorescein diacetate and analyzed by flow cytometry produced a bimodal fluorescence distribution that was shown to be attributable to the differential staining of T and B lymphocytes. Lymphocytes were fractionated into rosetting (T cell) and nonrosetting (B cell) populations. Both subfractions were reacted with fluorescein diacetate and analyzed by flow cytometry. The rosetting fraction was more fluorescent than the nonrosetting fraction, and the analysis of an appropriate mixture of the subfractionated populations produced a fluorescence distribution very similar to that obtained with unfractionated lymphocytes.

Light scattering with stream-in-air flow systems.

Both forward angle and 90 degrees light-scattering measurements have been used for cell sizing with stream-in-air flow systems with very little theoretical base for the measurements. Mie theory calculations are compared with measurements on plastic microspheres. Detector response for homogeneous spheres is shown to be sensitive to refractive index.

Prevalence of chronic skin and musculoskeletal conditions, United States-1969.

During 1969 the prevalence of chronic skin ,and musculoskeletal diseases among members of the civilian noninstitutionalized population was measured in the Health Interview Survey. Prevalence estimates and measures of impact of these conditions on the population covered by the Survey are presented in this report. This is the first report on the prevalence of chronic skin and musculoskeletal conditions based on data collected in health interviews to be published in the series, Vital and Health Statistics, and is one in a series of reports on specific condition groups (Series 10, Nos. 83,84, and 94). Methodological studies have shown that chronic conditions are generally underreported in interview surveys. Respondents in health interviews can report only those conditions of which they are aware and which they are willing to report to the interviewer. Reporting is better for those conditions which have made an impact on the affected individual and his family. Conditions that are severe, costly, or require treatment tend to be better reported than conditions having lesser impact. For instance, a condition which has caused hospitalization, limitation of activity, visits to the doctor, or days in bed is more likely to be reported in the interview than a condition which has little or no impact on the person.

Characteristics of persons with corrective lenses, United States -1971.

This report from the Health Interview Survey presents data on the use of corrective lenses for persons 3 years of age and over. Estimates are derived from a survey during 1971 of the civilian, noninstitutionalized population of the United States. Corrective lenses include eyeglasses and contact lenses. The term "corrective Ienses" is limited to visual aids worn to correct or improve vision and therefore excludes sunglasses worn only to filter light, safety glasses worn only for protection of the eyes, hand magnifying glasses, and other such devices. However, if the safety glasses are worn also for correction or improvement of vision, they are considered corrective lenses, as are prescription glasses. This report analyzes use of corrective Ienses by various demographic characteristics. An earlier report from the Health Interview Survey presented data on corrective lenses based on the July 1965-June 1966 survey. It contained information on age at which persons first obtained corrective lenses, type of prescription, usage, and the source of the optical examination if the individual was examined during the 2-year period prior to interview ("Characteristics of Persons with Corrective Lenses: United States, July 1965-June 1966," Vital and Health Statistics, Series 10, Number 53). A later section of the present report compares demographic differences in the proportion of the population with corrective lenses for the two time periods.

Disability days, United States-1968.

Included in this report are data on the total number and annual rates per person for disability days experienced by the civilian, noninstitutional population for data year 1968. Disability days included are restricted-activity days, beddays, and school-loss days. A day of restricted activity is defined as a day on which a person reduced his normal activities for the entire day because of illness or injury. Bed days, school-loss days, and work-loss days are included in the total number of restricted-activity days. Demographic characteristics used to describe the persons with disability days are age, sex, place of residence, geographic region, family income, usual activity, and color. Previous reports of data from the Health Interview Survey on disability days are published for July 1961-June 1962, July 1963-June 1964, and July 1965-June 1966 (vital and Health Statistics, SerieslO, Numbers 4,24, and 47).

Disability days, United States, 1971.

