Mass Spectrometric Fingerprints of Bacteria and Archaea for Life Detection on Icy Moons.

The icy moons of the outer Solar System display evidence of subsurface liquid water and, therefore, potential habitability for life. Flybys of Saturn's moon Enceladus by the Cassini spacecraft have provided measurements of material from plumes that suggest hydrothermal activity and the presence of organic matter. Jupiter's moon Europa may have similar plumes and is the target for the forthcoming Europa Clipper mission that carries a high mass resolution and high sensitivity mass spectrometer, called the MAss Spectrometer for Planetary EXploration (MASPEX), with the capability for providing detailed characterization of any organic materials encountered. We have performed a series of experiments using pyrolysis-gas chromatography-mass spectrometry to characterize the mass spectrometric fingerprints of microbial life. A range of extremophile Archaea and Bacteria have been analyzed and the laboratory data converted to MASPEX-type signals. Molecular characteristics of protein, carbohydrate, and lipid structures were detected, and the characteristic fragmentation patterns corresponding to these different biological structures were identified. Protein pyrolysis fragments included phenols, nitrogen heterocycles, and cyclic dipeptides. Oxygen heterocycles, such as furans, were detected from carbohydrates. Our data reveal how mass spectrometry on Europa Clipper can aid in the identification of the presence of life, by looking for characteristic bacterial fingerprints that are similar to those from simple Earthly organisms.

Oral and Dermal Bioavailability Studies of Polycyclic Aromatic Hydrocarbons from Soils Containing Weathered Fragments of Clay Shooting Targets.

The relative oral bioavailability and dermal absorption of chemical substances from environmental media are key factors that are needed to accurately estimate site-specific risks and manage human exposures. This study evaluated the in vivo relative oral bioavailability and in vitro dermal absorption of several polycyclic aromatic hydrocarbons (PAHs) found in soils collected from two formerly used Department of Defense sites impacted by weathered fragments of clay shooting targets. Concentrations of individual carcinogenic PAHs in the ≤250 µm fraction of soil ranged from approximately 0.1 to 100 mg/kg. A novel sample preparation method was developed to produce accurate and precise test diets for oral studies. The resulting test diets showed consistent concentrations of PAHs in soil- and soil-extract-amended diets and a consistent PAH concentration profile. Mean oral relative bioavailability factors (RBAFs) and dermal absorption fractions (ABSd) for benzo(a)pyrene ranged from 8 to 14% and 0.58 to 1.3%, respectively. Using the RBAF and ABSd values, measured here, for benzo(a)pyrene in USEPA's regional screening level equations yields concentrations for residential soils that are approximately eight times higher than those when default values are used (e.g., 9.6 vs 1.2 mg/kg at a target excess risk of 1 × 10-5).

Testing the validity of a proposed dermal cancer slope factor for Benzo[a]pyrene.

In 2014, the United States Environmental Protection Agency (EPA) proposed a Dermal Slope Factor (DSF) for benzo[a]pyrene (BaP) of 0.006 (µg/day)-1 (USEPA 2014a). It would make cancer risk estimates associated with soil contact 100 times greater than those from soil ingestion and would predict that a large fraction of skin Basal Cell Carcinomas (BCCs) and Squamous Cell Carcinomas (SCCs) worldwide are caused by low level dermal exposures to PAHs, such as BaP. This is not logical given that sunlight (ultraviolet radiation (UV)) exposure is the generally recognized cause of BCCs and SCCs. This paper critically evaluates the proposed DSF. First, a reality check is performed using EPA standard risk assessment methods and comparing the results to actual BCC and SCC rates in the U.S. population. Then, the biological plausibility of the mechanism by which PAHs might cause human skin cancer is evaluated by exploring the generally recognized etiology of human skin cancer and comparing the genetic mutation signatures of rodent skin tumors caused by PAH exposures to those of human skin cancers. It is concluded that scientific flaws resulted in a proposed DSF value that greatly overestimates the skin cancer risk for humans dermally exposed to BaP in soil.

