Exploiting the adenoassociated virus Rep protein to mediate site-specific integration into the human genome and optimisation of Ad/AAV vector design

Gene therapy is an approach to treating diseases in which an exogenous gene is introduced to correct for a defective or missing protein or to affect a biochemical pathway. Few successes have been reported in humans (0), as several technical issues limit its broader application. One question is how to deliver DNA to the appropriate cells. Nature provides one solution in the form of viruses, which are in essence protected gene delivery packages with native ability to introduce their genomes into cells. Once the desired gene is delivered to target cells, another issue that arises is the fate of the DNA. Some strategies rely on long-term expression from extra-chromosomal DNA, but there are cases, such as dividing cells, where it would be highly beneficial to permanently insert the gene into chromosomes. Certain viral genomes can be integrated into host DNA by non-homologous recombination or, in the case of retroviruses, by virally encoded integrases. While integration seems to be not dependent on target sequence, in vivo, retroviruses, such as HIV and murine leukemia virus, integrate preferentially into active genes (1, 2), introducing the possibility of insertional mutagenesis. The theoretical danger inherent in retrovirus-based gene therapy has been concretely demonstrated in a recent clinical trial in which the modified retrovirus integrated into the LMO2 locus, causing leukemia in three of the patients (4). A powerful system to circumvent this critical issue could be to develop a system to site-specific integrate the exogenous gene into a safety zone into the genome. To date, only one animal virus, the adenoassociated virus (AAV), has been identified that integrates its genome into a particular location into human chromosomal DNA. When cells are infected in the absence of helper virus, AAV establishes a latent infection in which the AAV genome integrates into a locus known as AAVS1 on the q arm of chromosome 19 (4). Recombinant AAV (rAAV) vectors too have a series of limitations as gene therapy vectors: they can accommodate only small genes and moreover eliminating most of wild-type AAV (wt AAV) sequences they have lost almost all the site-specific integration capability. On the basis of AAV site-specific integration machinery a series of effort to re-introduce wt AAV’s integration efficiency have been done. Different viruses have been engineered using the AAV’s integration machinery to transform them to target and integrate site-specifically large genes into the chr 19. Development of a maximized integrating, large capacity DNA viral vector is still an unmet goal of gene transfer technology. The initial aim of this project was to characterize the combination of the attributes of both the AAV and adenovirus (Ad) gene therapy vectors to develop an Ad/AAV hybrid virus system able to target site-specifically a large fragment of DNA into the host cell genome. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional rAAV integrating elements. The finding that traditional rAAV plasmid vectors lack integration potency compared to wt AAV plasmid constructs led recently to the discovery of an AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. This study has addressed both of these problems. Moreover the project aimed also the capability of such vectors to target a large integrating cassette and the differences in the system integration efficiency compairing the dimension of the integrating cassettes. We demonstrated that an Ad can be generated that expresses Rep proteins and that Rep-mediated AAV persistence can occur in the presence of Ad vectors. We exploited the size limit capability introducing a large integrating cassette (12 kb) into the Ad/AAV vector and we obtained a good level of integration into the human genome. Specifically we succeeded in integrating it without any recombination event and in a site-specific fashion at good level. The model we extensively tested in human cell lines was also used successfully into human primary cells where we obtained site-specific integration into human chromosome 19 as expected. Another problem analysed was the flexibility of Ad vector system. Adenoviral vectors maintain the cellular specificity of adenoviruses from which they derive. A good gene therapy vector should have a broad tropism to ensure a good transduction efficiency, and moreover should be able to transduce cells of interest, such as CD34+ cells and other primary cells. We tried to expand Ad tropism engineering the commonly used Ad 5 vector (not able to transduce very well CD34+ and hematopoietic cells), transforming it to an Ad 5/35 modified vector. This approach permitted us to have better CD34+ cell transduction and a very good efficiency with hematopoietic cell lines

Understanding preferences for tree attributes: the relative effects of socio-economic and local environmental factors

Urban plant biodiversity is influenced by both the physical environment and attitudes and preferences of urban residents for specific plant types. Urban residents are assumed to be disconnected from their immediate environment, and cultural and societal factors have been emphasized over environmental factors in studies of landscaping choices. However, we postulate that local climatic and environmental factors can also affect preferences for plant attributes. Therefore, spatial and temporal patterns in urban tree biodiversity may be driven not only by the direct effect of environmental variables on plant function, but also by the effect of environmental variables on attitudes toward trees and associated choices about which types of trees to plant. Here, we tested the relative effects of socio-economic and local environmental factors on preferences toward tree attributes in five counties in southern California in and surrounding Los Angeles, based on 1,029 household surveys. We found that local environmental factors have as strong an effect on preferences for tree attributes as socio-economic factors. Specifically, people located in hotter climates (average maximum temperature 25.1 °C) were more likely to value shade trees than those located in cooler regions (23.1 °C). Additionally, people located in desert areas were less likely to consider trees to be important in their city compared with people located in naturally forested areas. Overall, our research demonstrates the inherent connections between local environmental factors and perceptions of nature, even in large modern cities. Accounting for these factors can contribute to the growing interest in understanding patterns of urban biodiversity

