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The relationships between DNA damage from UV radiation, alkylating drugs and the methylated xanthines (MX) have been studied in normal and malignant rodent and human cells. A comparison of the level of DNA excision repair (repair replication and unscheduled DNA synthesis) confirms that some forms of alkylating-agent damage (probably mono-filar DNA adducts) are less completely removed by both normal and malignant rodent cells than by their human counterparts, rendering rodent cells more susceptible to the toxic potential of unexcised lesions. The toxicity of alkylating agents can be increased by the presence of several MXs during the period of DNA replication which follows infliction of the damage. Human cells appear capable of excising more DNA damage, rendering them somewhat less susceptible to enhancement of cytotoxicity by MX. This resistance of human cells is only quantitative, however, since 2 human cancer cell lines (HeLa and HT-29) could be sensitized to a variety of alkylating agents by appropriate concentrations of MX. Trimethylxanthine (caffeine) and the 2 clinically useful dimethylxanthines (theophylline and theobromine) appeared equally effective in sensitizing cells. The sensitization was dependent upon a slightly cytotoxic concentration of the MX and a suitably prolonged period of post-damage MX exposure. Of these 3 classic MXs, only theobromine might be clinically useful. The levels required for alkylating-agent sensitization exceed the clinically tolerable level of theophylline, and probably approach the tolerance of man to caffeine. The most likely mechanism by which MX sensitization is achieved is reversal of the inhibition of DNA replicon initiation which follows the infliction of significant DNA damage. Through the selection of suitable clinically useful alkylating agents (those dependent on active cellular transport for cell penetration) and appropriate MX scheduling, an enhanced therapeutic ratio might be achieved, potentially increasing the clinical usefulness of these alkylating agents. MX would thus form a useful class of agents adjuvant to conventional anti-cancer drugs.

Ectopic expression of telomerase results in an immortal phenotype in various types of normal cells, including primary human fibroblasts. In addition to its role in telomere lengthening, telomerase has now been found to have various functions, including the control of DNA repair, chromatin modification, and the control of expression of genes involved in cell cycle regulation. The investigations on the long-term effects of telomerase expression in normal human fibroblast highlighted that these cells show low frequencies of chromosomal aberrations. In this paper, we describe the karyotypic stability of human fibroblasts immortalized by expression of hTERT. The ectopic overexpression of telomerase is associated with unusual spontaneous as well as radiation-induced chromosome stability. In addition, we found that irradiation did not enhance plasmid integration in cells expressing hTERT, as has been reported for other cell types. Long-term studies illustrated that human fibroblasts immortalized by telomerase show an unusual stability for chromosomes and for plasmid integration sites, both with and without exposure to ionizing radiation. These results confirm a role for telomerase in genome stabilisation by a telomere-independent mechanism and point to the possibility for utilizing hTERT-immortalized normal human cells for the study of gene targeting. Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Telomeric repeat sequences, located at the end of eukaryotic chromosomes, have been detected at intrachromosomal locations in many species. Large blocks of telomeric sequences are located near the centromeres in hamster cells, and have been reported to break spontaneously or after exposure to ionizing radiation, leading to chromosome aberrations. In human cells, interstitial telomeric sequences (ITS) can be composed of short tracts of telomeric repeats (less than twenty), or of longer stretches of exact and degenerated hexanucleotides, mainly localized at subtelomeres. In this paper, we analyzed the radiation sensitivity of a naturally occurring short ITS localized in 2q31 and we found that this region is not a hot spot of radiation-induced chromosome breaks. We then selected a human cell line in which approximately 800 bp of telomeric DNA had been introduced by transfection into an internal euchromatic chromosomal region in chromosome 4q. In parallel, a cell line containing the plasmid without telomeric sequences was also analyzed. Both regions containing the transfected plasmids showed a higher frequency of radiation-induced breaks than expected, indicating that the instability of the regions containing the transfected sequences is not due to the presence of telomeric sequences. Taken together, our data show that ITS themselves do not enhance the formation of radiation-induced chromosome rearrangements in these human cell lines. Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Although most telomere repeat sequences are found at the ends of chromosomes, some telomeric repeat sequences are also found at intrachromosomal locations in mammalian cells. Several studies have found that these interstitial telomeric repeat sequences can promote chromosome instability in rodent cells, either spontaneously or following ionizing radiation. In the present study we describe the extensive cytogenetic analysis of three different human cell lines with plasmids containing telomeric repeat sequences integrated at interstitial sites. In two of these cell lines, Q18 and P8SX, instability has been detected in the chromosome containing the integrated plasmid, involving breakage/fusion/bridge cycles or amplification of the plasmid DNA, respectively. However, the data suggest that the instability observed is characteristic of the general instability in these cell lines and that the telomeric repeat sequences themselves are not responsible. Consistent with this interpretation, the chromosome containing an integrated plasmid with 500 bp of telomeric repeat sequences is highly stable in the third cell line, SNG28, which has a relatively stable genome. The stability of the chromosome containing the integrated plasmid sequences in SNG28 makes this an excellent cell line to study the effect of ionizing radiation on the stability of interstitial telomeric sequences in human cells. [ABSTRACT FROM AUTHOR]

Background: In 2014, we developed a QI-directed Morbidity and Mortality (M&M) Conference, prioritizing discussion of individual and system failures, as well as development of action items to prevent failure recurrence. However, due to a reliance on individual electronic documents to store M&M data, our ability to assess trends in failures and action item implementation was hindered. To address this issue, in 2019, we created a secure electronic health record (EHR)-integrated web application (web app) to store M&M data., Study Design: In this study, we assessed the impact of our web app on efficient review and tracking of M&M data, including system failure occurrence and closure of action items. Additionally, in 2021, it was discovered that a backlog of action items existed. To address this issue, we implemented a QI initiative to reduce the backlog, and used the web app to compare action item closure over time., Results: Use of the web app dramatically improved review of M&M data. During the study period, there was a 67.0% reduction in the occurrence of the most common system failures. Additionally, our QI initiative resulted in a 97.7% reduction in the duration of time to complete a single action item and a 61.1% increase in the on-time closure rate for action items., Conclusions: Integration of a web app into a QI-directed M&M Conference enhanced our ability to track system level failures and action item closure over time. Using this web app, we demonstrated that our M&M Conference achieved its intended goal of improving the quality of patient care., Level of Evidence: IV., Competing Interests: Conflicts of interest None., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Constructed wetlands (CWs) have been demonstrated as a cost-effective alternative to chemical treatment systems for mine waters, with the microbial communities attributed to promoting carbonation and aiding pH neutralization. However, few data are available for the long-term use of CWs treating alkaline leachates nor the activity of microbes within them. To investigate the feasibility of CW to buffer alkaline pH, a pilot-scale wetland was implemented in 2015 to treat alkaline bauxite residue leachate. After 5.5 years, samples of supernatant water and sediment were taken at 0.5 m increments along the 11 m long wetland. Waters were analysed for pH, EC and metal(loid) content, while sediment was subjected to physico-chemical assessment and element fractionation. Microbial biomass and community were assessed by phospholipid fatty acid analysis (PLFA) and functionality by the Rapid Automated Bacterial Impedance Technique (RABIT). Evidence presented demonstrates that the CW operating for 66 months effectively treats bauxite residue leachate, with reduced influent pH from 11.5 to 7.8. Trace element analysis revealed effective reduction in Al (94.9 %), As (86.7 %) and V (57.6 %) with substrate analysis revealing a frontloading of elevated pH and trace element content in the first 5 m of the wetland. Sediment Al, As and V were present mostly (>94 % of total) in recalcitrant forms. Sediment Na was mostly soluble (48-62 %), but soils were not sodic (ESP < 15 %). Investigations into the microbial community revealed greatest biomass was in the first 5 m of the wetland, where pH, EC and metal contents were greatest. Microbial respiration using endemic Phragmites australis as a substrate demonstrates an ability