Specialization of rabbit reticulocyte transfer RNA content for hemoglobin synthesis.

The amount of acceptance of each amino acid per absorbancy unit of rabbit reticulocyte transfer RNA was determined. The results were compared with the amino acid composition of rabbit hemoglobin and with a similar determination of the transfer RNA content of rabbit liver. The histidine and isoleucine transfer RNA content of reticulocytes is specialized for the synthesis of hemoglobin, in which histidine is unusually common and isoleucine unusually scarce compared to most proteins.

TOPAS-nBio: An Extension to the TOPAS Simulation Toolkit for Cellular and Sub-cellular Radiobiology.

The TOPAS Monte Carlo (MC) system is used in radiation therapy and medical imaging research, having played a significant role in making Monte Carlo simulations widely available for proton therapy related research. While TOPAS provides detailed simulations of patient scale properties, the fundamental unit of the biological response to radiation is a cell. Thus, our goal was to develop TOPAS-nBio, an extension of TOPAS dedicated to advance understanding of radiobiological effects at the (sub-)cellular, (i.e., the cellular and sub-cellular) scale. TOPAS-nBio was designed as a set of open source classes that extends TOPAS to model radiobiological experiments. TOPAS-nBio is based on and extends Geant4-DNA, which extends the Geant4 toolkit, the basis of TOPAS, to include very low-energy interactions of particles down to vibrational energies, explicitly simulates every particle interaction (i.e., without using condensed histories) and propagates radiolysis products. To further facilitate the use of TOPAS-nBio, a graphical user interface was developed. TOPAS-nBio offers full track-structure Monte Carlo simulations, integration of chemical reactions within the first millisecond, an extensive catalogue of specialized cell geometries as well as sub-cellular structures such as DNA and mitochondria, and interfaces to mechanistic models of DNA repair kinetics. We compared TOPAS-nBio simulations to measured and published data of energy deposition patterns and chemical reaction rates (G values). Our simulations agreed well within the experimental uncertainties. Additionally, we expanded the chemical reactions and species provided in Geant4-DNA and developed a new method based on independent reaction times (IRT), including a total of 72 reactions classified into 6 types between neutral and charged species. Chemical stage simulations using IRT were a factor of 145 faster than with step-by-step tracking. Finally, we applied the geometric/chemical modeling to obtain initial yields of double-strand breaks (DSBs) in DNA fibers for proton irradiations of 3 and 50 MeV and compared the effect of including chemical reactions on the number and complexity of DSB induction. Over half of the DSBs were found to include chemical reactions with approximately 5% of DSBs caused only by chemical reactions. In conclusion, the TOPAS-nBio extension to the TOPAS MC application offers access to accurate and detailed multiscale simulations, from a macroscopic description of the radiation field to microscopic description of biological outcome for selected cells. TOPAS-nBio offers detailed physics and chemistry simulations of radiobiological experiments on cells simulating the initially induced damage and links to models of DNA repair kinetics.

A New Standard DNA Damage (SDD) Data Format.

Our understanding of radiation-induced cellular damage has greatly improved over the past few decades. Despite this progress, there are still many obstacles to fully understand how radiation interacts with biologically relevant cellular components, such as DNA, to cause observable end points such as cell killing. Damage in DNA is identified as a major route of cell killing. One hurdle when modeling biological effects is the difficulty in directly comparing results generated by members of different research groups. Multiple Monte Carlo codes have been developed to simulate damage induction at the DNA scale, while at the same time various groups have developed models that describe DNA repair processes with varying levels of detail. These repair models are intrinsically linked to the damage model employed in their development, making it difficult to disentangle systematic effects in either part of the modeling chain. These modeling chains typically consist of track-structure Monte Carlo simulations of the physical interactions creating direct damages to DNA, followed by simulations of the production and initial reactions of chemical species causing so-called "indirect" damages. After the induction of DNA damage, DNA repair models combine the simulated damage patterns with biological models to determine the biological consequences of the damage. To date, the effect of the environment, such as molecular oxygen (normoxic vs. hypoxic), has been poorly considered. We propose a new standard DNA damage (SDD) data format to unify the interface between the simulation of damage induction in DNA and the biological modeling of DNA repair processes, and introduce the effect of the environment (molecular oxygen or other compounds) as a flexible parameter. Such a standard greatly facilitates inter-model comparisons, providing an ideal environment to tease out model assumptions and identify persistent, underlying mechanisms. Through inter-model comparisons, this unified standard has the potential to greatly advance our understanding of the underlying mechanisms of radiation-induced DNA damage and the resulting observable biological effects when radiation parameters and/or environmental conditions change.

