Irradiated Esophageal Cells are Protected from Radiation-Induced Recombination by MnSOD Gene Therapy

Radiation-induced DNA damage is a precursor to mutagenesis and cytotoxicity. During radiotherapy, exposure of healthy tissues can lead to severe side effects. We explored the potential of mitochondrial SOD (MnSOD) gene therapy to protect esophageal, pancreatic and bone marrow cells from radiation-induced genomic instability. Specifically, we measured the frequency of homologous recombination (HR) at an integrated transgene in the Fluorescent Yellow Direct Repeat (FYDR) mice, in which an HR event can give rise to a fluorescent signal. Mitochondrial SOD plasmid/liposome complex (MnSOD-PL) was administered to esophageal cells 24 h prior to 29 Gy upper-body irradiation. Single cell suspensions from FYDR, positive control FYDR-REC, and negative control C57BL/6NHsd (wild-type) mouse esophagus, pancreas and bone marrow were evaluated by flow cytometry. Radiation induced a statistically significant increase in HR 7 days after irradiation compared to unirradiated FYDR mice. MnSOD-PL significantly reduced the induction of HR by radiation at day 7 and also reduced the level of HR in the pancreas. Irradiation of the femur and tibial marrow with 8 Gy also induced a significant increase in HR at 7 days. Radioprotection by intraesophageal administration of MnSOD-PL was correlated with a reduced level of radiation-induced HR in esophageal cells. These results demonstrate the efficacy of MnSOD-PL for suppressing radiation-induced HR in vivo.National Institutes of Health (U.S.) (NIH Grant R01-CA83876-8)National Institute of Allergy and Infectious Diseases (U.S.) (NIH grant U19A1068021)National Institutes of Health (U.S.) (Grant T32-ES07020)United States. Dept. of Energy (DOE DE-FG01-04ER04)National Institutes of Health (U.S.) (NIH P01-CA26735

The impact of age, exposure and genetics on homologous recombination at the engineered repeat sequence in mice

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.Includes bibliographical references.Mitotic homologous recombination is a critical pathway for the repair of DNA double-strand breaks and broken replication forks. Although homologous recombination is generally error-free, recombination between misaligned sequences can lead to deleterious sequence rearrangements, and conditions that stimulate homologous recombination are associated with an increased risk of cancer. To study homologous recombination in vivo, we used Fluorescent Yellow Direct Repeat (FYDR) mice in which a homologous recombination event at a transgene yields a fluorescent cell. To study homologous recombination using FYDR mice, we developed one- and two-photon in situ imaging techniques that reveal both the frequency and the sizes of isolated recombinant cell clusters within intact pancreatic tissue. We then applied these tools to analyze the effects of cancer risk factors such as exposure, genetic predisposition and age on homologous recombination in vivo. To determine the effect of exposure to exogenous carcinogens on homologous recombination, FYDR mice were treated with two different chemotherapeutic agents, cisplatin and mitomycin-C.(cont.) Results show that exposure to these DNA damaging agents causes an induction of recombinant pancreatic cells in vivo, indicating that homologous recombination is an active repair pathway in adult pancreatic cells and that exposure to certain carcinogens stimulates recombinational repair. As a first step towards exploring the effect of genetic predisposition to genomic instability on homologous recombination in vivo, FYDR mice were crossed with mice carrying a defect in p53, a critical tumor suppressor that is mutated in almost 50% of all human tumors. Although loss of p53 is known to promote genomic instability, results show that p53 status does not significantly affect the spontaneous recombinant cell frequency in the pancreas in vivo or the rate of homologous recombination in cultured fibroblasts in vitro. Age is a risk factor for many types of cancers. Here we examined the effect of age on homologous recombination in two tissues of FYDR mice, pancreas and skin. In the pancreas, a dramatic accumulation of recombinant cells is seen with age, resulting from both de novo recombination events and clonal expansion of recombinant cells. In contrast, the skin shows no increase in recombinant cell frequency with age.(cont.) In vitro studies using primary fibroblasts indicate that the ability to undergo homologous recombination in response to endogenous and exogenous DNA damage does not significantly change with age, suggesting that these skin cells are able to undergo de novo homologous recombination events in aged mice. Thus, we propose that tissue-specific differences in the accumulation of recombinant cells with age result from differences in the ability of these cells to persist and clonally expand within the tissue. To further characterize the FYDR mice as a tool for studying homologous recombination, we exploited positive control FYDR-Recombined mice in which all cells carry the full-length coding sequence for enhanced yellow fluorescent protein. Studies show that expression of the FYDR transgene varies among mice, among tissues, and even among cells within a tissue. However, the variation in FYDR expression does not significantly change with age or exposure to exogenous carcinogens. Furthermore, positive control mice reveal that several tissues, in addition to the pancreas and skin, may be amenable for studying homologous recombination in the FYDR mice.(cont.) Thus, our studies demonstrate that FYDR mice combined with in situ imaging technology provide powerful tools to study the effects of cancer risk factors on homologous recombination in vivo. Ultimately, by applying these techniques to study additional cancer risk factors, we may better understand the relationship between DNA damage, homologous recombination and cancer.by Dominika M. Wiktor-Brown.Ph.D