to cycle recalcitrant carbon sources within a CW system. These novel microbial findings highlight the need for further investigation into the microbial communities in alkaline CWs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Farm roadways are an important sub-component of the nutrient transfer continuum (NTC) and roadway runoff (RR), leading to nutrient pressures in receiving waters at different times of the year at catchment scale. This study developed a semi-quantitative risk assessment model for dairy farms that once populated with data identifies roadway sections where RR enters waters. The model contains parameters that represent source, mobilisation and transport-connectivity stages of the NTC defined as continuous or categorical variables. Each parameter has a corresponding scoring system in terms of connectivity likelihood to waters (L) and the associated impact on water quality (I) from which field data can be converted to a risk score (RS). The connectivity or impact risk of any roadway section is a sum of all parameter scores, i.e. 'Total Risk Score' (TRS). The risk scores were classified into 5 categories (very low, low, moderate, high and very high). Field data from seven farms enabled five equal interval risk score classifications to be developed (very low (110-134), low (135-158), moderate (159-182), high (183-206), very high (207-230)). Fieldwork data showed differences between the number of mapped roadway sections ranging from 35 to 76, with the lowest and highest risk scores being 110 and 230, respectively. Out of all sections scored 25.9 %, 45.6 %, 20.4 %, 6.4 %, and 2 % were in very low, low, moderate, high and very high categories, respectively. In terms of management, only 8.4 % (i.e. high or very high scores) had all components of the NTC and required RR mitigation. An examination of the mobilisation parameter showed that the % of roadway sections needing mitigation is likely to increase if rainfall increases on these farms. An uncertainty assessment limiting the model to different levels of connectivity confirmed that all components of the NTC and those with greater than moderate risk should only be considered in future mitigation plans. Future work should concentrate on adapting this methodology to a wide range of farm enterprises., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Chronic pain in multiple sclerosis is common and difficult to treat. Its mechanisms remain incompletely understood. Dysfunction of the descending pain modulatory system is known to contribute to human chronic pain conditions. However, it is not clear how alterations in executive function influence this network, despite healthy volunteer studies linking function of the descending pain modulatory system, to cognition. In adults with multiple sclerosis-associated chronic neuropathic limb pain, compared to those without pain, we hypothesized altered functional connectivity of the descending pain modulatory system, coupled to executive dysfunction. Specifically we hypothesized reduced mental flexibility, because of potential importance in stimulus reappraisal. To investigate these hypotheses, we conducted a case-control cross-sectional study of 47 adults with relapsing remitting multiple sclerosis (31 with chronic neuropathic limb pain, 16 without pain), employing clinical, neuropsychological, structural, and functional MRI measures. We measured brain lesions and atrophy affecting descending pain modulatory system structures. Both cognitive and affective dysfunctions were confirmed in the chronic neuropathic limb pain group, including reduced mental flexibility (Delis Kaplan Executive Function System card sorting tests P  < 0.001). Functional connectivity of rostral anterior cingulate and ventrolateral periaqueductal gray, key structures of the descending pain modulatory system, was significantly lower in the group experiencing chronic neuropathic pain. There was no significant between-group difference in whole-brain grey matter or lesion volumes, nor lesion volume affecting white matter tracts between rostral anterior cingulate and periaqueductal gray. Brainstem-specific lesion volume was higher in the chronic neuropathic limb pain group ( P  = 0.0017). Differential functional connectivity remained after correction for brainstem-specific lesion volume. Gabapentinoid medications were more frequently used in the chronic pain group. We describe executive dysfunction in people with multiple sclerosis affected by chronic neuropathic pain, along with functional and structural MRI evidence compatible with dysfunction of the descending pain modulatory system. These findings extend understanding of close inter-relationships between cognition, function of the descending pain modulatory system, and chronic pain, both in multiple sclerosis and more generally in human chronic pain conditions. These findings could support application of pharmacological and cognitive interventions in chronic neuropathic