Creation and validation of a ligation-independent cloning (LIC) retroviral vector for stable gene transduction in mammalian cells

<p>Abstract</p> <p>Background</p> <p>Cloning vectors capable of retroviral transduction have enabled stable gene overexpression in numerous mitotic cell lines. However, the relatively small number of feasible restriction enzyme sequences in their cloning sites can hinder successful generation of overexpression constructs if these sequences are also present in the target cDNA insert.</p> <p>Results</p> <p>Utilizing ligation-independent cloning (LIC) technology, we have modified the highly efficient retroviral transduction vector, pBABE, to eliminate reliance on restriction enzymes for cloning. Instead, the modified plasmid, pBLIC, utilizes random 12/13-base overhangs generated by T4 DNA polymerase 3' exonuclease activity. PCR-based introduction of the complementary sequence into any cDNA of interest enables universal cloning into pBLIC. Here we describe creation of the pBLIC plasmid, and demonstrate successful cloning and protein overexpression from three different cDNAs, Bax, catalase, and p53 through transduction into the human prostate cancer cell line, LNCaP or the human lung cancer line, H358.</p> <p>Conclusions</p> <p>Our results show that pBLIC vector retains the high transduction efficiency of the original pBABE while eliminating the requirement for checking individual cDNA inserts for internal restriction sites. Thus it comprises an effective retroviral cloning system for laboratory-scale stable gene overexpression or for high-throughput applications such as creation of retroviral cDNA libraries. To our knowledge, pBLIC is the first LIC vector for retroviral transduction-mediated stable gene expression in mammalian cells.</p

Formación del profesorado y desarrollo profesional en los Estados Unidos

Los encargados de la enseñanza de las matemáticas desde preescolar hasta undécimo año, tienen una tarea muy importante e influyente en el desarrollo de la sociedad. Son los docentes quienes deben formar a los estudiantes en esta área. Sin embargo, algunos de los docentes encargados de la formación, en matemáticas, no tienen la preparación adecuada para hacerlo, principalmente, los docentes de los niveles más básicos como desde preescolar hasta primaria. Conscientes de la necesidad de formación y capacitación de docentes en servicio, y en formación, es que algunas organizaciones de los Estados Unidos han trabajado tanto en diagnosticar la magnitud del problema como de plantear posibles soluciones al mismo dentro de su nación

Early Regeneration of Thymic Progenitors in Rhesus Macaques Infected with Simian Immunodeficiency Virus

The thymus plays a critical role in the maturation and production of T lymphocytes and is a target of infection by human immunodeficiency virus (HIV) and the related simian immunodeficiency virus (SIV). Using the SIV/macaque model of AIDS, we examined the early effects of SIV on the thymus. We found that thymic infection by SIV resulted in increased apoptosis 7–14 d after infection, followed by depletion of thymocyte progenitors by day 21. A marked rebound in thymocyte progenitors occurred by day 50 and was accompanied by increased levels of cell proliferation in the thymus. Our results demonstrate a marked increase in thymic progenitor activity very early in the course of SIV infection, long before marked declines in peripheral CD4+ T cell counts

Macondo crude oil from the Deepwater Horizon oil spill disrupts specific developmental processes during zebrafish embryogenesis

Background: The Deepwater Horizon disaster was the largest marine oil spill in history, and total vertical exposure of oil to the water column suggests it could impact an enormous diversity of ecosystems. The most vulnerable organisms are those encountering these pollutants during their early life stages. Water-soluble components of crude oil and specific polycyclic aromatic hydrocarbons have been shown to cause defects in cardiovascular and craniofacial development in a variety of teleost species, but the developmental origins of these defects have yet to be determined. We have adopted zebrafish, Danio rerio, as a model to test whether water accumulated fractions (WAF) of the Deepwater Horizon oil could impact specific embryonic developmental processes. While not a native species to the Gulf waters, the developmental biology of zebrafish has been well characterized and makes it a powerful model system to reveal the cellular and molecular mechanisms behind Macondo crude toxicity. Results: WAF of Macondo crude oil sampled during the oil spill was used to treat zebrafish throughout embryonic and larval development. Our results indicate that the Macondo crude oil causes a variety of significant defects in zebrafish embryogenesis, but these defects have specific developmental origins. WAF treatments caused defects in craniofacial development and circulatory function similar to previous reports, but we extend these results to show they are likely derived from an earlier defect in neural crest cell development. Moreover, we demonstrate that exposure to WAFs causes a variety of novel deformations in specific developmental processes, including programmed cell death, locomotor behavior, sensory and motor axon pathfinding, somitogenesis and muscle patterning. Interestingly, the severity of cell death and muscle phenotypes decreased over several months of repeated analysis, which was correlated with a rapid drop-off in the aromatic and alkane hydrocarbon components of the oil. Conclusions: Whether these teratogenic effects are unique to the oil from the Deepwater Horizon oil spill or generalizable for most crude oil types remains to be determined. This work establishes a model for further investigation into the molecular mechanisms behind crude oil mediated deformations. In addition, due to the high conservation of genetic and cellular processes between zebrafish and other vertebrates, our work also provides a platform for more focused assessment of the impact that the Deepwater Horizon oil spill has had on the early life stages of native fish species in the Gulf of Mexico and the Atlantic Ocean

