Targeted Chromosomal Insertion of Large DNA into the Human Genome by a Fiber-Modified High-Capacity Adenovirus-Based Vector System

A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant full-length DMD genes)

Haemophilus influenzae Infection Drives IL-17-Mediated Neutrophilic Allergic Airways Disease

A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12–15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses

Biology of human hair: Know your hair to control it

Hair can be engineered at different levels—its structure and surface—through modification of its constituent molecules, in particular proteins, but also the hair follicle (HF) can be genetically altered, in particular with the advent of siRNA-based applications. General aspects of hair biology are reviewed, as well as the most recent contributions to understanding hair pigmentation and the regulation of hair development. Focus will also be placed on the techniques developed specifically for delivering compounds of varying chemical nature to the HF, indicating methods for genetic/biochemical modulation of HF components for the treatment of hair diseases. Finally, hair fiber structure and chemical characteristics will be discussed as targets for keratin surface functionalization

Quantum Spacetime Phenomenology

I review the current status of phenomenological programs inspired by quantum-spacetime research. I stress in particular the significance of results establishing that certain data analyses provide sensitivity to effects introduced genuinely at the Planck scale. And my main focus is on phenomenological programs that managed to affect the directions taken by studies of quantum-spacetime theories.Comment: 125 pages, LaTex. This V2 is updated and more detailed than the V1, particularly for quantum-spacetime phenomenology. The main text of this V2 is about 25% more than the main text of the V1. Reference list roughly double

Large-scale unit commitment under uncertainty: an updated literature survey

The Unit Commitment problem in energy management aims at finding the optimal production schedule of a set of generation units, while meeting various system-wide constraints. It has always been a large-scale, non-convex, difficult problem, especially in view of the fact that, due to operational requirements, it has to be solved in an unreasonably small time for its size. Recently, growing renewable energy shares have strongly increased the level of uncertainty in the system, making the (ideal) Unit Commitment model a large-scale, non-convex and uncertain (stochastic, robust, chance-constrained) program. We provide a survey of the literature on methods for the Uncertain Unit Commitment problem, in all its variants. We start with a review of the main contributions on solution methods for the deterministic versions of the problem, focussing on those based on mathematical programming techniques that are more relevant for the uncertain versions of the problem. We then present and categorize the approaches to the latter, while providing entry points to the relevant literature on optimization under uncertainty. This is an updated version of the paper "Large-scale Unit Commitment under uncertainty: a literature survey" that appeared in 4OR 13(2), 115--171 (2015); this version has over 170 more citations, most of which appeared in the last three years, proving how fast the literature on uncertain Unit Commitment evolves, and therefore the interest in this subject

Down the line from genome-wide association studies in inflammatory bowel disease:the resulting clinical benefits and the outlook for the future

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis, is a chronic inflammatory disease of the gut. The etiology of IBD is complex, involving genetic as well as environmental factors. Genetic studies have identified 163 genetic risk loci for IBD, which have led to new insights into the biological mechanisms of the disease. The currently known IBD risk loci show an almost 75% overlap with genetic risk loci for other immune mediated diseases. Current studies are focused on the translation of the identified risk loci to clinical practice. The first steps towards this translation are being taken with the identification of genetic risk factors for drugs toxicity, specific disease course and response to therapy. In this review we will discuss how the IBD genetic risk loci were identified and how this knowledge can be translated towards clinical practice