pain associated with multiple sclerosis., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Among the pleotropic roles of transforming growth factor-β (TGFβ) signaling in cancer, its impact on genomic stability is least understood. Inhibition of TGFβ signaling increases use of alternative end joining (alt-EJ), an error-prone DNA repair process that typically functions as a "backup" pathway if double-strand break repair by homologous recombination or nonhomologous end joining is compromised. However, the consequences of this functional relationship on therapeutic vulnerability in human cancer remain unknown. Here, we show that TGFβ broadly controls the DNA damage response and suppresses alt-EJ genes that are associated with genomic instability. Mechanistically based TGFβ and alt-EJ gene expression signatures were anticorrelated in glioblastoma, squamous cell lung cancer, and serous ovarian cancer. Consistent with error-prone repair, more of the genome was altered in tumors classified as low TGFβ and high alt-EJ, and the corresponding patients had better outcomes. Pan-cancer analysis of solid neoplasms revealed that alt-EJ genes were coordinately expressed and anticorrelated with TGFβ competency in 16 of 17 cancer types tested. Moreover, regardless of cancer type, tumors classified as low TGFβ and high alt-EJ were characterized by an insertion-deletion mutation signature containing short microhomologies and were more sensitive to genotoxic therapy. Collectively, experimental studies revealed that loss or inhibition of TGFβ signaling compromises the DNA damage response, resulting in ineffective repair by alt-EJ. Translation of this mechanistic relationship into gene expression signatures identified a robust anticorrelation that predicts response to genotoxic therapies, thereby expanding the potential therapeutic scope of TGFβ biology., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The secondary use of P-sorbing industrial by-products as a fertilizer or soil conditioner is gaining increased attention, particularly in light of diminishing reserves of rock phosphate traditionally used to manufacture P fertilizer. This study examined applications of red mud (RM) and water treatment residuals (WTR) at two levels of P saturation (i.e. 'as received' and partially saturated) in a soil incubation and runoff plot study. When incubated with soils ranging in texture and initial P concentration, P-sorbing residuals that were less enriched with P decreased water-extractable soil P (WEP) concentration to a greater extent than more P saturated residuals. In contrast to WTR treatments, not all of the RM applications decreased soil WEP concentrations below those of the control soils. The runoff study investigated soil P dynamics when partially P-saturated RM and WTR's were surface applied to grass plots at 2 t ha -1 on Day 0, followed by three rainfall simulations (7 cm h -1 for 30 min, Days 2, 7 and 28) and at 3 t ha -1 on Day 70 followed by two more rainfall simulations (Days 77 and 96). Application of residuals at these rates did not significantly increase dissolved reactive P (DRP) in runoff compared with unamended controls during the study. Forage cuttings taken 90 days after the first rainfall simulation indicated that nutrient uptake was not compromised by the application of the residuals. Overall results indicate that WTRs may be a more suitable soil amendment than RM residuals given their greater ability to reduce soil WEP across a range of soils without simultaneously increasing Mehlich-3 extractable soil P concentrations above the upper threshold limit (150 mg P kg -1 ), and their minimal impact on plant nutrient uptake., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Repair of DNA damage protects genomic integrity, which is key to tissue functional integrity. In cancer, the type and fidelity of DNA damage response is the fundamental basis for clinical response to cytotoxic therapy. Here we consider the contribution of transforming growth factor-beta (TGFβ), a ubiquitous, pleotropic cytokine that is abundant in the tumor microenvironment, to therapeutic response. The action of TGFβ is best illustrated in head and neck squamous cell carcinoma (HNSCC). Survival of HNSCC patients with human papilloma virus (HPV) positive cancer is more than double compared to those with HPV-negative HNSCC. Notably, HPV infection profoundly impairs TGFβ signaling. HPV blockade of TGFβ signaling, or pharmaceutical TGFβ inhibition that phenocopies HPV infection, shifts cancer cells from error-free homologous-recombination DNA double-strand-break (DSB) repair to error-prone alternative end-joining (altEJ). Cells using altEJ are more sensitive to standard of care radiotherapy and cisplatin, and are sensitized to PARP inhibitors. Hence, HPV-positive HNSCC is an experiment of nature that provides a strong rationale for the use of TGFβ inhibitors for optimal therapeutic combinations that improve patient outcome.