Risk assessment implications of site-specific oral relative bioavailability factors and dermal absorption fractions for polycyclic aromatic hydrocarbons in surface soils impacted by clay skeet target fragments.

Risk assessment conclusions for a site may differ when using site-specific versus default values for the relative bioavailability factor (RBAF) and dermal absorption fraction (ABS.d), because these inputs affect both surface soil screening levels and risk/hazard estimates. Indeed, our case study demonstrates that different conclusions may be reached as to regulatory need for remedial action to protect human health when evaluating soil sampling data for seven carcinogenic polycyclic aromatic hydrocarbons (PAHs) using site-specific versus default TCEQ and USEPA residential soil screening levels. Use of site-specific RBAF and ABS.d values increased carcinogenicity-based TCEQ and USEPA surface soil screening levels for PAHs by 4.4- and 6-fold on average, respectively. Soil screening levels for PAHs were more sensitive to changes in ingestion exposure route parameters than to changes in dermal exposure route parameters. Accordingly, site-specific RBAF and ABS.d information has important implications for screening chemicals at PAH-impacted sites, and in addition provides more realistic estimates of risks/hazards posed by PAHs in soil with reduced uncertainty compared to estimates based on default RBAF and ABS.d values. Although default values are generally deemed acceptable by regulatory agencies, use of risk/hazard estimates based on these default values may compel insufficiently justified remedial action in some instances.

Macromolecular organic compounds from the depths of Enceladus.

Saturn's moon Enceladus harbours a global water ocean 1 , which lies under an ice crust and above a rocky core 2 . Through warm cracks in the crust 3 a cryo-volcanic plume ejects ice grains and vapour into space4-7 that contain materials originating from the ocean8,9. Hydrothermal activity is suspected to occur deep inside the porous core10-12, powered by tidal dissipation 13 . So far, only simple organic compounds with molecular masses mostly below 50 atomic mass units have been observed in plume material6,14,15. Here we report observations of emitted ice grains containing concentrated and complex macromolecular organic material with molecular masses above 200 atomic mass units. The data constrain the macromolecular structure of organics detected in the ice grains and suggest the presence of a thin organic-rich film on top of the oceanic water table, where organic nucleation cores generated by the bursting of bubbles allow the probing of Enceladus' organic inventory in enhanced concentrations.

Cassini finds molecular hydrogen in the Enceladus plume: Evidence for hydrothermal processes.

Saturn's moon Enceladus has an ice-covered ocean; a plume of material erupts from cracks in the ice. The plume contains chemical signatures of water-rock interaction between the ocean and a rocky core. We used the Ion Neutral Mass Spectrometer onboard the Cassini spacecraft to detect molecular hydrogen in the plume. By using the instrument's open-source mode, background processes of hydrogen production in the instrument were minimized and quantified, enabling the identification of a statistically significant signal of hydrogen native to Enceladus. We find that the most plausible source of this hydrogen is ongoing hydrothermal reactions of rock containing reduced minerals and organic materials. The relatively high hydrogen abundance in the plume signals thermodynamic disequilibrium that favors the formation of methane from CO2 in Enceladus' ocean.

Retroperitoneal hemorrhage after ureteroscopy without laser lithotripsy: an extreme example of an underreported event?

Retroperitoneal hemorrhage and an associated hematoma are uncommon but potentially serious complications following ureteroscopy with laser lithotripsy. However, no reports of serious bleeding complications have been published regarding ureteroscopy without laser lithotripsy in the management of stone disease. We report of such a case here and then review the current literature in order to discuss the incidence, risk factors, and management of such events.

Quantitative determination of hydroxy polycylic aromatic hydrocarbons as a biomarker of exposure to carcinogenic polycyclic aromatic hydrocarbons.

