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Study Purpose: The incidence of breast cancer (BC) in young women under age 40 years has increased substantially over time, underscoring the need for a better understanding of early life modifiers of risk. We examined the association of recreational physical activity (RPA) with BC risk before age 40 years, focusing on RPA during the adolescent window of susceptibility. Methods: We used data from the ProF-SC Study, which is enriched for young women at increased risk for BC. We conducted an ambidirectional cohort analysis that included women, aged = 55 years at baseline, who were enrolled within 5 years of their first primary BC diagnosis or had no history of BC at baseline (n = 14,865). Women reported by baseline questionnaire their average levels of moderate and strenuous RPA during adolescence (12-17 years), which we converted to total metabolic equivalents per week (METs-per-week) and categorized into quartiles after adjusting for baseline age. We conducted attained age analyses until 40 years using multivariable Cox proportional hazards regression models adjusted for baseline sociodemographic and lifestyle factors. Follow-up started at age 18 years and ended at age at first invasive BC diagnosis, last follow-up, or 40 years, whichever came first. Results: There were 1,646 BC diagnoses before age 40 during 30,5594 person-years of follow-up. Being in the highest vs. lowest quartile of adolescent RPA was significantly associated with a 16% reduced risk of BC until age 40 before adjustment for baseline RPA (HR = 0.84, 95% CI = 0.73-0.97). This association was attenuated and no longer statistically significant after adjustment for baseline RPA (HR = 0.89, 95% CI = 0.77-1.04). We identified a statistically significant multiplicative interaction between adolescent and baseline RPA (interaction P value = 0.03). A one standard deviation increase in METs-per-week of baseline RPA was associated with a 5% reduced BC risk for women in the 10th percentile of adolescent RPA (HR = 0.95, 95% CI = 0.90-1.00) and an 11% reduced risk in the 90th percentile (HR = 0.89, 95% CI = 0.84-0.95). Conclusions: These findings suggest that adolescent physical activity might reduce breast cancer risk in young women and underscore the importance of maintain or increasing RPA levels across the lifecourse. [ABSTRACT FROM AUTHOR]

DNA repair phenotype can be measured in blood and may be a potential biomarker of cancer risk. We conducted a systematic review and meta-analysis of epidemiological studies of DNA repair phenotype and cancer through March 2021. We used random-effects models to calculate pooled odds ratios (ORs) of cancer risk for those with the lowest DNA repair capacity compared with those with the highest capacity. We included 55 case-control studies that evaluated 12 different cancers using 10 different DNA repair assays. The pooled OR of cancer risk (all cancer types combined) was 2.92 (95% Confidence Interval (CI) 2.49, 3.43) for the lowest DNA repair. Lower DNA repair was associated with all studied cancer types, and pooled ORs (95% CI) ranged from 2.02 (1.43, 2.85) for skin cancer to 7.60 (3.26, 17.72) for liver cancer. All assays, except the homologous recombination repair assay, showed statistically significant associations with cancer. The effect size ranged from 1.90 (1.00, 3.60) for the etoposide-induced double-strand break assay to 5.06 (3.67, 6.99) for the γ-H2AX assay. The consistency and strength of the associations support the use of these phenotypic biomarkers; however large-scale prospective studies will be important for understanding their use related to age and screening initiation., (© 2022. The Author(s).)

Generation of reactive oxygen species and related oxidants is an inevitable consequence of life. Proteins are major targets for oxidation reactions, because of their rapid reaction rates with oxidants and their high abundance in cells, extracellular tissues, and body fluids. Additionally, oxidative stress is able to degrade lipids and carbohydrates to highly reactive intermediates, which eventually attack proteins at various functional sites. Consequently, a wide variety of distinct posttranslational protein modifications is formed by protein oxidation, glycoxidation, and lipoxidation. Reversible modifications are relevant in physiological processes and constitute signaling mechanisms ("redox signaling"), while non-reversible modifications may contribute to pathological situations and several diseases. A rising number of publications provide evidence for their involvement in the onset and progression of diseases as well as aging processes. Certain protein oxidation products are chemically stable and formed in large quantity, which makes them promising candidates to become biomarkers of oxidative damage. Moreover, progress in the development of detection and quantification methods facilitates analysis time and effort and contributes to their future applicability in clinical routine. The present review outlines the most important classes and selected examples of oxidative protein modifications, elucidates the chemistry beyond their formation and discusses available methods for detection and analysis. Furthermore, the relevance and potential of protein modifications as biomarkers in the context of disease and aging is summarized., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The number of people above the age of 60 years is raising world-wide being associated with an increase in the prevalence of aging-associated impairments and even diseases. Recent studies suggest that aging is associated with alterations in bacterial endotoxin levels and that these changes may add to low-grade inflammation, the so-called 'inflammaging', and aging-associated liver degeneration. However, mechanisms involved, and especially, the interaction of intestinal microbiota and barrier in the development of aging-associated inflammation and liver degeneration have not been fully understood., Objective: The aim of the present study was to determine if intestinal microbiota composition changes with age and if these alterations are associated with changes of markers of intestinal barrier function and the development of inflammation and liver degeneration., Methods: Blood, liver, small and large intestinal tissue of male 2-, 15-, 24- and 30-months old C57BL/6 mice fed standard chow were obtained. Intestinal microbiota composition, expression levels of antimicrobial peptides in small intestine and markers of intestinal barrier function were measured. Furthermore, indices of liver damage, inflammation and expression levels of lipopolysaccharide binding protein (Lbp) as well as of toll-like receptors (Tlr) 1-9 in liver tissue were assessed., Results: Pairwise comparisons of the microbial community in the small intestine showed differences between 2- and 24-, 15- and 24-, as well as 15- and 30-months old animals while Shannon's diversity, species richness and evenness indexes did not differ in both small and large intestine, respectively, between age groups. Concentrations of nitric oxide were significantly lower in small intestine of 15-, 24- and 30-months old mice compared to 2-months old mice while mRNA expression of the antimicrobial peptides defensin alpha 1 and lysozyme 1 was unchanged. In contrast, in liver tissue, older age of animals was associated with increasing inflammation and the development of fibrosis in 24- and 30-months old mice. Numbers of inflammatory foci and neutrophils in livers of 24- and 30-months old mice were significantly higher compared to 2-months old mice. These alterations were also associated with higher endotoxin levels in plasma as well as an increased mRNA expression of Lbp and Tlr1, Tlr2, Tlr4, Tlr6 and Tlr9 in livers in older mice., Conclusion: Despite no consistent and robust changes of microbiota composition in small and/or large intestine of mice of different age were observed, our data suggest that alterations of markers of intestinal barrier function in small intestine are associated with an induction of several Tlrs and beginning hepatic inflammation in older mice and increase with age., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Early-life body size has been consistently associated with breast cancer risk. The direction of the association changes over time, with high birth weight, smaller adolescent body size, and adult weight gain all increasing breast cancer risk. There is also a clear positive association between larger body size and increased breast adipose tissue measured by mammograms, but less is known about how body size changes across life stages affect stromal and epithelial breast tissue. Using breast tissue slides from women with benign breast disease, Oh and colleagues applied machine learning methods to evaluate body size across the life course and adipose, epithelial, and stromal tissue concentrations in adulthood. They found consistent patterns for higher adipose and lower stromal tissue concentrations with larger childhood and adult body size at age 18 years. They reported lower levels of epithelial tissue with larger body size at 18 years, but not at other time periods. Additional studies examining how body size at different life stages may affect breast tissue composition will be important. Noninvasive methods that can provide measures of breast tissue composition may offer potential ways forward to ensure generalizability, and repeated measurements by life stage. See related article by Oh et al., p. 608 ., (©2021 American Association for Cancer Research.)