Objective: To inform feasibility and design of a future randomised controlled trial (RCT) using brain functional MRI (fMRI) to determine the mechanism of action of gabapentin in managing chronic pelvic pain (CPP) in women., Design: Mechanistic study embedded in pilot RCT., Setting: University Hospital., Participants: Twelve women (18-50 years) with CPP and no pelvic pathology (follow-up completed March 2014)., Intervention: Oral gabapentin (300-2700 mg) or matched placebo., Outcome Measures: After 12 weeks of treatment, participants underwent fMRI of the brain (Verio Siemens 3T MRI) during which noxious heat and punctate stimuli were delivered to the pelvis and arm. Outcome measures included pain (visual analogue scale), blood oxygen level dependent signal change and a semi-structured acceptability questionnaire at study completion prior to unblinding., Results: Full datasets were obtained for 11 participants. Following noxious heat to the abdomen, the gabapentin group (GG) had lower pain scores (Mean: 3.8 [SD 2.2]) than the placebo group (PG) (Mean: 5.8 [SD 0.9]). This was also the case for noxious heat to the arm with the GG having lower pain scores (Mean: 2.6 [SD 2.5]) than the PG (Mean: 6.2 [SD 1.1]). Seven out of 12 participants completed the acceptability questionnaire. 71% (five out of seven) described their participation in the fMRI study as positive; the remaining two rated it as a negative experience., Conclusions: Incorporating brain fMRI in a future RCT to determine the mechanism of action of gabapentin in managing CPP in women was feasible and acceptable to most women., Trial Registration Number: ISRCTN70960777., Competing Interests: Competing interests: The funders did not have any influence on the study design, data collection, analysis or interpretation of results., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. Published by BMJ.)

Purpose: Following cytotoxic therapy, 70% of patients with human papillomavirus (HPV)-positive oropharyngeal head and neck squamous cell carcinoma (HNSCC) are alive at 5 years compared with 30% of those with similar HPV-negative cancer. Loss of TGFβ signaling is a poorly studied consequence of HPV that could contribute to patient outcome by compromising DNA repair., Experimental Design: Human HNSCC cell lines ( n = 9), patient-derived xenografts ( n = 9), tissue microarray ( n = 194), TCGA expression data ( n = 279), and primary tumor specimens ( n = 10) were used to define the relationship between TGFβ competency, response to DNA damage, and type of DNA repair., Results: Analysis of HNSCC specimens in situ and in vitro showed that HPV associated with loss of TGFβ signaling that increased response to radiation or cisplatin. TGFβ suppressed miR-182, which inhibited both BRCA1, necessary for homologous recombination repair (HRR), and FOXO3, required for ATM kinase activity. TGFβ signaling blockade by either HPV or inhibitors released miR182 control, compromised HRR and increased response to PARP inhibition. Antagonizing miR-182 rescued the HRR deficit in HPV-positive cells. Loss of TGFβ signaling unexpectedly increased repair by error prone, alternative end-joining (alt-EJ)., Conclusions: HPV-positive HNSCC cells are unresponsive to TGFβ. Abrogated TGFβ signaling compromises repair by HRR and increases reliance on alt-EJ, which provides a mechanistic basis for sensitivity to PARP inhibitors. The effect of HPV in HNSCC provides critical validation of TGFβ's role in DNA repair proficiency and further raises the translational potential of TGFβ inhibitors in cancer therapy., (©2018 American Association for Cancer Research.)