A high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) method was developed for quantitative analysis of hydroxy polycyclic aromatic hydrocarbons (OH-PAHs). Four hydroxy metabolites of known and suspected carcinogenic PAHs (benzo[a]pyrene (B[a]P), benz[a]anthracene (B[a]A), and chrysene (CRY)) were selected as suitable biomarkers of PAH exposure and associated risks to human health. The analytical method included enzymatic deconjugation, liquid - liquid extraction, followed by derivatization with methyl-N-(trimethylsilyl) trifluoroacetamide and instrumental analysis. Photo-induced oxidation of target analytes - which has plagued previously published methods - was controlled by a combination of minimizing exposure to light, employing an antioxidant (2-mercaptoethanol) and utilizing a nitrogen atmosphere. Stability investigations also indicated that conjugated forms of the analytes are more stable than the non-conjugated forms. Accuracy and precision of the method were 77.4-101% (<4.9% RSD) in synthetic urine and 92.3-117% (<15% RSD) in human urine, respectively. Method detection limits, determined using eight replicates of low-level spiked human urine, ranged from 13 to 24pg/mL. The method was successfully applied for analysis of a pooled human urine sample and 78 mouse urine samples collected from mice fed with PAH-contaminated diets. In mouse urine, greater than 94% of each analyte was present in its conjugated form.

A quantitative assessment of risks of heavy metal residues in laundered shop towels and their use by workers.

This paper presents a risk assessment of exposure to metal residues in laundered shop towels by workers. The concentrations of 27 metals measured in a synthetic sweat leachate were used to estimate the releasable quantity of metals which could be transferred to workers' skin. Worker exposure was evaluated quantitatively with an exposure model that focused on towel-to-hand transfer and subsequent hand-to-food or -mouth transfers. The exposure model was based on conservative, but reasonable assumptions regarding towel use and default exposure factor values from the published literature or regulatory guidance. Transfer coefficients were derived from studies representative of the exposures to towel users. Contact frequencies were based on assumed high-end use of shop towels, but constrained by a theoretical maximum dermal loading. The risk estimates for workers developed for all metals were below applicable regulatory risk benchmarks. The risk assessment for lead utilized the Adult Lead Model and concluded that predicted lead intakes do not constitute a significant health hazard based on potential worker exposures. Uncertainties are discussed in relation to the overall confidence in the exposure estimates developed for each exposure pathway and the likelihood that the exposure model is under- or overestimating worker exposures and risk.

The UK Standardisation of Breast Radiotherapy (START) trials of radiotherapy hypofractionation for treatment of early breast cancer: 10-year follow-up results of two randomised controlled trials.

