Universal platform for quantitative analysis of DNA transposition

<p>Abstract</p> <p>Background</p> <p>Completed genome projects have revealed an astonishing diversity of transposable genetic elements, implying the existence of novel element families yet to be discovered from diverse life forms. Concurrently, several better understood transposon systems have been exploited as efficient tools in molecular biology and genomics applications. Characterization of new mobile elements and improvement of the existing transposition technology platforms warrant easy-to-use assays for the quantitative analysis of DNA transposition.</p> <p>Results</p> <p>Here we developed a universal <it>in vivo </it>platform for the analysis of transposition frequency with class II mobile elements, i.e., DNA transposons. For each particular transposon system, cloning of the transposon ends and the cognate transposase gene, in three consecutive steps, generates a multifunctional plasmid, which drives inducible expression of the transposase gene and includes a mobilisable <it>lacZ</it>-containing reporter transposon. The assay scores transposition events as blue microcolonies, papillae, growing within otherwise whitish <it>Escherichia coli </it>colonies on indicator plates. We developed the assay using phage Mu transposition as a test model and validated the platform using various MuA transposase mutants. For further validation and to illustrate universality, we introduced IS<it>903 </it>transposition system components into the assay. The developed assay is adjustable to a desired level of initial transposition via the control of a plasmid-borne <it>E. coli </it>arabinose promoter. In practice, the transposition frequency is modulated by varying the concentration of arabinose or glucose in the growth medium. We show that variable levels of transpositional activity can be analysed, thus enabling straightforward screens for hyper- or hypoactive transposase mutants, regardless of the original wild-type activity level.</p> <p>Conclusions</p> <p>The established universal papillation assay platform should be widely applicable to a variety of mobile elements. It can be used for mechanistic studies to dissect transposition and provides a means to screen or scrutinise transposase mutants and genes encoding host factors. In succession, improved versions of transposition systems should yield better tools for molecular biology and offer versatile genome modification vehicles for many types of studies, including gene therapy and stem cell research.</p

Ancient Migratory Events in the Middle East: New Clues from the Y-Chromosome Variation of Modern Iranians

Knowledge of high resolution Y-chromosome haplogroup diversification within Iran provides important geographic context regarding the spread and compartmentalization of male lineages in the Middle East and southwestern Asia. At present, the Iranian population is characterized by an extraordinary mix of different ethnic groups speaking a variety of Indo-Iranian, Semitic and Turkic languages. Despite these features, only few studies have investigated the multiethnic components of the Iranian gene pool. In this survey 938 Iranian male DNAs belonging to 15 ethnic groups from 14 Iranian provinces were analyzed for 84 Y-chromosome biallelic markers and 10 STRs. The results show an autochthonous but non-homogeneous ancient background mainly composed by J2a sub-clades with different external contributions. The phylogeography of the main haplogroups allowed identifying post-glacial and Neolithic expansions toward western Eurasia but also recent movements towards the Iranian region from western Eurasia (R1b-L23), Central Asia (Q-M25), Asia Minor (J2a-M92) and southern Mesopotamia (J1-Page08). In spite of the presence of important geographic barriers (Zagros and Alborz mountain ranges, and the Dasht-e Kavir and Dash-e Lut deserts) which may have limited gene flow, AMOVA analysis revealed that language, in addition to geography, has played an important role in shaping the nowadays Iranian gene pool. Overall, this study provides a portrait of the Y-chromosomal variation in Iran, useful for depicting a more comprehensive history of the peoples of this area as well as for reconstructing ancient migration routes. In addition, our results evidence the important role of the Iranian plateau as source and recipient of gene flow between culturally and genetically distinct population

Dietary Reference Values for riboflavin

Following a request from the European Commission, the EFSA Panelon Dietetic Products, Nutrition and Allergies (NDA) derives dietary reference values (DRVs) for riboflavin. The Panelconsiders that the inflection point in the urinary riboflavin excretion curve in relation to riboflavin intake reflects body saturation and can be used as a biomarker of adequate riboflavin status. The Panelalso considers that erythrocyte glutathione reductase activation coefficient is a useful biomarker, but has limitations. For adults, the Panelconsiders that average requirements (ARs) and population reference intakes (PRIs) can be determined from the weighted mean of riboflavin intake associated with the inflection point in the urinary riboflavin excretion curve reported in four intervention studies. PRIs are derived for adults and children assuming a coefficient of variation of 10%, in the absence of information on the variability in the requirement and to account for the potential effect of physical activity and the methylenetetrahydrofolate reductase 677TT genotype. For adults, the AR and PRI are set at 1.3and 1.6mg/day. For infants aged 7-11months, an adequate intake of 0.4mg/day is set by upward extrapolation from the riboflavin intake of exclusively breastfed infants aged 0-6months. For children, ARs are derived by downward extrapolation from the adult AR, applying allometric scaling and growth factors and considering differences in reference body weight. For children of both sexes aged 1-17years, ARs range between 0.5 and 1.4mg/day, and PRIs between 0.6 and 1.6mg/day. For pregnant or lactating women, additional requirements are considered, to account for fetal uptake and riboflavin accretion in the placenta during pregnancy or the losses through breast milk, and PRIs of 1.9 and 2.0mg/day, respectively, are derived