This study aimed to quantify leaching losses of nitrogen (N), phosphorus (P) and carbon (C), as well as carbon dioxide (CO 2 ) and methane (CH 4 ) emissions from stored slurry, and from packed soil columns surface applied with unamended and chemically amended dairy and pig slurries, and dairy soiled water (DSW). The amendments to the slurries, which were applied individually and together, were: polyaluminum chloride (PAC) and zeolite for pig and dairy slurry, and liquid aluminium sulfate (alum) and zeolite for DSW. Application of pig slurry resulted in the highest total nitrogen (TN) and nitrate-nitrogen (NO 3 -N) fluxes (22 and 12 kg ha -1 ), whereas corresponding fluxes from dairy slurries and DSW were not significantly (p < 0.05) higher than those from the control soil. There were no significant (p < 0.05) differences in leachate N losses between unamended and amended dairy slurries, unamended and amended pig slurries, and unamended and amended DSW. There were no leachate P losses measured over the experimental duration. Total cumulative organic (TOC) and inorganic C (TIC) losses in leachate were highest for unamended dairy slurry (82 and 142 kg ha -1 ), and these were significantly (p < 0.05) reduced when amended with PAC (38 and 104 kg ha -1 ). The highest average cumulative CO 2 emissions for all treatments were measured for pig slurries (680 kg CO 2 -C ha -1 ) followed by DSW (515 kg CO 2 -C ha -1 ) and dairy slurries (486 kg CO 2 -C ha -1 ). The results indicate that pig slurry, either in raw or chemically amended form, poses the greatest environmental threat of leaching losses and gaseous emissions of CO 2 and CH 4 and, in general, amendment of wastewater with PAC, alum or zeolite, does not mitigate the risk of these losses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Land application of dairy soiled water (DSW) is expensive relative to its nutrient replacement value. The use of aerobic filters is an effective alternative method of treatment and potentially allows the final effluent to be reused on the farm. Knowledge gaps exist concerning the optimal design and operation of filters for the treatment of DSW. To address this, 18 laboratory-scale filters, with depths of either 0.6 m or 1 m, were intermittently loaded with DSW over periods of up to 220 days to evaluate the impacts of depth (0.6 m versus 1 m), organic loading rates (OLRs) (50 versus 155 g COD m(-2) d(-1)), and media type (woodchip versus sand) on organic, nutrient and suspended solids (SS) removals. The study found that media depth was important in contaminant removal in woodchip filters. Reductions of 78% chemical oxygen demand (COD), 95% SS, 85% total nitrogen (TN), 82% ammonium-nitrogen (NH4N), 50% total phosphorus (TP), and 54% dissolved reactive phosphorus (DRP) were measured in 1 m deep woodchip filters, which was greater than the reductions in 0.6 m deep woodchip filters. Woodchip filters also performed optimally when loaded at a high OLR (155 g COD m(-2) d(-1)), although the removal mechanism was primarily physical (i.e. straining) as opposed to biological. When operated at the same OLR and when of the same depth, the sand filters had better COD removals (96%) than woodchip (74%), but there was no significant difference between them in the removal of SS and NH4N. However, the likelihood of clogging makes sand filters less desirable than woodchip filters. Using the optimal designs of both configurations, the filter area required per cow for a woodchip filter is more than four times less than for a sand filter. Therefore, this study found that woodchip filters are more economically and environmentally effective in the treatment of DSW than sand filters, and optimal performance may be achieved using woodchip filters with a depth of at least 1 m, operated at an OLR of 155 g COD m(-2) d(-1)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Stereotactic body radiation therapy (SBRT) to central lung tumors is associated with normal -tissue toxicity. Highly conformal technologies may reduce the risk of complications. This study compares physical dose characteristics and anticipated risks of radiation pneumonitis (RP) among three SBRT modalities: robotic radiosurgery (RR), helical tomotherapy (HT) and volumetric modulated arc therapy (VMAT). Nine patients with central lung tumors ≤5 cm were compared. RR, HT and VMAT plans were developed per RTOG 0831. Dosimetric comparisons included target coverage, conformity index, heterogeneity