Yield of Image-Guided Needle Biopsy for Infectious Discitis: A Systematic Review and Meta-Analysis.

BACKGROUND: Image-guided biopsy is routinely conducted in patients with suspected discitis, though the sensitivity reported in the literature ranges widely. PURPOSE: We applied a systematic review and meta-analysis to estimate the yield of image-guided biopsy for infectious discitis. DATA SOURCES: We performed a literature search of 4 data bases: PubMed, Cochrane CENTRAL Register of Controlled Trials, Embase.com, and Scopus from data base inception to March 2016. STUDY SELECTION: A screen of 1814 articles identified 88 potentially relevant articles. Data were extracted for 33 articles, which were eligible if they were peer-reviewed publications of patients with clinical suspicion of discitis who underwent image-guided biopsy. DATA ANALYSIS: Patients with positive cultures out of total image-guided biopsy procedures were pooled to estimate yield with 95% confidence intervals. Hypothesis testing was performed with an inverse variance method after logit transformation. DATA SYNTHESIS: Image-guided biopsy has a yield of approximately 48% (793/1763), which is significantly lower than the open surgical biopsy yield of 76% (152/201; P < .01). Biopsy in patients with prior antibiotic exposure had a yield of 32% (106/346), which was not significantly different from the yield of 43% (336/813; P = .08) in patients without prior antibiotic exposure. LIMITATIONS: The conclusions of this meta-analysis are primarily limited by the heterogeneity of the included studies. CONCLUSIONS: Image-guided biopsy has a moderate yield for the diagnosis of infectious discitis, which is significantly lower than the yield of open surgical biopsy. This yield is not significantly affected by prior antibiotic use.

Comparing stochastic proton interactions simulated using TOPAS-nBio to experimental data from fluorescent nuclear track detectors.

Whilst Monte Carlo (MC) simulations of proton energy deposition have been well-validated at the macroscopic level, their microscopic validation remains lacking. Equally, no gold-standard yet exists for experimental metrology of individual proton tracks. In this work we compare the distributions of stochastic proton interactions simulated using the TOPAS-nBio MC platform against confocal microscope data for Al2O3:C,Mg fluorescent nuclear track detectors (FNTDs). We irradiated [Formula: see text] mm3 FNTD chips inside a water phantom, positioned at seven positions along a pristine proton Bragg peak with a range in water of 12 cm. MC simulations were implemented in two stages: (1) using TOPAS to model the beam properties within a water phantom and (2) using TOPAS-nBio with Geant4-DNA physics to score particle interactions through a water surrogate of Al2O3:C,Mg. The measured median track integrated brightness (IB) was observed to be strongly correlated to both (i) voxelized track-averaged linear energy transfer (LET) and (ii) frequency mean microdosimetric lineal energy, [Formula: see text], both simulated in pure water. Histograms of FNTD track IB were compared against TOPAS-nBio histograms of the number of terminal electrons per proton, scored in water with mass-density scaled to mimic Al2O3:C,Mg. Trends between exposure depths observed in TOPAS-nBio simulations were experimentally replicated in the study of FNTD track IB. Our results represent an important first step towards the experimental validation of MC simulations on the sub-cellular scale and suggest that FNTDs can enable experimental study of the microdosimetric properties of individual proton tracks.

Polarisation-based coincidence event discrimination: an in silico study towards a feasible scheme for Compton-PET.