Overnutrition contributes to insulin resistance, obesity and metabolic stress, initiating a loss of functional beta-cells and diabetes development. Whether these damaging effects are amplified in advanced age is barely investigated. Therefore, New Zealand Obese (NZO) mice, a well-established model for the investigation of human obesity-associated type 2 diabetes, were fed a metabolically challenging diet with a high-fat, carbohydrate restricted period followed by a carbohydrate intervention in young as well as advanced age. Interestingly, while young NZO mice developed massive hyperglycemia in response to carbohydrate feeding, leading to beta-cell dysfunction and cell death, aged counterparts compensated the increased insulin demand by persistent beta-cell function and beta-cell mass expansion. Beta-cell loss in young NZO islets was linked to increased expression of thioredoxin-interacting protein (TXNIP), presumably initiating an apoptosis-signaling cascade via caspase-3 activation. In contrast, islets of aged NZOs exhibited a sustained redox balance without changes in TXNIP expression, associated with higher proliferative potential by cell cycle activation. These findings support the relevance of a maintained proliferative potential and redox homeostasis for preserving islet functionality under metabolic stress, with the peculiarity that this adaptive response emerged with advanced age in diabetes-prone NZO mice., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

This study investigated whether changes in neighborhood context induced by neighborhood relocation mediated the impact of the Moving to Opportunity (MTO) housing voucher experiment on adolescent mental health. Mediators included participant-reported neighborhood safety, social control, disorder, and externally-collected neighborhood collective efficacy. For treatment group members, improvement in neighborhood disorder and drug activity partially explained MTO's beneficial effects on girls' distress. Improvement in neighborhood disorder, violent victimization, and informal social control helped counteract MTO's adverse effects on boys' behavioral problems, but not distress. Housing mobility policy targeting neighborhood improvements may improve mental health for adolescent girls, and mitigate harmful effects for boys., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Adipogenesis is a fundamental process of white adipose tissue function, supporting lipid storage and release, while avoiding its spillover and ectopic accumulation in tissues and organs. During aging adipogenesis is impaired and among other factors, oxidative stress contributes to this process. Adipogenesis requires functional and dynamic mitochondria; however, this organelle itself becomes dysfunctional during aging and accounts for most of reactive oxygen species (ROS) production. Here, we evaluated whether oxidative stress impairs adipogenesis through functional impairment of mitodynamics by utilizing hyperoxia as a continuous source of oxidative stress while maintaining cellular viability. This negatively impacted mitochondrial function, including respiration and dynamics and ultimately blocked adipogenesis. Interestingly, this state was reversible by using the antidiabetic drug, Rosiglitazone, which reduced oxidative stress, restored mitochondrial dynamics and respiration and augmented adipogenesis. Moreover, in vitro results were in agreement with in vivo models of oxidative stress and aging, in which mice depleted of the superoxide dismutase enzyme 1 (SOD1) and old wild-type C57BL/6JRj mice demonstrated the same trend of adipogenic potential. Importantly, in humans the results follow the same pattern, showing a downregulation of adipogenic markers during aging. Since the levels of oxidative stress and peripheral insulin resistance increase with age, while adipogenesis decreases during aging, our model helps to understand a possible way to overcome physiologically low, steady stress conditions and restore adipogenesis, avoiding accumulation of deleterious hypertrophic adipocytes in favor of beneficial hyperplasia., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Aging is a risk factor in the development of many diseases, including liver-related diseases. The two aims of the present study were 1 ) to determine how aging affects liver health in mice in the absence of any interventions and 2 ) if degenerations observed in relation to blood endotoxin levels are critical in aging-associated liver degeneration. Endotoxin levels and markers of liver damage, mitochondrial dysfunction, insulin resistance, and apoptosis as well as the Toll-like receptor 4 (Tlr-4) signaling cascade were studied in liver tissue and blood, respectively, of 3- and 24-mo-old male C57BL/6J mice. In a second set of experiments, 3- to 4-mo-old and 14-mo-old female lipopolysaccharide-binding protein (LBP) -/- mice and littermates fed standard chow, markers of liver damage, insulin resistance, and mitochondrial dysfunction were assessed. Plasma activity of aspartate aminotransferase and histological signs of hepatic inflammation and fibrosis were significantly higher in old C57BL/6J mice than in young animals. The number of neutrophils, CD8α-positive cells, and mRNA expression of markers of apoptosis were also significantly higher in livers of old C57BL/6J mice compared with young animals, being also associated with a significant induction of hepatic Tlr-4 and LBP expression as well as higher endotoxin levels in peripheral blood. Compared with age-matched littermates, LBP -/- mice display less signs of senescence in liver. Taken together, our data suggest that, despite being fed standard chow, old mice developed liver inflammation and beginning fibrosis and that bacterial endotoxin may play a critical role herein. NEW & NOTEWORTHY Old age in mice is associated with marked signs of liver degeneration, hepatic inflammation, and fibrosis. Aging-associated liver degeneration is associated with elevated bacterial endotoxin levels and an induction of lipopolysaccharide-binding protein (LBP) and Toll-like receptor 4-dependent signaling cascades in liver tissue. Furthermore, in old aged LBP -/- mice, markers of senescence seem to be lessened, supporting the hypothesis that bacterial endotoxin levels might be critical in aging-associated decline of liver.