Medullary and papillary carcinoma of the thyroid gland occurring as a collision tumor with lymph node metastasis: A case report

<p>Abstract</p> <p>Introduction</p> <p>Papillary thyroid carcinoma and medullary thyroid carcinoma are two different thyroid neoplasia. The simultaneous occurrence of medullary thyroid carcinoma and papillary thyroid carcinoma as a collison tumor with metastases from both lesions in the regional lymph nodes is a rare phenomenon.</p> <p>Case presentation</p> <p>A 32-year-old Iranian man presented with a fixed anterior neck mass. Ultrasonography revealed two separate thyroid nodules as well as a suspicious neck mass that appeared to be a metastatic lesion. The results of thyroid function tests were normal, but the preoperative calcitonin serum value was elevated. Our patient underwent a total thyroidectomy with neck exploration. Two separate and ill-defined solid lesions grossly in the right lobe were noticed. Histological and immunohistochemical studies of these lesions suggested the presence of medullary thyroid carcinoma and papillary thyroid carcinoma. The lymph nodes isolated from a neck dissection specimen showed metastases from both lesions.</p> <p>Conclusions</p> <p>The concomitant occurrence of papillary thyroid carcinoma and medullary thyroid carcinoma and the exact diagnosis of this uncommon event are important. The treatment strategy should be reconsidered in such cases, and genetic screening to exclude multiple endocrine neoplasia 2 syndromes should be performed. For papillary thyroid carcinoma, radioiodine therapy and thyroid-stimulating hormone suppressive therapy are performed. However, the treatment of medullary thyroid carcinoma is mostly radical surgery with no effective adjuvant therapy.</p

Primary Prevention of Gestational Diabetes Mellitus and Large-for-Gestational-Age Newborns by Lifestyle Counseling: A Cluster-Randomized Controlled Trial

In a cluster-randomized trial, Riitta Luoto and colleagues find that counseling on diet and activity can reduce the birthweight of babies born to women at risk of developing gestational diabetes mellitus (GDM), but fail to find an effect on GDM

Influence of wood species on toxicity of log-wood stove combustion aerosols: A parallel animal and air-liquid interface cell exposure study on spruce and pine smoke

Background Wood combustion emissions have been studied previously either by in vitro or in vivo models using collected particles, yet most studies have neglected gaseous compounds. Furthermore, a more accurate and holistic view of the toxicity of aerosols can be gained with parallel in vitro and in vivo studies using direct exposure methods. Moreover, modern exposure techniques such as air-liquid interface (ALI) exposures enable better assessment of the toxicity of the applied aerosols than, for example, the previous state-of-the-art submerged cell exposure techniques. Methods We used three different ALI exposure systems in parallel to study the toxicological effects of spruce and pine combustion emissions in human alveolar epithelial (A549) and murine macrophage (RAW264.7) cell lines. A whole-body mouse inhalation system was also used to expose C57BL/6 J mice to aerosol emissions. Moreover, gaseous and particulate fractions were studied separately in one of the cell exposure systems. After exposure, the cells and animals were measured for various parameters of cytotoxicity, inflammation, genotoxicity, transcriptome and proteome. Results We found that diluted (1:15) exposure pine combustion emissions (PM1 mass 7.7 ± 6.5 mg m− 3, 41 mg MJ$^{Zahl}$) contained, on average, more PM and polycyclic aromatic hydrocarbons (PAHs) than spruce (PM1 mass 4.3 ± 5.1 mg m− 3, 26 mg MJ− 1) emissions, which instead showed a higher concentration of inorganic metals in the emission aerosol. Both A549 cells and mice exposed to these emissions showed low levels of inflammation but significantly increased genotoxicity. Gaseous emission compounds produced similar genotoxicity and a higher inflammatory response than the corresponding complete combustion emission in A549 cells. Systems biology approaches supported the findings, but we detected differing responses between in vivo and in vitro experiments. Conclusions Comprehensive in vitro and in vivo exposure studies with emission characterization and systems biology approaches revealed further information on the effects of combustion aerosol toxicity than could be achieved with either method alone. Interestingly, in vitro and in vivo exposures showed the opposite order of the highest DNA damage. In vitro measurements also indicated that the gaseous fraction of emission aerosols may be more important in causing adverse toxicological effects. Combustion aerosols of different wood species result in mild but aerosol specific in vitro and in vivo effects