Expiratory airflow in late adolescence and early adulthood in individuals born very preterm or with very low birthweight compared with controls born at term or with normal birthweight : a meta-analysis of individual participant data

Background Maximal expiratory airflow peaks early in the third decade of life, then gradually declines with age. The pattern of airflow through adulthood for individuals born very preterm (at 2499 g) or at term. Methods We did a meta-analysis of individual participant data from cohort studies, mostly from the pre-surfactant era. Studies were identified through the Adults born Preterm International Collaboration and by searching PubMed and Embase (search date May 25, 2016). Studies were eligible if they reported on expiratory flow rates beyond 16 years of age in individuals born very preterm or with very low birthweight, as well as controls born at term or with normal birthweight. Studies with highly selected cohorts (eg, only participants with bronchopulmonary dysplasia) or in which few participants were born very preterm or with very low birthweight were excluded. De-identified individual participant data from each cohort were provided by the holders of the original data to a central site, where all the data were pooled into one data file. Any data inconsistencies were resolved by discussion with the individual sites concerned. Individual participant data on expiratory flow variables (FEV1, forced vital capacity [FVC], FEV1/FVC ratio, and forced expiratory flow at 25-75% of FVC [FEF25-75%]) were converted to Z scores and analysed with use of generalised linear mixed models in a one-step approach. Findings Of the 381 studies identified, 11 studies, comprising a total of 935 participants born very preterm or with very low birthweight and 722 controls, were eligible and included in the analysis. Mean age at testing was 21 years (SD 3.4; range 16-33). Mean Z scores were close to zero (as expected) in the control group, but were reduced in the very preterm or very low birthweight group for FEV1 (-0.06 [SD 1.03] vs -0.81 [1.33], mean difference -0.78 [95% CI -0.96 to -0.61], p Interpretation Individuals born very preterm or with very low birthweight are at risk of not reaching their full airway growth potential in adolescence and early adulthood, suggesting an increased risk of chronic obstructive pulmonary disease in later adulthood. Copyright (C) 2019 Elsevier Ltd. All rights reserved.Peer reviewe

Predicting eating disorder and anxiety symptoms using disorder-specific and transdiagnostic polygenic scores for anorexia nervosa and obsessive-compulsive disorder

BACKGROUND: Clinical, epidemiological, and genetic findings support an overlap between eating disorders, obsessive-compulsive disorder (OCD), and anxiety symptoms. However, little research has examined the role of genetics in the expression of underlying phenotypes. We investigated whether the anorexia nervosa (AN), OCD, or AN/OCD transdiagnostic polygenic scores (PGS) predict eating disorder, OCD, and anxiety symptoms in a large developmental cohort in a sex-specific manner. METHODS: Using summary statistics from Psychiatric Genomics Consortium AN and OCD genome-wide association studies, we conducted an AN/OCD transdiagnostic genome-wide association meta-analysis. We then calculated AN, OCD, and AN/OCD PGS in participants from the Avon Longitudinal Study of Parents and Children to predict eating disorder, OCD, and anxiety symptoms, stratified by sex (combined N = 3212-5369 per phenotype). RESULTS: The PGS prediction of eating disorder, OCD, and anxiety phenotypes differed between sexes, although effect sizes were small. AN and AN/OCD PGS played a more prominent role in predicting eating disorder and anxiety risk than OCD PGS, especially in girls. AN/OCD PGS provided a small boost over AN PGS in the prediction of some anxiety symptoms. All three PGS predicted higher compulsive exercise across different developmental timepoints [β = 0.03 (s.e. = 0.01) for AN and AN/OCD PGS at age 14; β = 0.05 (s.e. = 0.02) for OCD PGS at age 16] in girls. CONCLUSIONS: Compulsive exercise may have a transdiagnostic genetic etiology, and AN genetic risk may play a role in the presence of anxiety symptoms. Converging with prior twin literature, our results also suggest that some of the contribution of genetic risk may be sex-specific