The antidiabetic drug metformin has been proposed to affect non-alcoholic fatty liver disease (NAFLD) through its effects on intestinal microbiota and barrier function. However, so far most studies focused on long-term effects and more progressed disease stages. The aim of this study was to assess in two experimental settings, if the onset of NAFLD is associated with changes of intestinal microbiota and barrier function and to determine effects of metformin herein. C57Bl/6J mice were fed a liquid control diet (C) or fat-, fructose- and cholesterol-rich diet (FFC) for four days or six weeks ±300 mg/kg BW/day metformin (Met). Markers of liver health, intestinal barrier function and microbiota composition were assessed. Metformin treatment markedly attenuated FFC-induced NAFLD in both experiments with markers of inflammation and lipidperoxidation in livers of FFC + Met-fed mice being almost at the level of controls. Metformin treatment attenuated the loss of tight junction proteins in small intestine and the increase of bacterial endotoxin levels in portal plasma. Changes of intestinal microbiota found in FFC-fed mice were also significantly blunted in FFC + Met-fed mice. Taken together, protective effects of metformin on the onset of NAFLD are associated with changes of intestinal microbiota composition and lower translocation of bacterial endotoxins.

Diet-induced hyperglycemia is described as one major contributor to the formation of advanced glycation end products (AGEs) under inflammatory conditions, crucial in type 2 diabetes progression. Previous studies have indicated high postprandial plasma AGE-levels in diabetic patients and after long-term carbohydrate feeding in animal models. Pancreatic islets play a key role in glucose metabolism; thus, their susceptibility to glycation reactions due to high amounts of dietary carbohydrates is of special interest. Therefore, diabetes-prone New Zealand Obese (NZO) mice received either a carbohydrate-free, high-fat diet (CFD) for 11 weeks or were additionally fed with a carbohydrate-rich diet (CRD) for 7 days. In the CRD group, hyperglycemia and hyperinsulinemia were induced accompanied by increasing plasma 3-nitrotyrosine (3-NT) levels, higher amounts of 3-NT and inducible nitric oxide synthase (iNOS) within pancreatic islets. Furthermore, N-ε-carboxymethyllysine (CML) was increased in the plasma of CRD-fed NZO mice and substantially higher amounts of arg-pyrimidine, pentosidine and the receptor for advanced glycation end products (RAGE) were observed in pancreatic islets. These findings indicate that a short-term intervention with carbohydrates is sufficient to form endogenous AGEs in plasma and pancreatic islets of NZO mice under hyperglycemic and inflammatory conditions.

Population, intervention, comparison and outcome (PICO) facets of clinical studies are required both for physicians in a clinical setting and for reviewers as they compare the effectiveness of different treatment strategies. Automated methods developed for the first three of these facets identify entities, but outcome detection has been limited to identifying the entire sentence. We frame outcome detection as a noun phrase prediction task and use semi-supervised learning to detect new outcomes (aka endpoints) from the method section of 88 K MEDLINE abstracts. A manual analysis showed that 96.7% of all outcomes can be captured using a noun phrase representation. With respect to the machine learning classifiers, the Support Vector Machine produced higher precision, F1-score, and accuracy than the General Linear Model when evaluated with respect to the initial gold standard of survivorship seed terms and a manual gold standard that considered all outcomes. However, the best model does not employ machine learning, but rather leverages list structure and resulted in 90.14 precision, 60.69 recall, 75.41 F1-score, and 92.60 accuracy with respect to the manual gold standard of all outcomes. Finally we developed a silver standard with a precision of 89.28 and recall of 86.77 compared to the manual gold standard and used the silver standard to identify all outcomes reported for five breast cancer treatments. The increased precision afforded by this approach reveals that in contrast to chemotherapy and targeted therapy, the surrogate outcome disease free survival (DFS) is reported more frequently than the clinically relevant outcome overall survival (OS) for hormone therapies, which is consistent with findings that DFS translates into firm OS improvements in a hormone therapy setting., (Published by Elsevier Inc.)

Large amounts of data from multi-channel, high resolution, fluorescence microscopic images require tools that provide easy, customizable, and reproducible high-throughput analysis. The freeware "ImageJ" has become one of the standard tools for scientific image analysis. Since ImageJ offers recording of "macros," even a complex multi-step process can be easily applied fully automated to large numbers of images, saving both time and reducing human subjective evaluation. In this work, we present "Cyt/Nuc," an ImageJ macro, able to recognize and to compare the nuclear and cytosolic areas of tissue samples, in order to investigate distributions of immunostained proteins between both compartments, while it documents in detail the whole process of evaluation and pattern recognition. As practical example, the redistribution of the 20S proteasome, the main intracellular protease in mammalian cells, is investigated in NZO-mouse liver after feeding the animals different diets. A significant shift in proteasomal distribution between cytosol and nucleus in response to metabolic stress was revealed using "Cyt/Nuc" via automatized quantification of thousands of nuclei within minutes. "Cyt/Nuc" is easy to use and highly customizable, matches the precision of careful manual evaluation and bears the potential for quick detection of any shift in intracellular protein distribution., (© 2018 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Aged tissues usually show a decreased regenerative capacity accompanied by a decline in functionality. During aging pancreatic islets also undergo several morphological and metabolic changes. Besides proliferative and regenerative limitations, endocrine cells lose their secretory capacity, contributing to a decline in functional islet mass and a deregulated glucose homeostasis. This is linked to several features of aging, such as induction of cellular senescence or the formation of modified proteins, such as advanced glycation end products (AGEs) - the latter mainly examined in relation to hyperglycemia and in disease models. However, age-related changes of endocrine islets under normoglycemic and non-pathologic conditions are poorly investigated. Therefore, a characterization of pancreatic tissue sections as wells as plasma samples of wild-type mice (C57BL/6J) at various age groups (2.5, 5, 10, 15, 21 months) was performed. Our findings reveal that mice at older age are able to secret sufficient amounts of insulin to maintain normoglycemia. During aging the pancreatic islet area increased and the islet size doubled in 21 months old mice when compared to 2.5 months old mice, whereas the islet number was unchanged. This was accompanied by an age-dependent decrease in Ki-67 levels and pancreatic duodenal homeobox-1 (PDX-1), indicating a decline in proliferative and regenerative capacity of pancreatic islets with advancing age. In contrast, the number of p16 Ink4a -positive nuclei within the islets was elevated starting from 10 months of age. Interestingly, AGEs accumulated exclusively in the islet blood vessels of old mice associated with increased amounts of inflammatory markers, such as the inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine (3-NT). In summary, the age-related increase in islet size and area was associated with the induction of senescence, accompanied by an accumulation of non-enzymatically modified proteins in the islet vascular system., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The purpose of this study was to further explore the rural school food environment. This study assessed trends in prevalence of vending machines and vending items within and between Minnesota schools located in 3 rural subtypes: town/rural fringe, town/rural distant, and remote rural. Generalized estimating equation models were employed to analyze data from the 2006 through 2012 School Health Profiles Principal's Surveys (Profiles). All 3 rural subtypes had a statistically significant decrease in the prevalence of low nutrient energy dense (LNED) vending items between 2006 and 2012, with the exception of sports drinks. However, different vending practices were observed between rural subtypes, with town/rural fringe schools providing more LNED vending options and experiencing less positive change over time compared to town/rural distant and remote rural schools. Differences in vending machine practices emerge when rural schools are subtyped., Competing Interests: Conflicts of Interest None of the authors have any conflicts of interest to disclose.