Photocatalytic Decomposition of Formic Acid on Mo2C-Containing Catalyst

Soluble components in the peripheral blood from experimental exposure of 14 healthy subjects to filtered air and wood smoke. Samples were collected before (pre), at 24 h and 44 h after exposure, to air and wood smoke. Data are given as medians with interquartile range. (DOCX 62 kb

HIV Replication Enhances Production of Free Fatty Acids, Low Density Lipoproteins and Many Key Proteins Involved in Lipid Metabolism: A Proteomics Study

BACKGROUND: HIV-infected patients develop multiple metabolic abnormalities including insulin resistance, lipodystrophy and dyslipidemia. Although progression of these disorders has been associated with the use of various protease inhibitors and other antiretroviral drugs, HIV-infected individuals who have not received these treatments also develop lipid abnormalities albeit to a lesser extent. How HIV alters lipid metabolism in an infected cell and what molecular changes are affected through protein interaction pathways are not well-understood. RESULTS: Since many genetic, epigenetic, dietary and other factors influence lipid metabolism in vivo, we have chosen to study genome-wide changes in the proteomes of a human T-cell line before and after HIV infection in order to circumvent computational problems associated with multiple variables. Four separate experiments were conducted including one that compared 14 different time points over a period of >3 months. By subtractive analyses of protein profiles overtime, several hundred differentially expressed proteins were identified in HIV-infected cells by mass spectrometry and each protein was scrutinized for its biological functions by using various bioinformatics programs. Herein, we report 18 HIV-modulated proteins and their interaction pathways that enhance fatty acid synthesis, increase low density lipoproteins (triglycerides), dysregulate lipid transport, oxidize lipids, and alter cellular lipid metabolism. CONCLUSIONS: We conclude that HIV replication alone (i.e. without any influence of antiviral drugs, or other human genetic factors), can induce novel cellular enzymes and proteins that are significantly associated with biologically relevant processes involved in lipid synthesis, transport and metabolism (p = <0.0002-0.01). Translational and clinical studies on the newly discovered proteins may now shed light on how some of these proteins may be useful for early diagnosis of individuals who might be at high risk for developing lipid-related disorders. The target proteins could then be used for future studies in the development of inhibitors for preventing lipid-metabolic anomalies. This is the first direct evidence that HIV-modulates production of proteins that are significantly involved in disrupting the normal lipid-metabolic pathways

Dying well with reduced agency: a scoping review and thematic synthesis of the decision-making process in dementia, traumatic brain injury and frailty

Background In most Anglophone nations, policy and law increasingly foster an autonomy-based model, raising issues for large numbers of people who fail to fit the paradigm, and indicating problems in translating practical and theoretical understandings of ‘good death’ to policy. Three exemplar populations are frail older people, people with dementia and people with severe traumatic brain injury. We hypothesise that these groups face some over-lapping challenges in securing good end-of-life care linked to their limited agency. To better understand these challenges, we conducted a scoping review and thematic synthesis. Methods To capture a range of literature, we followed established scoping review methods. We then used thematic synthesis to describe the broad themes emerging from this literature. Results Initial searches generated 22,375 references, and screening yielded 49, highly heterogeneous, studies that met inclusion criteria, encompassing 12 countries and a variety of settings. The thematic synthesis identified three themes: the first concerned the processes of end-of-life decision-making, highlighting the ambiguity of the dominant shared decision-making process, wherein decisions are determined by families or doctors, sometimes explicitly marginalising the antecedent decisions of patients. Despite this marginalisation, however, the patient does play a role both as a social presence and as an active agent, by whose actions the decisions of those with authority are influenced. The second theme examined the tension between predominant notions of a good death as ‘natural’ and the drive to medicalise death through the lens of the experiences and actions of those faced with the actuality of death. The final theme considered the concept of antecedent end-of-life decision-making (in all its forms), its influence on policy and decision-making, and some caveats that arise from the studies. Conclusions Together these three themes indicate a number of directions for future research, which are likely to be applicable to other conditions that result in reduced agency. Above all, this review emphasises the need for new concepts and fresh approaches to end of life decision-making that address the needs of the growing population of frail older people, people with dementia and those with severe traumatic brain injury