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

Toxocariasis: a silent threat with a progressive public health impact

Background: Toxocariasis is a neglected parasitic zoonosis that afflicts millions of the pediatric and adolescent populations worldwide, especially in impoverished communities. This disease is caused by infection with the larvae of Toxocara canis and T. cati, the most ubiquitous intestinal nematode parasite in dogs and cats, respectively. In this article, recent advances in the epidemiology, clinical presentation, diagnosis and pharmacotherapies that have been used in the treatment of toxocariasis are reviewed. Main text: Over the past two decades, we have come far in our understanding of the biology and epidemiology of toxocariasis. However, lack of laboratory infrastructure in some countries, lack of uniform case definitions and limited surveillance infrastructure are some of the challenges that hindered the estimation of global disease burden. Toxocariasis encompasses four clinical forms: visceral, ocular, covert and neural. Incorrect or misdiagnosis of any of these disabling conditions can result in severe health consequences and considerable medical care spending. Fortunately, multiple diagnostic modalities are available, which if effectively used together with the administration of appropriate pharmacologic therapies, can minimize any unnecessary patient morbidity. Conclusions: Although progress has been made in the management of toxocariasis patients, there remains much work to be done. Implementation of new technologies and better understanding of the pathogenesis of toxocariasis can identify new diagnostic biomarkers, which may help in increasing diagnostic accuracy. Also, further clinical research breakthroughs are needed to develop better ways to effectively control and prevent this serious disease

A coarsened multinomial regression model for perinatal mother to child transmission of HIV

Background: In trials designed to estimate rates of perinatal mother to child transmission of HIV, HIV assays are scheduled at multiple points in time. Still, infection status for some infants at some time points may be unknown, particularly when interim analyses are conducted. Methods: Logistic regression models are commonly used to estimate covariate-adjusted transmission rates, but their methods for handling missing data may be inadequate. Here we propose using coarsened multinomial regression models to estimate cumulative and conditional rates of HIV transmission. Through simulation, we compare the proposed models to standard logistic models in terms of bias, mean squared error, coverage probability, and power. We consider a range of treatment effect and visit process scenarios, while including imperfect sensitivity of the assay and contamination of the endpoint due to early breastfeeding transmission. We illustrate the approach through analysis of data from a clinical trial designed to prevent perinatal transmission. Results: The proposed cumulative and conditional models performed well when compared to their logistic counterparts. Performance of the proposed cumulative model was particularly strong under scenarios where treatment was assumed to increase the risk of in utero transmission but decrease the risk of intrapartum and overall perinatal transmission and under scenarios designed to represent interim analyses. Power to estimate intrapartum and perinatal transmission was consistently higher for the proposed models. Conclusion: Coarsened multinomial regression models are preferred to standard logistic models for estimation of perinatal mother to child transmission of HIV, particularly when assays are missing or occur off-schedule for some infants.U.S. National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), and Dept. of Health and Human Services (DHHS)

Identification of C 25 highly branched isoprenoid (HBI) alkenes in diatoms of the genus Rhizosolenia in polar and sub-polar marine phytoplankton.