Background: The Empty Spiracles Homeobox (EMX-) 2 gene has been associated with regulation of growth and differentiation in neuronal development. While recent studies provide evidence that EMX2 regulates tumorigenesis of various solid tumors, its role in colorectal cancer remains unknown. We aimed to assess the prognostic significance of EMX2 expression in stage III colorectal adenocarcinoma., Methods: Expression levels of EMX2 in human colorectal cancer and adjacent mucosa were assessed by qRT-PCR technology, and results were correlated with clinical and survival data. siRNA-mediated knockdown and adenoviral delivery-mediated overexpression of EMX2 were performed in order to investigate its effects on the migration of colorectal cancer cells in vitro., Results: Compared to corresponding healthy mucosa, colorectal tumor samples had decreased EMX2 expression levels. Furthermore, EMX2 down-regulation in colorectal cancer tissue was associated with distant metastasis (M1) and impaired overall patient survival. In vitro knockdown of EMX2 resulted in increased tumor cell migration. Conversely, overexpression of EMX2 led to an inhibition of tumor cell migration., Conclusions: EMX2 is frequently down-regulated in human colorectal cancer, and down-regulation of EMX2 is a prognostic marker for disease-free and overall survival. EMX2 might thus represent a promising therapeutic target in colorectal cancer.

Aging is a complex phenomenon and its impact is becoming more relevant due to the rising life expectancy and because aging itself is the basis for the development of age-related diseases such as cancer, neurodegenerative diseases and type 2 diabetes. Recent years of scientific research have brought up different theories that attempt to explain the aging process. So far, there is no single theory that fully explains all facets of aging. The damage accumulation theory is one of the most accepted theories due to the large body of evidence found over the years. Damage accumulation is thought to be driven, among others, by oxidative stress. This condition results in an excess attack of oxidants on biomolecules, which lead to damage accumulation over time and contribute to the functional involution of cells, tissues and organisms. If oxidative stress persists, cellular senescence is a likely outcome and an important hallmark of aging. Therefore, it becomes crucial to understand how senescent cells function and how they contribute to the aging process. This review will cover cellular senescence features related to the protein pool such as morphological and molecular hallmarks, how oxidative stress promotes protein modifications, how senescent cells cope with them by proteostasis mechanisms, including antioxidant enzymes and proteolytic systems. We will also highlight the nutritional status of senescent cells and aged organisms (including human clinical studies) by exploring trace elements and micronutrients and on their importance to develop strategies that might increase both, life and health span and postpone aging onset., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The 2012 FAV-S pilot study was developed as a dietary intervention program for low-income Somali mothers grounded in the health belief model. The intervention was geared toward increasing fruit and vegetable intake among participants' children. The purpose of this analysis was to determine the impact of the FAV-S program on participants' (1) self-efficacy in ability to serve more fruits and vegetables, (2) knowledge and beliefs about healthy eating, and (3) perceived barriers to accessing healthy foods. Furthermore, this study assessed change in fruit and vegetable intake among participants and their children. The intervention consisted of two small group education sessions addressing nutrition, serving size, and label reading; a cooking session incorporating fruits and vegetables into traditional Somali dishes; and a grocery store session demonstrating best purchasing practices. Self-efficacy, knowledge and beliefs, and perceived barriers were assessed via surveys administered verbally in Somali pre- and postintervention. Paired t tests were used to compare pre- and postintervention survey responses. Twenty-five women participated in the pilot study; mean age was 43.6 years ( SD = 12.4). Self-efficacy significantly increased among participants postintervention ( p = .01), though there were no significant changes in knowledge and beliefs or perceived barriers. Following intervention, daily servings of fruits and vegetables significantly increased among both women and children ( p = .01 to p < .01). Findings suggest that a multistage, culturally tailored, approach is effective at increasing self-efficacy and fruit and vegetable intake in the Somali community. Continued and expanded research is needed to further develop culturally focused dietary interventions.

Early life exposures influence numerous social determinants of health, as distal causes or confounders of later health outcomes. Although a growing literature is documenting how early life socioeconomic position affects later life health, few epidemiologic studies have tested measures for operationalizing early life neighborhood context, or examined their effects on later life health. In the Life-course Influences on Fetal Environments (LIFE) Study, a retrospective cohort study among Black women in Southfield, Michigan (71% response rate), we tested the validity and reliability of retrospectively-reported survey-based subjective measures of early life neighborhood context(N=693). We compared 3 subjective childhood neighborhood measures (disorder, informal social control, victimization), with 3 objective childhood neighborhood measures derived from 4 decades of historical census tract data 1970-2000, linked through geocoded residential histories (tract % poverty, tract % black, tract deprivation score derived from principal components analysis), as well as with 2 subjective neighborhood measures in adulthood. Our results documented that internal consistency reliability was high for the subjective childhood neighborhood scales (Cronbach's α =0.89, 0.93). Comparison of subjective with objective childhood neighborhood measures found moderate associations in hypothesized directions. Associations with objective variables were strongest for neighborhood disorder (rhos=.40), as opposed to with social control or victimization. Associations between subjective neighborhood context in childhood versus adulthood were moderate and stronger for residentially-stable populations. We lastly formally tested for, but found little evidence of, recall bias of the retrospective subjective reports of childhood context. These results provide evidence that retrospective reports of subjective neighborhood context may be a cost-effective, valid, and reliable method to operationalize early life context for health studies.