BACKGROUND: 5-year results of the UK Standardisation of Breast Radiotherapy (START) trials suggested that lower total doses of radiotherapy delivered in fewer, larger doses (fractions) are at least as safe and effective as the historical standard regimen (50 Gy in 25 fractions) for women after primary surgery for early breast cancer. In this prespecified analysis, we report the 10-year follow-up of the START trials testing 13 fraction and 15 fraction regimens. METHODS: From 1999 to 2002, women with completely excised invasive breast cancer (pT1-3a, pN0-1, M0) were enrolled from 35 UK radiotherapy centres. Patients were randomly assigned to a treatment regimen after primary surgery followed by chemotherapy and endocrine treatment (where prescribed). Randomisation was computer-generated and stratified by centre, type of primary surgery (breast-conservation surgery or mastectomy), and tumour bed boost radiotherapy. In START-A, a regimen of 50 Gy in 25 fractions over 5 weeks was compared with 41·6 Gy or 39 Gy in 13 fractions over 5 weeks. In START-B, a regimen of 50 Gy in 25 fractions over 5 weeks was compared with 40 Gy in 15 fractions over 3 weeks. Eligibility criteria included age older than 18 years and no immediate surgical reconstruction. Primary endpoints were local-regional tumour relapse and late normal tissue effects. Analysis was by intention to treat. Follow-up data are still being collected. This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN59368779. FINDINGS: START-A enrolled 2236 women. Median follow-up was 9·3 years (IQR 8·0-10·0), after which 139 local-regional relapses had occurred. 10-year rates of local-regional relapse did not differ significantly between the 41·6 Gy and 50 Gy regimen groups (6·3%, 95% CI 4·7-8·5 vs 7·4%, 5·5-10·0; hazard ratio [HR] 0·91, 95% CI 0·59-1·38; p=0·65) or the 39 Gy (8·8%, 95% CI 6·7-11·4) and 50 Gy regimen groups (HR 1·18, 95% CI 0·79-1·76; p=0·41). In START-A, moderate or marked breast induration, telangiectasia, and breast oedema were significantly less common normal tissue effects in the 39 Gy group than in the 50 Gy group. Normal tissue effects did not differ significantly between 41·6 Gy and 50 Gy groups. START-B enrolled 2215 women. Median follow-up was 9·9 years (IQR 7·5-10·1), after which 95 local-regional relapses had occurred. The proportion of patients with local-regional relapse at 10 years did not differ significantly between the 40 Gy group (4·3%, 95% CI 3·2-5·9) and the 50 Gy group (5·5%, 95% CI 4·2-7·2; HR 0·77, 95% CI 0·51-1·16; p=0·21). In START-B, breast shrinkage, telangiectasia, and breast oedema were significantly less common normal tissue effects in the 40 Gy group than in the 50 Gy group. INTERPRETATION: Long-term follow-up confirms that appropriately dosed hypofractionated radiotherapy is safe and effective for patients with early breast cancer. The results support the continued use of 40 Gy in 15 fractions, which has already been adopted by most UK centres as the standard of care for women requiring adjuvant radiotherapy for invasive early breast cancer. FUNDING: Cancer Research UK, UK Medical Research Council, UK Department of Health.

Screening-level population risk assessment of nasal tumors in the US due to naphthalene exposure.

Several naphthalene Unit Risk Factors (URFs) were proposed by the US Environmental Protection Agency in 2004 using data on the development of olfactory epithelial neuroblastomas and nasal respiratory epithelial adenomas in rats, but these URFs may be inappropriate and unnecessarily conservative for estimating human cancer risks. The purpose of the present exercise was to perform a screening-level population risk assessment of the US population to compare the observed number of naphthalene-induced nasal tumors in the US to the number that would be predicted if the URFs for naphthalene were as proposed. Nine scenarios were evaluated to represent the range of exposures individuals have typically experienced. Results indicate that the total predicted burden of naphthalene-induced nasal tumors per year in the US (65,905 rare nasal tumors, of which 29,121 are olfactory epithelial neuroblastomas) is much greater than the number of these tumors actually observed per year (910 total nasal tumors, of which 66 are olfactory neuroblastomas) over the period 1973-2006. This suggests that using rat nasal tumor data to derive a naphthalene URF for humans should be re-evaluated.

Inter-fraction motion and dosimetric consequences during breast intensity-modulated radiotherapy (IMRT).

BACKGROUND AND PURPOSE: Intensity-modulated radiotherapy (IMRT) can improve dose homogeneity within the breast planned target volume (PTV), but may be more susceptible to patient/organ motion than standard tangential radiotherapy (RT). We used daily cone-beam CT (CBCT) imaging to assess inter-fraction motion during breast IMRT and its subsequent impact on IMRT and standard RT dose homogeneity. MATERIALS AND METHODS: Ten breast cancer patients selected for IMRT were studied. CBCT images were acquired immediately after daily treatment. Automatic image co-registration was used to determine patient positioning variations. Daily PTV contours were used to calculate PTV variations and daily delivered IMRT and theoretically planned tangential RT dose. RESULTS: Group systematic (and random) setup errors detected by CBCT were 5.7 (3.9)mm laterally, 2.8 (3.5)mm vertically and 2.3 (3.2)mm longitudinally. Rotations >2 degrees in any axis occurred on 53/106 (50%) occasions. Daily PTV volume varied up to 23%. IMRT dose homogeneity was superior at planning and throughout the treatment compared with standard RT (1.8% vs. 15.8% PTV received >105% planned mean dose), despite increased motion sensitivity. CONCLUSIONS: CBCT revealed inadequacies of current patient positioning and verification procedures during breast RT and confirmed improved dose homogeneity using IMRT for the patients studied.