index, gradient index, maximal dose at 2 cm from target (D2 cm), and dose-volume parameters for organs at risk (OARs). Efficiency endpoints included total beam-on time and monitor units. RP risk was derived from Lyman-Kutcher-Burman modeling on in-house software. The average GTV and PTV were 11.6 ± 7.86 cm(3) and 36.8 ± 18.1 cm(3). All techniques resulted in similar target coverage (p = 0.64) and dose conformity (p = 0.88). While RR had sharper fall-off gradient (p = 0.002) and lower D2 cm (p = 0.02), HT and VMAT produced greater homogeneity (p < 0.001) and delivery efficiency (p = 0.001). RP risk predicted from whole or contralateral lung volumes was less than 10%, but was 2-3 times higher using ipsilateral volumes. Using whole (p = 0.04, p = 0.02) or ipsilateral (p = 0.004, p = 0.0008) volumes, RR and VMAT had a lower risk of RP than HT. Using contralateral volumes, RR had the lowest RP risk (p = 0.0002, p = 0.0003 versus HT, VMAT). RR, HT and VMAT were able to provide clinically acceptable plans following the guidelines provided by RTOG 0813. All techniques provided similar coverage and conformity. RR seemed to produce a lower RP risk for a scenario of small PTV-OAR overlap and small PTV. VMAT and HT produced greater homogeneity, potentially desirable for a large PTV-OAR overlap. VMAT probably yields the lowest RP risk for a large PTV. Understanding subtle differences among these technologies may assist in situations where multiple choices of modality are available., (© The Author(s) 2014.)

A comprehensive understanding of cancer is predicated upon knowledge of the structure of malignant genomes underlying its many variant forms and the molecular mechanisms giving rise to them. It is well established that solid tumor genomes accumulate a large number of genome rearrangements during tumorigenesis. End Sequence Profiling (ESP) maps and clones genome breakpoints associated with all types of genome rearrangements elucidating the structural organization of tumor genomes. Here we extend the ESP methodology in several directions using the breast cancer cell line MCF-7. First, targeted ESP is applied to multiple amplified loci, revealing a complex process of rearrangement and co-amplification in these regions reminiscent of breakage/fusion/bridge cycles. Second, genome breakpoints identified by ESP are confirmed using a combination of DNA sequencing and PCR. Third, in vitro functional studies assign biological function to a rearranged tumor BAC clone, demonstrating that it encodes anti-apoptotic activity. Finally, ESP is extended to the transcriptome identifying four novel fusion transcripts and providing evidence that expression of fusion genes may be common in tumors. These results demonstrate the distinct advantages of ESP including: (1) the ability to detect all types of rearrangements and copy number changes; (2) straightforward integration of ESP data with the annotated genome sequence; (3) immortalization of the genome; (4) ability to generate tumor-specific reagents for in vitro and in vivo functional studies. Given these properties, ESP could play an important role in a tumor genome project.

Chromosome instability plays an important role in cancer by promoting the alterations in the genome required for tumor cell progression. The loss of telomeres that protect the ends of chromosomes and prevent chromosome fusion has been proposed as one mechanism for chromosome instability in cancer cells, however, there is little direct evidence to support this hypothesis. To investigate the relationship between spontaneous telomere loss and chromosome instability in human cancer cells, clones of the EJ-30 tumor cell line were isolated in which a herpes simplex virus thymidine kinase (HSV-tk) gene was integrated immediately adjacent to a telomere. Selection for HSV-tk-deficient cells with ganciclovir demonstrated a high rate of loss of the end these "marked" chromosomes (10-4 events/cell per generation). DNA sequence and cytogenetic analysis suggests that the loss of function of the HSV-tk gene most often involves telomere loss, sister chromatid fusion, and prolonged periods of chromosome instability. In some HSV-tk-deficient cells, telomeric repeat sequences were added on to the end of the truncated HSV-tk gene at a new location, whereas in others, no telomere was detected on the end of the marked chromosome. These results suggest that spontaneous telomere loss is a mechanism for chromosome instability in human cancer cells.