Current positron emission tomography (PET) systems use temporally localised coincidence events discriminated by energy and time-of-flight information. The two annihilation photons are in an entangled polarisation state and, in principle, additional information from the polarisation correlation of photon pairs could be used to improve the accuracy of coincidence classification. In a previous study, we demonstrated that in principle, the polarisation correlation information could be transferred to an angular correlation in the distribution of scattered photon pairs in a planar Compton camera system. In the present study, we model a source-phantom-detector system using Geant4 and we develop a coincidence classification scheme that exploits the angular correlation of scattered annihilation quanta to improve the accuracy of coincidence detection. We find a [Formula: see text] image quality improvement in terms of the peak signal-to-noise ratio when scattered coincidence events are discriminated solely by their angular correlation, thus demonstrating the feasibility of this novel classification scheme. By integrating scatter events (both single-single and single-only) with unscattered coincidence events discriminated using conventional methods, our results suggest that Compton-PET may be a promising candidate for optimal emission tomographic imaging.

Dose enhancement effects to the nucleus and mitochondria from gold nanoparticles in the cytosol.

Gold nanoparticles (GNPs) have shown potential as dose enhancers for radiation therapy. Since damage to the genome affects the viability of a cell, it is generally assumed that GNPs have to localise within the cell nucleus. In practice, however, GNPs tend to localise in the cytoplasm yet still appear to have a dose enhancing effect on the cell. Whether this effect can be attributed to stress-induced biological mechanisms or to physical damage to extra-nuclear cellular targets is still unclear. There is however growing evidence to suggest that the cellular response to radiation can also be influenced by indirect processes induced when the nucleus is not directly targeted by radiation. The mitochondrion in particular may be an effective extra-nuclear radiation target given its many important functional roles in the cell. To more accurately predict the physical effect of radiation within different cell organelles, we measured the full chemical composition of a whole human lymphocytic JURKAT cell as well as two separate organelles; the cell nucleus and the mitochondrion. The experimental measurements found that all three biological materials had similar ionisation energies ∼70 eV, substantially lower than that of liquid water ∼78 eV. Monte Carlo simulations for 10-50 keV incident photons showed higher energy deposition and ionisation numbers in the cell and organelle materials compared to liquid water. Adding a 1% mass fraction of gold to each material increased the energy deposition by a factor of ∼1.8 when averaged over all incident photon energies. Simulations of a realistic compartmentalised cell show that the presence of gold in the cytosol increases the energy deposition in the mitochondrial volume more than within the nuclear volume. We find this is due to sub-micron delocalisation of energy by photoelectrons, making the mitochondria a potentially viable indirect radiation target for GNPs that localise to the cytosol.

The cytoplasm as a radiation target: an in silico study of microbeam cell irradiation.

We performed in silico microbeam cell irradiation modelling to quantitatively investigate ionisations resulting from soft x-ray and alpha particle microbeams targeting the cytoplasm of a realistic cell model. Our results on the spatial distribution of ionisations show that as x-rays are susceptible to scatter within a cell that can lead to ionisations in the nucleus, soft x-ray microbeams may not be suitable for investigating the DNA damage response to radiation targeting the cytoplasm alone. In contrast, ionisations from an ideal alpha microbeam are tightly confined to the cytoplasm, but a realistic alpha microbeam degrades upon interaction with components upstream of the cellular target. Thus it is difficult to completely rule out a contribution from alpha particle hits to the nucleus when investigating DNA damage response to cytoplasmic irradiation. We find that although the cytoplasm targeting efficiency of an alpha microbeam is better than that of a soft x-ray microbeam (the probability of stray alphas hitting the nucleus is 0.2% compared to 3.6% for x-rays), stray alphas produce more ionisations in the nucleus and thus have greater potential for initiating damage responses therein. Our results suggest that observed biological responses to cytoplasmic irradiation include a small component that can be attributed to stray ionisations in the nucleus resulting from the stochastic nature of particle interactions that cause out-of-beam scatter. This contribution is difficult to isolate experimentally, thus demonstrating the value of the in silico approach.