Cassini ion and neutral mass spectrometer: Enceladus plume composition and structure.

The Cassini spacecraft passed within 168.2 kilometers of the surface above the southern hemisphere at 19:55:22 universal time coordinated on 14 July 2005 during its closest approach to Enceladus. Before and after this time, a substantial atmospheric plume and coma were observed, detectable in the Ion and Neutral Mass Spectrometer (INMS) data set out to a distance of over 4000 kilometers from Enceladus. INMS data indicate that the atmospheric plume and coma are dominated by water, with significant amounts of carbon dioxide, an unidentified species with a mass-to-charge ratio of 28 daltons (either carbon monoxide or molecular nitrogen), and methane. Trace quantities (<1%) of acetylene and propane also appear to be present. Ammonia is present at a level that does not exceed 0.5%. The radial and angular distributions of the gas density near the closest approach, as well as other independent evidence, suggest a significant contribution to the plume from a source centered near the south polar cap, as distinct from a separately measured more uniform and possibly global source observed on the outbound leg of the flyby.

Experience of external beam radiotherapy given adjuvantly or at relapse following surgery for craniopharyngioma.

BACKGROUND AND PURPOSE: Evaluation of effect of timing of external beam radiation therapy (EBRT) following surgery for craniopharyngioma. MATERIALS AND METHODS: Between 1976 and 2002, 87 patients (28 children) received EBRT in a regional referral centre. Forty-four patients received EBRT adjuvantly and 43 on relapse. The median total dose was 42.5Gy (range 34.7-52.5Gy) in 2.25-2.83Gy fractions over a median of 20 days (range 17-32). Effect of EBRT timing, type of original surgery, age on survival, progression-free survival (PFS) and quality of life (QOL) was studied. RESULTS: Survival from diagnosis was 86 and 76% and PFS was 78 and 66% at 10 and 20 years, respectively, with no significant difference seen between those treated adjuvantly or at relapse or according to age. QOL deteriorated significantly from diagnosis to last follow-up. Excluding patients who relapsed following EBRT, QOL did not deteriorate significantly overall (P=0.35). Children had worse QOL and greater morbidity at all timepoints compared to adults. CONCLUSIONS: EBRT is effective both adjuvantly and at relapse. QOL deteriorates over time-relapse following EBRT was the only significant factor. Children have greater morbidity compared to adults, but no evidence for greater EBRT-induced toxicity was seen.

Ion neutral mass spectrometer results from the first flyby of Titan.

The Cassini Ion Neutral Mass Spectrometer (INMS) has obtained the first in situ composition measurements of the neutral densities of molecular nitrogen, methane, molecular hydrogen, argon, and a host of stable carbon-nitrile compounds in Titan's upper atmosphere. INMS in situ mass spectrometry has also provided evidence for atmospheric waves in the upper atmosphere and the first direct measurements of isotopes of nitrogen, carbon, and argon, which reveal interesting clues about the evolution of the atmosphere. The bulk composition and thermal structure of the moon's upper atmosphere do not appear to have changed considerably since the Voyager 1 flyby.

Dermal irritation assessment of three benzene sulfonate compounds.