Telomere maintenance is essential in immortal cancer cells to compensate for DNA lost from the ends of chromosomes, to prevent chromosome fusion, and to facilitate chromosome segregation. However, the high rate of fusion of chromosomes near telomeres, termed telomere association, in many cancer cell lines has led to the proposal that some cancer cells may not efficiently perform telomere maintenance. Deficient telomere maintenance could play an important role in cancer because telomere associations and nondisjunction have been demonstrated to be mechanisms for genomic instability. To investigate this possibility, we have analyzed the telomeres of the human squamous cell carcinoma cell line SQ-9G, which has telomere associations in approximately 75% of the cells in the population. The absence of detectable telomeric repeat sequences at the sites of these telomere associations suggests that they result from telomere loss. The analysis of telomere length by quantitative in situ hybridization demonstrated that, compared to the human squamous cell carcinoma cell line SCC-61 which has few telomere associations, SQ-9G has more extensive heterogeneity in telomere length and more telomeres without detectable telomeric repeat sequences. The dynamics of the changes in telomere length also demonstrated a higher rate of fluctuation in telomere length, both on individual telomeres and coordinately on all telomeres. These results demonstrate that telomere maintenance can play a role in the genomic instability seen in cancer cells.

Objectives: Citibank, N.A., initiated a comprehensive health, demand, and disease management program in 1994, using program services offered by Healthtrac, Inc., of Menlo Park, California. Program components included an initial screening of employees, computerized triage of subjects into higher and lower risk intervention programs, extensive follow-up with the higher risk subjects, and general health education and awareness building. The objective of this study was to estimate the financial impact of this program on medical expenditures., Methods: A quasiexperimental design was applied comparing medical expenditures before vs. after the intervention for program participants and nonparticipants. The 22,838 subjects (11,194 program participants and 11,644 nonparticipants) were followed for an average of 38 months before and after administration of a Healthtrac health risk appraisal (HRA) instrument that triggered the start of the program. To adjust for selection bias to the extent possible with these data, multiple regression models were used to estimate the savings in medical expenditures associated with program participation. The resulting dollar savings were compared to program costs to estimate the economic return on the company's investment in the program., Results: The return on investment (ROI) was estimated to be between $4.56 and $4.73 saved per dollar spent on the program, depending on the discount rate applied. These results are similar to published evaluations of Healthtrac programs implemented with other populations., Conclusions: Despite limitations inherent in any retrospective observational study, the strong, positive ROI shown here suggests that a well-designed health management program (HMP), which focuses interventions on high risk populations, can result in monetary savings to an organization.

The proteins encoded by the SIR1, SIR2, SIR3 and SIR4 genes in yeast repress transcription at the mating type loci and telomeres. Among the SIR genes, SIR2 is the most evolutionarily conserved, and a number of genes with homology to SIR2 have been identified. In addition to transcriptional silencing, the product of SIR2 gene (Sir2p) has been shown to be involved in DNA repair and suppression of rDNA recombination. In the present study, the complete sequence of a human gene, SIR2L, with homology to the yeast SIR2 gene is presented. Comparison of the predicted sequence of the protein encoded by the SIR2L gene (SIR2Lp) with Sir2p or other proteins with homology to Sir2p reveals 20-33% overall identity and four highly conserved regions, the significance of which is unknown. SIR2L codes for a 2.1kb transcript which is expressed in various human tissues. The expression level of the transcript is found to be relatively high in the heart, brain and skeletal muscle tissues and low in lung and placenta. The intracellular location of SIR2Lp was visualized by fusion to the Green Fluorescent Protein or with a FLAG-tag. The results indicate that unlike Sir2p in yeast, SIR2Lp in human cells is found primarily in the cytoplasm. Using a mammalian inducible expression system, we also observed that unlike SIR2 in yeast, overexpression of SIR2L in human cancer cells has no effect on cell growth. Thus, although the human SIR2L gene appears to be related to the yeast SIR2 gene, it does not appear to have similar functions.