Detection of HIV-1 provirus in bronchoalveolar lavage cells by polymerase chain reaction.

This study was undertaken to evaluate whether HIV-seropositive individuals harbor HIV provirus in cells obtained by bronchoalveolar lavage (BAL). BAL cells were obtained from 14 HIV-positive patients undergoing bronchoscopy for evaluation of acute pulmonary symptoms. Cells were fractionated into macrophage-enriched and lymphocyte-enriched populations. The quantity of HIV-1 proviral DNA in the unfractionated BAL cells and in each population of fractionated cells was determined following polymerase chain reaction (PCR) amplification. Detectable quantities (3-90 copies/100,000 cells) of HIV-1 proviral DNA were found in unfractionated BAL cells in 12 of 14 patients. In the other two patients, provirus was detected after a sevenfold enrichment of lymphocytes. Provirus was also detected in BAL macrophages from 8/14 patients although proviral content was significantly higher in the lymphocyte fraction (133 +/- 72 vs. 35 +/- 22 proviral copies, p = 0.03). No correlation was seen with the ability to detect provirus in lymphocyte- or macrophage-enriched fractions and clinical diagnosis (e.g., Pneumocystis carinii pneumonia). The data suggest that lymphocytes are the predominant cells that contain provirus found in the lungs, although macrophages may be infected in some patients.

Optimisation of the imaging and dosimetric characteristics of an electronic portal imaging device employing plastic scintillating fibres using Monte Carlo simulations.

A Monte Carlo model of a novel electronic portal imaging device (EPID) has been developed using Geant4 and its performance for imaging and dosimetry applications in radiotherapy has been characterised. The EPID geometry is based on a physical prototype under ongoing investigation and comprises an array of plastic scintillating fibres in place of the metal plate/phosphor screen in standard EPIDs. Geometrical and optical transport parameters were varied to investigate their impact on imaging and dosimetry performance. Detection efficiency was most sensitive to variations in fibre length, achieving a peak value of 36% at 50 mm using 400 keV x-rays for the lengths considered. Increases in efficiency for longer fibres were partially offset by reductions in sensitivity. Removing the extra-mural absorber surrounding individual fibres severely decreased the modulation transfer function (MTF), highlighting its importance in maximising spatial resolution. Field size response and relative dose profile simulations demonstrated a water-equivalent dose response and thus the prototype's suitability for dosimetry applications. Element-to-element mismatch between scintillating fibres and underlying photodiode pixels resulted in a reduced MTF for high spatial frequencies and quasi-periodic variations in dose profile response. This effect is eliminated when fibres are precisely matched to underlying pixels. Simulations strongly suggest that with further optimisation, this prototype EPID may be capable of simultaneous imaging and dosimetry in radiotherapy.

Towards optimal imaging with PET: an in silico feasibility study.

The efficacy of Positron Emission Tomography (PET) imaging relies fundamentally on the ability of the system to accurately identify true coincidence events. With existing systems, this is currently accomplished with an energy acceptance criterion followed by correction techniques to remove suspected false coincidence events. These corrections generally result in signal and contrast loss and thus limit the PET system's ability to achieve optimum image quality. A key property of annihilation radiation is that the photons are polarised with respect to each other. This polarisation correlation offers a potentially powerful discriminator, independent of energy, to accurately identify true events. In this proof of concept study, we investigate how photon polarisation information can be exploited in PET imaging by developing a method to discriminate true coincidences using the polarisation correlation of annihilation pairs. We implement this method using a Geant4 PET simulation of a GE Advance/Discovery LS system and demonstrate the potential advantages of the polarisation coincidence selection method over a standard energy criterion method. Current PET ring detectors are not capable of exploiting the polarisation correlation of the photon pairs. Compton PET systems, however are promising candidates for this application. We demonstrate the feasibility of a two-component Compton camera system in identifying true coincidences with Monte Carlo simulations. Our study demonstrates the potential of improving signal gain using polarisation, particularly for high photon emission rates. We also demonstrate the ability of the Compton camera at exploiting this polarisation correlation in PET.