Migrazioni e networks urbani

Ripercorrendo l’ampio dibattito sul tema emerge quanto numerose siano le definizioni di integrazione elaborate dagli studiosi che si sono occupati di migrazioni. Soprattutto in anni più recenti, in forza dei rilevanti cambiamenti dei fenomeni migratori in atto, in molti concordano che questi processi sono aperti a molteplici esiti, in gran parte collegati a fattori di contesto politico, sociale, economico e culturale. Questi diversi fattori rappresentano altrettante dimensioni con cui si può guardare all’integrazione, che pertanto si configura come concetto multidimensionale, oltre che dinamico, e che può essere declinato a diversi livelli di analisi. Il livello relazionale (livello meso) rappresenta il punto di convergenza di fattori di integrazione macro e micro: i percorsi di inserimento urbano spesso dipendono dall’efficacia delle reti nelle quali si è inseriti. Questo contributo presenta i risultati di una ricerca condotta nel quartiere Mercato a Napoli, che ha avuto come oggetto di analisi l’integrazione della comunità cabardina, attraverso la metodologia e gli strumenti della Social Network Analysis.There are many definitions of integration developed by scholars of migration. They agree – especially in recent years, due to the significant changes in migration – that these processes are open to multiple outcomes, largely related political, social, economic and cultural factors. These different factors represent the different dimension which you can look to the integration; a term that appears as a multidimensional concept, as well as dynamic, and can be declined at different levels of analysis. The relational level (meso-level) represents the point of convergence between macro and micro factors of integration. In fact, the urban integration processes often depend on the effectiveness of their own social networks. This paper presents the results of a survey in the Mercato neighborhood (Naples). The aim is to analyze the integration of Kabardians community, through Social Network Analysis methods.Peer Reviewe

Rational treatment of metastatic colorectal cancer: A reverse tale of men, mice, and culture dishes

Stratification of colorectal cancer into subgroups with different response to therapy was initially guided by descriptive associations between specific biomarkers and treatment outcome. Recently, preclinical models based on propagatable patient-derived tumor samples have yielded an improved understanding of disease biology, which has facilitated the functional validation of correlative information and the discovery of novel response determinants, therapeutic targets, and mechanisms of tumor adaptation and drug resistance. We review the contribution of patient-derived models to advancing colorectal cancer characterization, discuss their influence on clinical decision-making, and highlight emerging challenges in the interpretation and clinical transferability of results obtainable with such approaches. SIGNIFICANCE: Association studies in patients with colorectal cancer have led to the identification of response biomarkers, some of which have been implemented as companion diagnostics for therapeutic decisions. By enabling biological investigation in a clinically relevant experimental context, patient-derived colorectal cancer models have proved useful to examine the causal role of such biomarkers in dictating drug sensitivity and are providing fresh knowledge on new actionable targets, dynamics of tumor evolution and adaptation, and mechanisms of drug resistance

Experimental determination of the frequency and field dependence of Specific Loss Power in Magnetic Fluid Hyperthermia

Magnetic nanoparticles are promising systems for biomedical applications and in particular for Magnetic Fluid Hyperthermia, a promising therapy that utilizes the heat released by such systems to damage tumor cells. We present an experimental study of the physical properties that influences the capability of heat release, i.e. the Specific Loss Power, SLP, of three biocompatible ferrofluid samples having a magnetic core of maghemite with different core diameter d= 10.2, 14.6 and 19.7 nm. The SLP was measured as a function of frequency f and intensity of the applied alternating magnetic field H, and it turned out to depend on the core diameter, as expected. The results allowed us to highlight experimentally that the physical mechanism responsible for the heating is size-dependent and to establish, at applied constant frequency, the phenomenological functional relationship SLP=cH^x, with 2<x<3 for all samples. The x-value depends on sample size and field frequency/ intensity, here chosen in the typical range of operating magnetic hyperthermia devices. For the smallest sample, the effective relaxation time Teff=19.5 ns obtained from SLP data is in agreement with the value estimated from magnetization data, thus confirming the validity of the Linear Response Theory model for this system at properly chosen field intensity and frequency

New nurses burnout and workplace wellbeing:The influence of authentic leadership and psychological capital

The detrimental effects of burnout on nurses’ health and wellbeing are well documented and positive leadership has been shown to be an important organizational resource for discouraging the development of burnout. Intrapersonal resources also play a protective role against workplace stressors. This study investigated the influence of authentic leadership, an organizational resource, and psychological capital, an intrapersonal resource, on new graduate burnout, occupational satisfaction, and workplace mental health over the first year of employment (n = 205). Results supported the protective role of organizational and intrapersonal resources against burnout, job dissatisfaction, and mental health. © 2014 The Authors. Published by Elsevier GmbH. This is an open access article under the CC-BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/)

Cellular-Automata model for dense-snow avalanches

This paper introduces a three-dimensional model for simulating dense-snow avalanches, based on the numerical method of cellular automata. This method allows one to study the complex behavior of the avalanche by dividing it into small elements, whose interaction is described by simple laws, obtaining a reduction of the computational power needed to perform a three-dimensional simulation. Similar models by several authors have been used to model rock avalanches, mud and lava flows, and debris avalanches. A peculiar aspect of avalanche dynamics, i.e., the mechanisms of erosion of the snowpack and deposition of material from the avalanche is taken into account in the model. The capability of the proposed approach has been illustrated by modeling three documented avalanches that occurred in Susa Valley (Western Italian Alps). Despite the qualitative observations used for calibration, the proposed method is able to reproduce the correct three-dimensional avalanche path, using a digital terrain model, and the order of magnitude of the avalanche deposit volume

Energy loss in calorimeters using muon spectrometer information at the 2004 ATLAS Combined Test Beam.

In 2004 an ATLAS Combined Test Beam (CTB) was performed in the CERN North area. A complete slice of the barrel detector and of the muon end-cap was tested, with the following goals: pre-commission the final elements and study the combined detector performance. In this note a combined analysis using calorimeter and muon spectrometer information, based on data samples collected during this test, is presented