We report the identification of a range of C25 highly branched isoprenoid (HBI) alkenes and certain sterols in filtered phytoplankton samples obtained from western Svalbard (Arctic) and near South Georgia (South Atlantic, sub-Antarctic) in 2016 and 2014, respectively. The C25 HBIs contained 3–5 double bonds and had structures identified previously from analysis of laboratory diatom cultures. The same HBIs were also identified in individual diatom taxa isolated from the mixed assemblages and with reasonably similar distributions. Thus, C25 HBIs were identified in Rhizosolenia setigera isolated from western Svalbard near-surface waters, while the same HBIs were also found in R. polydactyla f. polydactyla and R. hebetata f. semispina picked from seawater collected from a site in the South Atlantic. The main sterol composition was slightly different between the two locations, with cholesta-5,24-dien-3β-ol (desmosterol) identified as one of the major components in the sample from West Svalbard, consistent with the diatom assemblage being dominated by R. setigera. In contrast, the major sterol in the South Atlantic sample was cholesta-5,22-dien-3β-ol (22-dehydrocholesterol), likely reflecting the relatively high proportion of the genus Pseudo-nitzschia. For both locations, the suite of HBIs included a tri-unsaturated isomer (HBI III; 6Z-2,6,10,14-tetramethyl-9-(3'-methylpent-4-enylidene)-pentadec-6-ene), proposed in previous studies as a potential proxy measure of pelagic sea ice-edge conditions, and thus, a counterpart to the mono- and di-unsaturated HBIs IP25 and IPSO25, which have been used as seasonal sea ice proxies in the Arctic and Antarctic, respectively. HBI III has been reported previously in sediments from West Svalbard and we report here its occurrence in a small number of surface sediments from the South Atlantic. For both regions, HBI III was present as one of the major HBIs in sediments, which contrasts the HBI distributions in the filtered phytoplankton samples, where HBIs with four and five double bonds were the major components. Differences in HBI distributions between phytoplankton and sediment samples may potentially be due to the presence of other (unanalysed) diatoms in the filtered water samples, seasonal/annual variability in the production of HBIs by a range of diatoms, differential degradation of HBIs between sources and sediments, or a combination of these. Interestingly, we did not detect any C30 HBIs in the water samples, picked cells or sediments from either location, despite earlier reports of these lipids in laboratory cultures of R. setigera. This study represents the first source identification of certain C25 HBI lipids under in situ pelagic conditions

Sustainable Forest Management Preferences of Interest Groups in Three Regions with Different Levels of Industrial Forestry: An Exploratory Attribute-Based Choice Experiment

The challenge of sustainable forest management is to integrate diverse and sometimes conflicting management objectives. In order to achieve this goal, we need a better understanding of the aspects influencing the preferences of diverse groups and how these groups make trade-offs between different attributes of SFM. We compare the SFM preferences of interest groups in regions with different forest use histories based on the reasoning that the condition of the forest reflects the forest use history of the area. The condition of the forest also shapes an individual’s forest values and attitudes. These held values and attitudes are thought to influence SFM preferences. We tested whether the SFM preferences vary amongst the different interest groups within and across regions. We collected data from 252 persons using a choice experiment approach, where participants chose multiple times among different options described by a combination of attributes that are assigned different levels. The novelty of our approach was the use of choice experiments in the assessment of regional preference differences. Given the complexity of interregional comparison and the small sample size, this was an exploratory study based on a purposive rather than random sample. Nevertheless, our results suggest that the aggregation of preferences of all individuals within a region does not reveal all information necessary for forest management planning since opposing viewpoints could cancel each other out and lead to an interpretation that does not reflect possibly polarised views. Although based on a small\ud sample size, the preferences of interest groups within a region are generally statistically significantly different from each other; however preferences of interest groups across regions are also significantly different. This illustrates the potential importance of assessing heterogeneity by region and by group

Chromatin Structure Regulates Gene Conversion

Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors. We found that the immunoglobulin Vλ pseudogene array is characterized by histone modifications associated with active chromatin. We directly demonstrated the importance of chromatin structure for gene conversion, using a regulatable experimental system in which the heterochromatin protein HP1 (Drosophila melanogaster Su[var]205), expressed as a fusion to Escherichia coli lactose repressor, is tethered to polymerized lactose operators integrated within the pseudo-Vλ donor array. Tethered HP1 diminished histone acetylation within the pseudo-Vλ array, and altered the outcome of Vλ diversification, so that nontemplated mutations rather than templated mutations predominated. Thus, chromatin structure regulates homology-directed repair. These results suggest that histone modifications may contribute to maintaining genomic stability by preventing recombination between repetitive sequences

Genetic Variation Stimulated by Epigenetic Modification

Homologous recombination is essential for maintaining genomic integrity. A common repair mechanism, it uses a homologous or homeologous donor as a template for repair of a damaged target gene. Such repair must be regulated, both to identify appropriate donors for repair, and to avoid excess or inappropriate recombination. We show that modifications of donor chromatin structure can promote homology-directed repair. These experiments demonstrate that either the activator VP16 or the histone chaperone, HIRA, accelerated gene conversion approximately 10-fold when tethered within the donor array for Ig gene conversion in the chicken B cell line DT40. VP16 greatly increased levels of acetylated histones H3 and H4, while tethered HIRA did not affect histone acetylation, but caused an increase in local nucleosome density and levels of histone H3.3. Thus, epigenetic modification can stimulate genetic variation. The evidence that distinct activating modifications can promote similar functional outcomes suggests that a variety of chromatin changes may regulate homologous recombination, and that disregulation of epigenetic marks may have deleterious genetic consequences