Objectives: Successful childhood obesity intervention models that build sustainable behavioral change are needed, particularly in low-income, ethnic minority communities disparately affected by this problem., Method: Families were referred to Taking Steps Together (TST) by their primary care provider if at least one child had a body mass index ≥85%. The TST intervention comprised 16 weekly 2-hour classes including educational activities, group cooking/eating, and physical activities for parents and children. TST's approach emphasized building self-efficacy, targeting both children and parents for healthy change, and fostering intrinsic motivation for healthier living. Pre-post intervention data were collected on health-related behaviors using a survey, and trained staff measured weight and height., Results: Adults (n = 33) and children (n = 62) were largely Hispanic/Latino and low-income. Adults and children significantly increased their fruit and vegetable consumption and weekly physical activity, and adults significantly decreased sugared beverage consumption and screen time. No change in body mass index was observed for adults or children., Conclusions: This family-focused childhood obesity intervention integrated evidence-based principles with a nonprescriptive approach and produced significant improvements in key healthy behaviors for both adults and children., (© 2014 Society for Public Health Education.)

Background: Although there are several evidence-based recommendations directed at improving nutrition and physical activity standards in schools, these guidelines have not been uniformly adopted throughout the United States. Consequently, research is needed to identify facilitators promoting schools to implement these recommendations. Therefore, this study analyzed the 2008 School Health Profiles Principal Survey (Profiles) to explore the role of family and community involvement in school nutrition and physical activity standards., Methods: Survey data on nutrition and physical activity policies, as well as family and community involvement, were available for 28 states, representing 6732 secondary schools. One-factor analysis of variance (ANOVA), 2-sample t-tests, Pearson's chi-square tests, and multiple logistic and linear regression models were employed in this analysis., Results: Family and community involvement were associated with schools more frequently utilizing healthy eating strategies and offering students healthier food options. Further, involvement was associated with greater support for physical education staff and more intramural sports opportunities for students., Conclusions: Though family and community involvement have the potential to have a positive influence on school nutrition and physical activity policies and practices, involvement remains low in schools. Increased efforts are needed to encourage collaboration among schools, families, and communities to ensure the highest health standards for all students., (© 2015, American School Health Association.)

Objective: To determine the feasibility, acceptability, and impact of a parent-centered intervention to increase fruit and vegetable servings and consumption among Somali children living in the United States., Design: Pilot intervention that included Somali community health workers who organized groups of 3 or 4 women to participate in 2 initial educational sessions, including topics of health and nutrition, serving size, and label reading. A third session taught interactive cooking to increase skills in preparing fruits and vegetables and increasing the numbers of vegetables included in traditional Somali dishes. The final session was a trip to the grocery store to identify fruits and vegetables, determine ripeness, and explore frozen and canned fruits and vegetables for halal, low-sodium, and low-sugar products. Surveys were completed pre- and post-intervention., Setting: Study participants were recruited from a large housing complex in Minneapolis, Minnesota, with a high population of Somali immigrants., Participants: Somali women (N = 25) with children aged 3 to 10 years., Results: The intervention was feasible and well accepted. In comparing pre- and post-intervention surveys, mothers reported providing their children significantly more frequent servings of fruits and vegetables at dinner, lunch, snack, and breakfast (vegetable only). There was a statistically significant increase in parent-reported intake of fruits and vegetables for themselves and their children., Conclusion: The FAV-S study was feasible and acceptable, and it demonstrated potential for increasing fruit and vegetable servings and consumption among Somali children. A larger-scale randomized trial is needed to assess the impact of this intervention.

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro,the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.

A herpesvirus of smelt (Osmerus eperlanus) was identified by thin section electron microscopy. Degenerated cells of skin lesions located on the back fin of smelt showed either intranucleic- or cytoplasmic herpesvirus-specific structures. In the nuclei "naked" virus capsids with a diameter of about 100 nm were observed. The diameter of the complete virion including its unilaterally extended envelope ranged from 200 to 350 nm. Remarkably, in complete virions the electron-opaque tegument is completely filling the region between nucleocapsid and envelope and as another unique feature the virion shows a "comet-shape" due to a long unilateral extension of its envelope. This kind of shape had been not reported for any of herpesviruses known so far. Consequently this virus was termed herpesvirus of Osmerus eperlanus (HVOE1) or Comet herpesvirus of smelt. Due to the long time storage at the nonstandard temperature of smelt virus the biological and genomic analysis of the HVOE1 was hampered. All attempts to study host range of HVOE1 failed as no virus replication was observed, indicating that infectivity was lost or the suitable cell culture was missing. The genomic DNA of HVOE1 was analyzed by DNA restriction endonucleases.

The effect of anise oil, dwarf-pine oil and chamomile oil against different thymidine-kinase-positive (aciclovir-sensitive) and thymidine-kinase-negative (aciclovir-resistant) herpes simplex virus type 1 (HSV-1) strains was examined. Clinical HSV-1 isolates containing frameshift mutations in the thymidine kinase (TK) gene, an insertion or a deletion, yield a non-functional thymidine kinase enzyme resulting in phenotypical resistance against aciclovir. The inhibitory activity of three different essential oils against herpes simplex virus isolates was tested in-vitro using a plaque reduction assay. All essential oils exhibited high levels of antiviral activity against aciclovir-sensitive HSV strain KOS and aciclovir-resistant clinical HSV isolates as well as aciclovir-resistant strain Angelotti. At maximum noncytotoxic concentrations of the plant oils, plaque formation was significantly reduced by 96.6-99.9%, when herpesviruses were preincubated with drugs before attachment to host cells. No significant effect on viral infectivity could be achieved by adding these compounds during the replication phase. These results indicate that anise oil, dwarf-pine oil and chamomile oil affected the virus by interrupting adsorption of herpesviruses and in a different manner than aciclovir, which is effective after attachment inside the infected cells. Thus the investigated essential oils are capable of exerting a direct effect on HSV and might be useful in the treatment of drug-resistant viruses. Chamomile oil did not reveal any irritating potential on hen's egg chorioallantoic membrane, demonstrated the highest selectivity index among the oils tested and was highly active against clinically relevant aciclovir-resistant HSV-1 strains.