Radiation damage on sub-cellular scales: beyond DNA.

This study investigates a model cell as a target for low-dose radiation using Monte Carlo simulations. Mono-energetic electrons and photons are used with initial energies between 10 and 50 keV, relevant to out-of-field radiotherapy scenarios where modern treatment modalities expose relatively large amounts of healthy tissue to low-dose radiation, and also to microbeam cell irradiation studies which show the importance of the cytoplasm as a radiation target. The relative proportions of number of ionizations and total energy deposit in the nucleus and cytoplasm are calculated. We show that for a macroscopic dose of no more than 1 Gy only a few hundred ionizations occur in the nucleus volume whereas the number of ionizations in the cytoplasm is over a magnitude larger. We find that the cell geometry can have an appreciable effect on the energy deposit in the cell and can cause a nonlinear increase in energy deposit with cytoplasm density. We also show that changing the nucleus volume has negligible effect on the total energy deposit but alters the relative proportion deposited in the nucleus and cytoplasm; the nucleus volume must increase to approximately the same volume as the cytoplasm before the energy deposit in the nucleus matches that in the cytoplasm. Additionally we find that energy deposited by electrons is generally insensitive to spatial variations in chemical composition, which can be attributed to negligible differences in electron stopping power for cytoplasm and nucleus materials. On the other hand, we find that chemical composition can affect energy deposited by photons due to non-negligible differences in attenuation coefficients. These results are of relevance in considering radiation effects in healthy cells, which tend to have smaller nuclei. Our results further show that the cytoplasm and organelles residing therein can be important targets for low-dose radiation damage in healthy cells and warrant investigation as much as the conventional focus of a high-dose radiation DNA target in tumour cells.

The determination of the efficiency of a Compton suppressed HPGe detector using Monte Carlo simulations.

A Compton suppressed high-purity germanium (HPGe) detector is well suited to the analysis of low levels of radioactivity in environmental samples. The difference in geometry, density and composition of environmental calibration standards (e.g. soil) can contribute to excessive experimental uncertainty to the measured efficiency curve. Furthermore multiple detectors, like those used in a Compton suppressed system, can add complexities to the calibration process. Monte Carlo simulations can be a powerful complement in calibrating these types of detector systems, provided enough physical information on the system is known. A full detector model using the Geant4 simulation toolkit is presented and the system is modelled in both the suppressed and unsuppressed mode of operation. The full energy peak efficiencies of radionuclides from a standard source sample is calculated and compared to experimental measurements. The experimental results agree relatively well with the simulated values (within ∼5 - 20%). The simulations show that coincidence losses in the Compton suppression system can cause radionuclide specific effects on the detector efficiency, especially in the Compton suppressed mode of the detector. Additionally since low energy photons are more sensitive to small inaccuracies in the computational detector model than high energy photons, large discrepancies may occur at energies lower than ∼100 keV.

The function of the histidine tRNA isoaccepting species in hemoglobin synthesis.

Rabbit reticulocytes contain two RNA isoaccepting species for histidine as resolved by various chromatographic methods, while rabbit liver contains only one. These isoacceptors cannot be distinguished on the basis of coding properties, consistent with the "Wobble Hypothesis" (Smith, D.W.E., Meltzer, V.N., and McNamara, A.L. (1974) Biochim. Biophys. Acta 349, 366-375). Their function in hemoglobin synthesis in reticulocyte lysates has been investigated. Each of the tRNA isoacceptors of reticulocytes and the tRNA species of liver can incorporate histidine into positions in hemoglobin encoded by both of the histidine code words, CAC and CAU, and it is likely that each can incorporate histidine into all of the histidine-containing positions of hemoglobin. Even in experiments in which the two histidine tRNA species of reticulocytes are placed together in a lysate and are therefore in competition with each other, each incorporates histidine into all of the histidine-containing positions. There is no evidence that any residues are incorporated preferentially by either of the tRNA species. The two species are attached to reticulocyte ribosomes in the same proportion as they occur in the reticulocyte, also suggesting that neither of them is used preferentially in hemoglobin synthesis. The first of the two reticulocyte histidine isoacceptors and the histidine tRNA of rabbit liver contain Q base.