The total antioxidant content of more than 3100 foods, beverages, spices, herbs and supplements used worldwide

Background: A plant-based diet protects against chronic oxidative stress-related diseases. Dietary plants contain variable chemical families and amounts of antioxidants. It has been hypothesized that plant antioxidants may contribute to the beneficial health effects of dietary plants. Our objective was to develop a comprehensive food database consisting of the total antioxidant content of typical foods as well as other dietary items such as traditional medicine plants, herbs and spices and dietary supplements. This database is intended for use in a wide range of nutritional research, from in vitro and cell and animal studies, to clinical trials and nutritional epidemiological studies. Methods: We procured samples from countries worldwide and assayed the samples for their total antioxidant content using a modified version of the FRAP assay. Results and sample information (such as country of origin, product and/or brand name) were registered for each individual food sample and constitute the Antioxidant Food Table. Results: The results demonstrate that there are several thousand-fold differences in antioxidant content of foods. Spices, herbs and supplements include the most antioxidant rich products in our study, some exceptionally high. Berries, fruits, nuts, chocolate, vegetables and products thereof constitute common foods and beverages with high antioxidant values. Conclusions: This database is to our best knowledge the most comprehensive Antioxidant Food Database published and it shows that plant-based foods introduce significantly more antioxidants into human diet than non-plant foods. Because of the large variations observed between otherwise comparable food samples the study emphasizes the importance of using a comprehensive database combined with a detailed system for food registration in clinical and epidemiological studies. The present antioxidant database is therefore an essential research tool to further elucidate the potential health effects of phytochemical antioxidants in diet

Formación del Profesorado y Desarrollo Profesional en los Estados Unidos

Los encargados de la enseñanza de las matemáticas desde preescolar hasta undécimo año, tienen una tarea muy importante e influyente en el desarrollo de la sociedad. Son los docentes quienes deben formar a los estudiantes en esta área.Sin embargo, algunos de los docentes encargados de la formación, en matemáticas, no tienen la preparación adecuada para hacerlo, principalmente,los docentes de los niveles más básicos como desde preescolar hasta primaria.Conscientes de la necesidad de formación y capacitación de docentes en servicio, y en formación, es que algunas organizaciones de los Estados Unidos han trabajado tanto en diagnosticar la magnitud del problema como de plantear posibles soluciones al mismo dentro de su nación. Those involved in mathematics education from kindergarten to eleventh year, have a very important and influential role in the development of society. They are the teachers who must train students in this area.However, some of the teaching staff training in mathematics, are not adequately prepared to do so, mainly teachers as the most basic levels from kindergarten to primary school.Aware of the need for training and in-service teacher training, is that some organizations of the United States have worked so hard to diagnose the magnitude of the problem and pose possible solutions within their nation

Immunosuppression Cascade in the Florida Manatee (Trichechus manatus latirostris)

The Florida manatee (Trichechus manatus latirostris) is a federally endangered marine mammal that resides on both coasts of the Florida peninsula. The species faces many anthropogenic and natural threats, including boat strikes, exposure to harmful algal blooms, and cold water temperatures. We have developed a conceptual model that depicts how changes in the environment, such as reduced water temperature, can trigger an immunosuppressive cascade of interrelated diseases and pathological conditions ultimately leading to the death of the animal. Although the Florida manatee has a relatively robust immune system, rendering it resistant to several diseases, the onset of unfavorable environmental conditions has been shown to compromise the immune system, often leading to infections that make an animal more susceptible to opportunistic pathogens. In this paper, we review several common diseases of the Florida manatee and compare and contrast their symptoms. We then interrelate these diseases and generate a conceptual model of a cascade of immunosuppressive conditions originally triggered by adverse environmental conditions or one of the diseases. The end result of the cascade is the death of the animal