In the last decades a significant number of so far unknown or underestimated pathogens have emerged as fundamental health hazards of the human population despite intensive research and exceptional efforts of modern medicine to embank and eradicate infectious diseases. Almost all incidents caused by such emerging pathogens could be ascribed to agents that are zoonotic or expanded their host range and crossed species barriers. Many different factors influence the status of a pathogen to remain unnoticed or evolves into a worldwide threat. The ability of an infectious agent to adapt to changing environmental conditions and variations in human behavior, population development, nutrition, education, social, and health status are relevant factors affecting the correlation between pathogen and host. Hantaviruses belong to the emerging pathogens having gained more and more attention in the last decades. These viruses are members of the family Bunyaviridae and are grouped into a separate genus known as Hantavirus. The serotypes Hantaan (HTN), Seoul (SEO), Puumala (PUU), and Dobrava (DOB) virus predominantly cause hemorrhagic fever with renal syndrome (HFRS), a disease characterized by renal failure, hemorrhages, and shock. In the recent past, many hantavirus isolates have been identified and classified in hitherto unaffected geographic regions in the New World (North, Middle, and South America) with characteristic features affecting the lungs of infected individuals and causing an acute pulmonary syndrome. Hantavirus outbreaks in the United States of America at the beginning of the 10th decade of the last century fundamentally changed our knowledge about the appearance of the hantavirus specific clinical picture, mortality, origin, and transmission route in human beings. The hantavirus pulmonary syndrome (HPS) was first recognized in 1993 in the Four Corners Region of the United States and had a lethality of more than 50%. Although the causative virus was first termed in connection with the geographic name of its outbreak region the analysis of the individual viruses indicate that the causing virus of HPS was a genetically distinct hantavirus and consequently termed as Sin Nombre virus. Hantaviruses are distributed worldwide and are assumed to share a long time period of co-evolution with specific rodent species as their natural reservoir. The degree of relatedness between virus serotypes normally coincides with the relatedness between their respective hosts. There are no known diseases that are associated with hantavirus infections in rodents underlining the amicable relationship between virus and host developed by mutual interaction in hundreds of thousands of years. Although rodents are the major reservoir, antibodies against hantaviruses are also present in domestic and wild animals like cats, dogs, pigs, cattle, and deer. Domestic animals and rodents live jointly in a similar habitat. Therefore the transmission of hantaviruses from rodents to domestic animals seems to be possible, if the target organs, tissues, and cell parenchyma of the co-habitat domestic animals possess adequate virus receptors and are suitable for hantavirus entry and replication. The most likely incidental infection of species other than rodents as for example humans turns hantaviruses from harmless to life-threatening pathogenic agents focusing the attention on this virus group, their ecology and evolution in order to prevent the human population from a serious health risk. Much more studies on the influence of non-natural hosts on the ecology of hantaviruses are needed to understand the directions that the hantavirus evolution could pursue. At least, domestic animals that share their environmental habitat with rodents and humans particularly in areas known as high endemic hantavirus regions have to be copiously screened. Each transfer of hantaviruses from their original natural hosts to other often incidental hosts is accompanied by a change of ecology, a change of environment, a modulation of numerous factors probably influencing the pathogenicity and virulence of the virus. The new environment exerts a modified evolutionary pressure on the virus forcing it to adapt and probably to adopt a form that is much more dangerous for other host species compared to the original one.

The objective of this study was to investigate the molecular mechanisms of neurobiological processes involved in the degeneration of the central nervous system. The bovine spongiform encephalopathy (BSE) was used as experimental model system for investigation of transmissible spongiform encephalopathy (TSE). The experimental strategy was to evaluate the possibility for protection of bovine PrP(C) transgenic mice against a bovine PrP(Sc) infection by DNA vaccination using the complete or partial cDNA sequences of the bovine prion protein. Three recombinant plasmids pCR3.1-EX-PrP-BSE-C20 (C20), pCR3.1-EX-PrP-BSE-90-235-C4 (C4), and pCR3.1-EX-PrP-BSE-106-131-C14 (C14) were constructed. These mammalian expression vectors harbor complete (C20) or partial (C4 and C14) cDNA sequences of the Bos taurus PrP(C) (BTPrP(C)) encoding for amino acid residues 1-264 (C20), 90-235 (C4), and 106-131 (C14) of the BTPrP(C). Transgenic mice harboring and expressing BTPrP(C) were generated using the donor strain C57/CBA, receptor strain Swiss mouse, and recombinant plasmid MoPrPXho-boPrP. Crossing of positive transgenic mice to bovine PrP and negative to murine PrP with 129/OLA (murine PrP-/-) and C57BL6x129/OLA (murine PrP+/-) mice was carried out to amplify the colony of transgenic mice termed bovine PrP(C) transgenic Swiss mice (BTPrP-TgM). The capabilities of C20, C4, and C14 to express the corresponding cDNA sequence of BTPrP(C) in vitro and in vivo were confirmed prior to DNA vaccination of the BTPrP-TgM using NIH 3T3 cells and BALB/c mice, respectively. In order to prove the capability of the constructed expression vectors to protect BTPrP-TgM in vivo against a BSE infection 80 female BTPrP-TgM were vaccinated intramuscularly and subcutaneously with DNA of the plasmids C20, C4, C14, and parental vector pCR3.1 (100 microg DNA corresponding to about 26-30 pmol DNA/animal and application) in four groups (each consists of 20 animals). DNA vaccination was followed by three additional boosters. The vaccinated animals (15 animals of each group) were challenged twice per oral with homogenates of brain material obtained from BSE cattle containing the infectious PrP(Sc) (100 microl/animal which corresponds to 15 mg of a 15% brain homogenate). The first and second challenge experiments were performed 76-83 and 181 days post DNA vaccination, respectively. A part of the vaccinated animals (3-5 animals of each group) that served as internal negative control were mock infected using the brain homogenate of healthy cattle or Phosphate saline buffer (PBS). A variety of symptoms and clinical pictures were observed during the monitoring of DNA vaccinated animals. However, the observed diseases seem to be similar in all experimental animal groups. After an observation period of 14 months post the second challenge experiment the remaining animals (some animals died or were sacrificed when moribund during the study) were sacrificed after expiration of the experimental schedule. The right hemisphere of the brain and a half of the spleen tissue of the individual animals were used for detection of PrP(Sc) by Western blot analysis. The misfolded bovine PrP(Sc) was not detected in the brain or spleen tissues of those animals that were vaccinated with DNA of C20, which was able to express the complete bovine PrP(C) protein in vitro and in vivo. In contrast, the bovine PrP(Sc) was detected in the brain or spleen tissues of animals that were DNA vaccinated with DNA of the parental vector pCR3.1, with DNA of C4, or with DNA of C14. The results of these studies underline that the constructed expression vector C20 possesses the protective capacity to inhibit the formation of misfolded bovine PrP(Sc) in BTPrP-TgM under the conditions used. A delay of occurrence of TSE-specific symptoms in the majority of the vaccinated animals seems to be due to the prolonged incubation time of BSE infection.