Data are presented in this report on the total number and annual rates per person for disability days experienced by the civilian, noninstitutionalized population for 1971. Disability days included are restricted-activity days, bed days, work-loss days, and school-loss days. A day of restricted activity is defined as a day on which a person reduced his normal activities for the entire day because of illness or injury. Bed days, work-loss days, and school-loss days are included in the total number of restricted-activity days. Demographic characteristics used to described the persons with disability days are age, sex, place of residence, geographic region, famiIy income, usual activity, color, employment status, and for the currently employed population, industry and occupation. Previous reports of data from the Health Interview Survey on disability days are pubIished for July 1961-June 1962, July 1963-June 1964, July 1965-June 1966, and January-December 1968 in Vital and Health Statistics, Series 10, Numbers 4, 24, 47, and 67. Data on work-loss days for the currently employed during 1968 are discussed in Series 10, Number 71. Summary data from July 1965-June 1966 and January-December 1968 are also shown in this report for comparative purposes.

Flow cytometric determination of radiation-induced chromosome damage and its correlation with cell survival.

Chinese hamster M3-1 cells were irradiated with several doses of X rays or alpha particles from 238Pu. Propidium iodide-stained chromosome suspensions were prepared at different times after irradiation; cells were also assayed for survival. The DNA histograms of these chromosomes showed increased background counts with increased doses of radiation. This increase in background was cell-cycle dependent and was correlated with cell survival. The correlation between radiation-induced chromosome damage and cell survival was the same for X rays and alpha particles. Data are presented which indicate that flow cytometric analysis of chromosomes of irradiated cell populations can be a useful adjunct to classical cytogenic analysis of irradiation-induced chromosomal damage by virtue of its ability to express and measure chromosomal damage not seen by classical cytogenic methods.

Coulter volume cell sorting to improve the precision of radiation survival assays.

A new method for measuring cell survival at low doses of ionizing radiation has been developed through the use of flow cytometric cell sorting on the basis of Coulter volume signals. The cell sorter is capable of deflecting a precisely known number of cells directly into culture dishes, thus eliminating any errors associated with cell dilution and volume sampling. The use of Coulter volume signals as the sorting parameter is shown to be noncytotoxic for a variety of cell lines. Comparison of radiation survival curves measured above the 10% survival level by either the cell sorter or standard dilution assay demonstrates the increased precision of the cell sorter technique . Because of these advantages of cell sorting over conventional methods of plating cells, this technique has many applications in the field of radiation biology and other studies of cell survival.

Mutational extinction induced by sequential X irradiation and actinomycin D treatments in Chinese hamster ovary cells.

The interactions of sequential X irradiation and actinomycin D (AMD) treatments for mutagenesis to 6-thioguanine resistance were investigated in CHO cells. Cells were exposed to single doses of X rays followed immediately by 1-h treatments with 0.1 or 1 microgram/ml AMD. X Rays alone induced mutagenesis which increased monotonically with dose to at least 8 Gy. AMD-treated control cultures showed slight to moderate cytotoxicity and little induced mutation. X Rays followed by AMD treatment produced bell-shaped mutagenesis dose-response curves with maximal mutation at approximately 5 or 4 Gy for 0.1 or 1.0 microgram/ml AMD, respectively. Induced mutation frequencies then fell to a negligible level at fractional survival levels below 0.10 for either combination treatment. Application of a stochastic Poisson distribution model to these data led to the prediction that two possible components govern induced mutation frequencies. First, X ray +AMD induced mutations may be depleted progressively with dose from the surviving populations by selective lethality, which we term mutational extinction. Second, X ray +AMD treatments were calculated to induce potentially much greater than additive mutagenesis. However, due to the overriding mutational extinction effect, most of these mutations are not recovered as viable colonies. These studies suggest that AMD binding to DNA immediately following irradiation may cause considerably enhanced mutagenic and often lethal DNA damage, and that mutational extinction may occur because these types of damage are statistically correlated in a sensitive subpopulation of exponentially growing CHO cells.

Radiobiology of ultrasoft X rays. II. Cultured C3H mouse cells (10T1/2).