Three benzene sulfonate compounds, benzene sulfonate, benzene meta-disulfonate, and para-phenol sulfonate, were reported to be present in groundwater sampled from residential wells near a former disposal site. Concentrations ranged from < 0.005 mg/L to 474 mg/L. Acute dermal irritation studies were performed on rabbits for each of the three sulfonate compounds to determine if they had the potential to cause irritation to the skin of persons using this groundwater for bathing, showering, or other uses where skin would be exposed. The studies were performed by Toxikon Corporation (Bedford, MA) in accordance with the United States Environmental Protection Agency (USEPA)'s 1998 Health Effects Test Guidelines. At the highest concentration tested (5000 mg/L), all three sulfonate compounds were considered to be slight irritants, producing very slight to mild erythema (Draize Score 1). In all cases, the reactions were reversible. At 2000 mg/L, benzene meta-disulfonate and para-phenol sulfonate caused no irritation and were considered not to be irritants. At 2000 mg/L, only benzene sulfonate was considered to be a slight irritant, producing a mild erythema that was completely reversible within 24 hours. Benzene sulfonate was not considered an irritant at 1000 mg/L or at 500 mg/L. It is important to note that all three sulfonate compounds produced only a slight irritation at the highest concentration tested. None of the compounds produced a moderate to severe irritation response (i.e., severe erythema, edema). Furthermore, any irritation response observed at the highest concentrations tested was reversible within 72 hours. The only irritation response observed at the second highest dose was also reversible within 24 hours.

Relationship between residual radiation-induced DNA double-strand breaks in cultured fibroblasts and late radiation reactions: a comparison of training and validation cohorts of breast cancer patients.

BACKGROUND AND PURPOSE: Using pulsed field gel electrophoresis (PFGE) a significant correlation was demonstrated between residual DNA double-strand breaks (dsbs) and the development of late radiation fibrosis in a group of 39 breast cancer patients studied retrospectively. This group formed a training cohort generating a hypothesis that there is a relationship between residual radiation-induced DNA dsbs in cultured fibroblasts and late radiotherapy reactions in breast cancer patients. The aim of this study was to retest and validate the hypothesis. MATERIALS AND METHODS: The study was retrospective. Skin biopsies were taken from a validation cohort of 50 breast cancer patients and PFGE was used to examine residual radiation-induced dsbs in cultured fibroblasts. Late morbidity was measured clinically as fibrosis and using the late effects on normal tissues scales that incorporate subjective, objective management and analytic data (LENT SOMA). RESULTS: PFGE data were obtained for 49 biopsies. In the 49 patients there was no correlation between residual DNA damage and either fibrosis (r=-0.027, P=0.85) or LENT SOMA (r=-0.10, P=0.48) scores. There was no significant relationship between residual damage and fibrosis for the combined training and validation cohorts of 88 patients (r=0.20, P=0.063). CONCLUSIONS: This study did not validate the hypothesis that there is a relationship between fibroblast residual DNA damage and late morbidity in breast cancer patients. The PFGE assay on fibroblasts is not a suitable test of the degree of late radiation-induced fibrosis in the breast.

Differential expression of cytokine genes in fibroblasts derived from skin biopsies of patients who developed minimal or severe normal tissue damage after radiotherapy.

Curative radiotherapy for cancer patients requires the use of radiation doses that are limited by the tolerance of the surrounding normal tissues. Unfortunately, these tolerance doses vary not only between tissues but also between individuals. In a small proportion of sensitive patients, exposure to radiation can lead to severe irreversible morbidity and even death several months to years after treatment. At present these radiosensitive patients can be identified only retrospectively. Here we describe a cytokine microarray technique that was used to identify differentially expressed gene transcripts in fibroblasts obtained from a small group of patients who suffered either negligible or severe normal damage to tissues after radiotherapy. If our preliminary findings can be confirmed, the availability of such markers may eventually allow the prediction of outcome prior to commencement of radiotherapy, and thus allow modification of radiotherapy protocols to minimize adverse late effects, without compromising tumor control.