Mortality and morbidity rates remain high among patients with herpes simplex virus encephalitis (HSVE). Chemokine-mediated recruitment and activation of leukocytes to focal areas of viral CNS infection are crucial steps in antiviral response and clearance. However, the inflammatory reaction and cellular antiviral response may enhance collateral damage to neurons and account for chronic progressive brain damage. We identified a specific mRNA expression of the interferon-gamma-inducible chemokines (CXCL9, CXCL10 and CXCL11), and RANTES (CCL5) in the acute course and long-term of experimental HSVE. This pattern was substantially altered by anti-viral and anti-inflammatory treatment. Our findings indicate a pivotal role of these chemokines in the immunopathogenesis of HSVE.

Herpes simplex virus encephalitis (HSVE) causes elevated morbidity and mortality despite antiviral treatment. Virus-independent mechanisms may perpetuate brain damage. Matrix metalloproteinases (MMPs) target extracellular matrix components. This study describes the protein and mRNA expression of MMP2 and MMP9 in experimental HSVE in the short and long term. Ten SJL-NBOM mice were infected with neurovirulent HSV-1 and compared with nine controls. The presence of MMP2 and MMP9 in brain tissue was analyzed with sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gelatin zymography and mRNA expression of MMP2 and MMP9 with quantitative real-time PCR at days 7, 21 and 180 post-inoculation. Infected animals had a significantly elevated gelatinolytic activity of MMP2 at all time points, and of MMP9 at days 21 and 180. Increased presence of MMP2 and MMP9 in chronic HSVE may contribute to ongoing damage. Inhibition of MMP2 and MMP9 might be a suitable target for therapeutic intervention.

In the brain tissue of 36 mice infected with herpes simplex virus type 1, strain F, we determined the expression of inducible nitric oxide synthase (iNOS) with semiquantitative reverse transcription polymerase chain reaction. The viral burden was quantitated by polymerase chain reaction. Nitric oxide, induced by iNOS, may contribute to neuronal cell damage following virus infection. As the experimental therapeutic strategy in herpes simplex virus encephalitis (HSVE), we used: N-nitro-L-arginin (L-NA), a selective inhibitor of iNOS; and combination therapies of either methylprednisolone/acyclovir or L-NA/acyclovir. The viral burden peaked in acute disease, and then returned to a low baseline value, except in untreated controls. The expression of iNOS mRNA was suppressed by L-NA and by acyclovir/corticosteroids. INOS inhibition may provide an additional therapeutic strategy targeted specifically to suppress iNOS expression as a potential secondary mechanism of tissue damage in acute and chronic HSVE.

Molluscum contagiosum virus (MCV), a member of the family Poxviridae, can be isolated from skin-lesion material of patients with molluscum contagiosum infection. MCV replicates efficiently in human keratinocytes in vivo but viral replication has not been observed in vitro in cell or tissue culture systems. We investigated a variety of established cell lines for productive MCV infection and found that: (i) MCV induces a typical cytopathogenic effect (CPE) only in human primary fibroblast cells (MRC5 ATCC-CCL 171 and HEPM ATCC-CRL 1486) but not in permanent eucaryotic cell lines of human or simian origin; (ii) UV irradiated MCV virions and heat inactivated virions do not induce a CPE; (iii) decreasing amounts of MCV viral DNA are detectable in infected human embryonic fibroblasts for at least 14 days post infection (p.i.); (iv) MCV early mRNAs are detectable by RT-PCR between one and two hours p.i. and remain detectable upon passaging of the infected cells; (iv) transcripts of viral late genes (mc095L and mc106L) are detectable by RT-PCR from day 5 p.i.; (v) MCV viral antigens can be detected on the surface of infected cells using human and rabbit polyclonal antisera against MCV from 24 h p.i.; (vi) a CPE can not be observed if cell free supernatants or homogenizates of MCV infected cells are used to try passage of the virus onto uninfected human embryonic fibroblasts, indicating that infectious viral progeny is not produced. This is the first report demonstrating long time persistence of MCV viral DNA and expression of late proteins in an in vitro cell culture system.

Transgenic plants, expressing recombinant proteins, are suitable alternatives for the production of relevant immunogens. In the present study, the expression of Puumala virus nucleocapsid protein in tobacco and potato plants (Nicotiana tabacum and Solanum tuberosum) and its immunogenicity was investigated. After infection of leaf discs of SR1 tobacco and tuber discs of potato cv. "Desiree" with the Agrobacterium strain LBA4404 (pAL4404, pBinAR-PUU-S) containing the 1302 bp cDNA sequence of S-RNA segment of a Puumala virus, transgenic tobacco and potato plants expressed the Puumala virus nucleocapsid protein under control of the cauliflower 35S promoter. The recombinant proteins were found to be identical to the authentic Puumala virus nucleocapsid protein as analyzed by immunoblotting. Expression of the nucleocapsid protein was investigated over four plant generations (P to F4) and found to be stable (1 ng/3 microg dried leaf tissue). Transgenic tobacco plants were smaller compared to controls. The transformed potato plants were morphologically similar to control plants and produced tubers as the control potatoes. The S-antigen was expressed at a level of 1 ng protein/5 microg and 1 ng protein/4 microg dried leaf and root tissues, respectively, and remained stable in the first generation of vegetatively propagated potato plants. The immunogenicity of the Puumala virus nucleocapsid protein expressed in Nicotiana tabacum and Solanum tuberosum was investigated in New Zealand white rabbits. They were immunized with leaf extracts from transgenic tobacco and potato plants, and the serum recognized Puumala virus nucleocapsid protein. Transgenic plants expressing hantaviral proteins can thus be used for the development of cost-effective diagnostic systems and for alternative vaccination strategies.