In the first paper of this series (Radiat. Res. 110, 396-412 (1987], using V79 cells, we reported that the relative biological effectiveness (RBE) of ultrasoft X rays was found to increase with decreasing energy, and the oxygen enhancement ratio (OER) was found to decrease with decreasing energy. In this report, we present RBE and OER results for 10T1/2 cells that are known to grow uniformly flat and are considerably thinner than V79 cells. Thus the variation in dose across the cell nucleus is considerably reduced. The OER results agree well with our earlier V79 results. However, the RBE values for 10T1/2 cells compared to V79 cells are systematically less for all soft X rays and especially for 0.28 keV carbon-K (1.3 compared to 3.4 for V79 cells). Some plausible explanations are presented to reconcile the apparent discrepancy between V79 and 10T1/2 results.

Radiobiology of ultrasoft X rays. III. Normal human fibroblasts and the significance of terminal track structure in cell inactivation.

Ultrasoft characteristic X rays from carbon (0.28 keV) are severely attenuated as they pass through biological material, causing a nonuniform distribution of dose to cell nuclei. Complications of studying ultrasoft X rays can be minimized in this context by using cells with very thin cytoplasm and nuclei (e.g., less than the attenuation length of the X rays), and which exhibit a more nearly exponential dose response to cell killing, such as normal human fibroblasts compared with V79 cells. Using this cell system, we report the relative biological effectiveness (RBE) of A1-K and C-K X rays to be near unity. Previous studies of cell inactivation by characteristic carbon X rays gave RBEs of 3 to 4, supporting the idea that localized energy depositions from secondary electrons and primary track ends represent the principal mode of biological action for other low-LET radiations. In part, the reported high RBEs result from the use of mean dose to describe energy deposited within the cell nuclei by these poorly penetrating radiations. Implicit in the use of mean dose is that cellular damage varies linearly with dose within a critical target(s), an assumption that is of questionable validity for cells that exhibit pronounced curvilinear dose responses. The simplest interpretation of the present findings is that most energy depositions caused by track-end effects are not necessarily more damaging than the sparsely ionizing component.

Radiobiology of ultrasoft X rays. IV. Flat and round-shaped hamster cells (CHO-10B, HS-23).

The results reported earlier in this series indicated that the relative biological effectiveness (RBE) of ultrasoft X rays decreases with decreasing cell thickness, approaching unity for the thinnest cells used, plateau-phase human skin fibroblasts (HSF). The possible dependence of RBE on the configuration of the cell nucleus is investigated further in this paper using two CHO cell lines that attach well and have similar intrinsic radiosensitivities to 60Co gamma rays. One of the lines forms monolayers similar to V79 cells, while the other remains more spherical during growth. We find an increasing RBE with decreasing X-ray energy for both of these cell lines, consistent with our results using V79 cells. Also consistent with our results obtained with 10T1/2 and HSF cells, we find an increasing RBE with increasing cell thickness. The possible dependence of RBE on radiosensitivity and the use of the concept of mean dose for ultrasoft X rays is discussed.

Radiobiology of ultrasoft X rays. I. Cultured hamster cells (V79).

Ultrasoft X rays (approximately less than keV) provide a useful probe for the study of the physical parameters associated with the induction of biological lesions because the spatial scale of their energy depositions is of nanometer dimensions, comparable to that of critical structures within the cell. We report on cell-killing experiments using cultured hamster cells (V79) exposed to carbon K (0.28 keV), aluminum K (1.5 keV), copper K (8.0 keV), and 250 kVp X rays, under oxic and hypoxic conditions, and as a function of cell-cycle phase. Our principal results are: RBE increases with decreasing X-ray energy; OER decreases with decreasing X-ray energy; and cell-cycle response is similar for all X-ray energies. Our RBE results confirm earlier observations using ultrasoft X rays on mammalian cells. The shapes of fitted curves through the data for each energy are statistically indistinguishable from one another, implying that the enhanced effectiveness is purely dose modifying. The results reported herein generally support the view that single-track effects of radiation are predominantly due to very local energy depositions on the nanometer scale, which are principally responsible for observed radiobiological effects.