Lysine tRNAs from Bacillus subtilis 168: function of the isoacceptors in a rabbit reticulocyte cell-free protein-synthesizing system.

The two principal tRNA Lys isoaccepting species of Bacillus subtilis were compared in their functional activity in translating rabbit globin. Although neither species demonstrates any preference in reading either of the lysine codons, there is an overall preference for tRNa Lys 3 in lysine incorporation. The ratios of lysine incorporated by the two species into the different lysine-containing sites in the globin subunits vary over a more than two-fold range. As described in the accompanying paper, tRNA Lys 1 is a hypomodified form of tRNA Lys 3. Consistent with studies on other rRNA species, the fully modified isoacceptor functions preferentially. In contrast to these results, however, the fully modified isoacceptor (tRNA Lys 3) is found predominantly in rapidly dividing cells while the hypomodified isoacceptor (tRNA Lys 1) predominates in the stationary cells and spores of B.l subtilis.

The effects of a post-transcriptional modification on the function of tRNALys isoaccepting species in translation.

Isoacceptors of rabbit liver tRNALys which preferentially translate the codon AAG were compared for their function in several aspects of translation. As shown in other laboratories, Lys-tRNALys1,2 are two isoacceptors which differ from each other by a single base pair and are fully modified with N6-threonyl-adenosine adjacent to the anticodon. Lys-tRNALys4, which occurs commonly in rapidly dividing mammalian cells and tissues, is hypomodified at several bases and contains a precursor of N6-threonyl-adenosine next to its anticodon. These isoacceptors were incubated in cell-free protein synthesizing systems which contain rabbit globin mRNA. (Lys-tRNALys3 which translates AAA was also included.) The resulting globin was isolated and digested with trypsin, and the relative incorporation of lysine from Lys-tRNALys1,2 and from Lys-tRNALys4 into lysine-containing sites in the globin peptides as determined. Lys-tRNALys1,2 and Lys-tRNALys4 translate AAG preferentially, but Lys-tRNALys4 wobbles more than the former and translates AAA codons more efficiently. Overall, Lys-tRNALys1,2 is preferred in globin synthesis by about 30% compared to Lys-tRNALys4, and with one exception, the incorporation of lysine into the individual AAG lysine-containing sites in globin occurs more efficiently from Lys-tRNALys1,2. There is, however, considerable variation from site to site in the relative efficiencies of the Lys-tRNAs in incorporation.

Hepatitis B core antigen antibody as an indicator of a low grade carrier state for hepatitis B virus in a Saudi Arabian blood donor population.

Blood donor screening for anti-hepatitis B core antigen (anti-HBc) was introduced as a surrogate marker of non-A, non-B hepatitis prior to the availability of a specific test for hepatitis C. In areas endemic for hepatitis B virus (HBV), such as Saudi Arabia, earlier studies indicated that up to 30% of blood donors might disqualify if screened for anti-HBc. The issue was readdressed in a study of 6035 consecutive first-time Saudi national blood donors in an attempt to identify a subgroup of anti-HBc positive donors who might be at high risk of being low grade carriers of HBV. An isolated anti-HBc of high titer in a donor with a low or absent anti-hepatitis B surface antigen (anti-HBsAg) was taken as an indicator of increased risk of a low grade carrier state. Using this algorithm, an additional 125 (2%) donors would disqualify. HBsAg immune complex assays and polymerase chain reaction of donor samples with an isolated anti-HBc identified two donors with immune complexes and two donors with HBV DNA. All four donor samples expressed over 90% neutralization in the anti-HBc supplementary testing, indicating high titer anti-HBc. These findings seem to support the suggested policy of donor exclusion based on the anti-HBc and anti-HBsAg serology as a means to eliminate low grade carriers of HBV in endemic areas without jeopardizing the blood supply.