In 1997 TTV was detected using representational difference analysis (RDA) in serum of a patient with posttransfusion hepatitis unrelated to known hepatitis viruses. The genome of TTV is a circular single-stranded DNA molecule of 3852 nt with negative polarity. TTV possibly can be grouped either into the existing family Circoviridae or into a recently established virus family "Circinoviridae". Analysis of the complete DNA nucleotide sequence of TTV identified three partially overlapping open reading frames (ORFs). Neither DNA nucleotide nor corresponding amino acid sequences of TTV do show significant homologies to known sequences. TTV DNA nucleotide sequences amplified by PCR from sera of different patients show considerable sequence variations. Although the natural route of transmission of TTV is still unknown, there is clear evidence for a transmission of TTV through blood and blood products. TTV DNA can be detected in the feces of infected individuals suggesting that it may be possible to attract TTV infection from environmental sources. Since the discovery of TTV, numerous studies have investigated the prevalence of TTV infections in different human population groups all over the world. All these studies are based on PCR detection systems, but the technical aspects of the PCR systems vary significantly between the different investigators. The results of the epidemiological studies do not show a clear picture. The discovery of TTV as a viral agent and particularly the identification of a high percentage of infected carriers in the healthy human population raises the following questions: Firstly, what is the origin and molecular relatedness of TT virus. Secondly, what is the significance of TTV as a human pathogen. And thirdly, what are the exact molecular mechanisms of viral replication. To answer these questions it will be necessary to determine the primary structure and the coding capacity of several TTV patient isolates.

Cranial magnetic resonance imaging (MRI) is a sensitive diagnostic tool for the in vivo detection of morphological abnormalities in herpes simplex virus encephalitis (HSVE). We performed a long-term MRI study in a mouse model of HSVE. Cranial MRI findings were compared with the viral load within brain tissue, the presence of HSV DNA in the cerebrospinal fluid (CSF), a daily clinical assessment and post-mortem neurohistopathological studies. A 1.5 T cranial MRI scanner with standard spin-echo sequences was used. Viral load within the brain and the presence of HSV DNA in cerebrospinal fluid were determined by a polymerase chain reaction assay. Clinically, animals were severely affected within the first 2 weeks and recovered thereafter. Focal histopathological and MRI abnormalities involved predominantly limbic structures, a pattern that mimics human disease. Severity and extent of abnormalities had increased at 6 months despite clinical improvement. HSV DNA was present in CSF during the acute disease only. Brain viral load peaked at day 10 and declined thereafter. MRI as an in vivo monitoring approach may reveal chronic progressive changes in HSVE, despite clinical recovery and low viral load in the brain. Secondary, not directly virus-mediated, mechanisms of tissue damage may contribute to tissue damage of HSVE., (Copyright Lippincott Williams & Wilkins)

Iridoviruses are large cytoplasmic DNA viruses that are specific for different insect or vertebrate hosts. The major structural component of the non-enveloped icosahedral virus particles is the major capsid protein (MCP) which appears to be highly conserved among members of the family Iridoviridae, Phycodnaviridae, and African swine fever virus. The amino acid sequences of the known MCPs were used in comparative analyses to elucidate the phylogenic relationships between different cytoplasmic DNA viruses including three insect iridoviruses (Tipula iridescent virus, Simulium iridescent virus, Chilo iridescent virus), seven vertebrate iridoviruses isolated either from fish (lymphocystis disease virus, rainbow trout virus, European catfish virus, doctor fish virus), amphibians (frog virus 3), or reptiles (turtle virus 3, turtle virus 5), one member of the family Phycodnaviridae (Paramecium bursaria Chlorella virus type 1), and African swine fever virus. These analyses revealed that the amino acid sequence of the MCP is a suitable target for the study of viral evolution since it contains highly conserved domains, but is sufficiently diverse to distinguish closely related iridovirus isolates. Furthermore the results suggest that a substantial revision of the taxonomy of iridoviruses based on molecular phylogeny is required.

The development of an in vitro-system for the stable expression and the analysis of native hantavirus proteins using hantaviral cDNA is of particular interest. As a first step the expression of the hantavirus nucleocapsid (N) proteins in mammalian cells was studied in more detail. The cDNA of the S-RNA segment of Puumala virus strain CG-1820 and Hantaan virus strain 76-118 was used for the construction of eucaryotic expression vectors that allow the generation and selection of mammalian cells harboring and expressing the N protein genes of hantaviruses. A variety of conventional and novel expression vectors as well as different mammalian cell lines were screened. The expression of the N protein of Puumala virus using the pGRE5-1 vector in which the transcription is under control of inducible glucocorticoid responsive elements (GRE) revealed that the Puumala virus N protein can be expressed in Vero E6 cells efficiently without any detectable cell toxicity. From the variety of expression vectors tested, it was found that pCR3.1 is the vector of choice for stable expression of hantavirus N proteins. The successful establishment of different mammalian cell lines expressing considerable amounts of Puumala and Hantaan virus N protein indicates that the stable and efficient expression of this particular viral protein in the cell lines of three evolutionary distinct species (human, monkey, and mouse) is possible. The system described here represents the experimental basis for further studies of hantavirus infection, replication, and pathogenesis using a reverse genetics approach.

Recently the UL56 protein of herpes simplex virus type 1 (HSV-1) was shown to be associated with the virion of HSV-1 as determined by Western blot analysis. The detection of the UL56 protein in infected cells and its association with virions of HSV-1 is of particular importance, pointing to a possible involvement of UL56 protein in virus-host interactions. In order to investigate the properties of the UL56 protein further immuno-localization was performed using rabbit hyperimmune serum against fusion recombinant UL56 protein and purified virions of HSV-1 strain F. The UL56 protein was detected in the HSV-1 virions by immuno gold negative staining.