Cloning and functional characterisation of avian transcription factor E2A.

BACKGROUND: During B lymphocyte development the E2A gene is a critical regulator of cell proliferation and differentiation. With regards to the immunoglobulin genes the E2A proteins contribute to the regulation of gene rearrangement, expression and class switch recombination. We are now using the chicken cell line DT40 as a model system to further analyse the function of E2A. RESULTS: Here we report the cloning and functional analysis of the transcription factor E2A from chicken. Using RACE PCR on the chicken lymphoma cell line DT40 we have isolated full-length clones for the two E2A splice variants E12 and E47. Sequence conservation between the human and chicken proteins is extensive: the basic-helix-loop-helix DNA binding domain of human and chicken E47 and E12 are 93% and 92% identical, respectively. In addition high levels of conservation are seen in activation domain I, the potential NLS and the ubiquitin ligase interaction domain. E2A is expressed in a variety of tissues in chicken, with higher levels of expression in organs rich in immune cells. We demonstrate that chicken E12 and E47 proteins are strong transcriptional activators whose function depends on the presence of activation domain I. As in mammals, the dominant negative proteins Id1 and Id3 can inhibit the function of chicken E47. CONCLUSIONS: The potential for homologous recombination in DT40 allows the genetic dissection of biochemical pathways in somatic cells. With the cloning of avian E2A and the recent description of an in vitro somatic hypermutation assay in this cell line, it should now be possible to dissect the potential role of E2A in the regulation of somatic hypermutation and gene conversion.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

DDG 51 operational evaluation: measures of workload from Combat Information Center communication patterns

This thesis analyzes 2,700 verbal transmissions from an audio tap on DDG-51's CIC internal communication network during the ship's OPEVAL. The frequency and duration of these voice transmissions are analyzed to explore for systematic changes. these changes are associated with different workload levels and the levels of stress induced by eight simulated combat scenarios. The data shows that CIC team member communication patterns varied as a function of workload. The use of verbal communication patterns as unobtrusive, noninvasive measures of workload in operational settings is discussed and recommendations are made to further develop these measures.http://archive.org/details/ddg51operational00conlLieutenant, United States NavyApproved for public release; distribution is unlimited

Equine or porcine synovial fluid as a novel ex vivo model for the study of bacterial free-floating biofilms that form in human joint infections

Bacterial invasion of synovial joints, as in infectious or septic arthritis, can be difficult to treat in both veterinary and human clinical practice. Biofilms, in the form of free-floating clumps or aggregates, are involved with the pathogenesis of infectious arthritis and periprosthetic joint infection (PJI). Infection of a joint containing an orthopedic implant can additionally complicate these infections due to the presence of adherent biofilms. Because of these biofilm phenotypes, bacteria within these infected joints show increased antimicrobial tolerance even at high antibiotic concentrations. To date, animal models of PJI or infectious arthritis have been limited to small animals such as rodents or rabbits. Small animal models, however, yield limited quantities of synovial fluid making them impractical for in vitro research. Herein, we describe the use of ex vivo equine and porcine models for the study of synovial fluid induced biofilm aggregate formation and antimicrobial tolerance. We observed Staphylococcus aureus and other bacterial pathogens adapt the same biofilm aggregate phenotype with significant antimicrobial tolerance in both equine and porcine synovial fluid, analogous to human synovial fluid. We also demonstrate that enzymatic dispersal of synovial fluid aggregates restores the activity of antimicrobials. Future studies investigating the interaction of bacterial cell surface proteins with host synovial fluid proteins can be readily carried out in equine or porcine ex vivo models to identify novel drug targets for treatment of prevention of these difficult to treat infectious diseases

Rapid, widespread transduction of the murine myocardium using self-complementary Adeno-associated virus

Adeno-associated virus (AAV) has shown great promise as a gene transfer vector. However, the incubation time needed to attain significant levels of gene expression is often too long for some clinical applications. Self-complementary AAV (scAAV) enters the cell as double stranded DNA, eliminating the step of second-strand synthesis, proven to be the rate-limiting step for gene expression of single-stranded AAV (ssAAV). The aim of this study was to compare the efficiency of these two types of AAV vectors in the murine myocardium. Four day old CD-1 mice were injected with either of the two AAV constructs, both expressing GFP and packaged into the AAV1 capsid. The animals were held for 4, 6, 11 or 21 days, after which they were euthanized and their hearts were excised. Serial sections of the myocardial tissue were used for real-time PCR quantification of AAV genome copies and for confocal microscopy. Although we observed similar numbers of AAV genomes at each of the different time points present in both the scAAV and the ssAAV infected hearts, microscopic analysis showed expression of GFP as early as 4 days in animals injected with the scAAV, while little or no expression was observed with the ssAAV constructs until day 11. AAV transduction of murine myocardium is therefore significantly enhanced using scAAV constructs

Axions In String Theory

In the context of string theory, axions appear to provide the most plausible solution of the strong CP problem. However, as has been known for a long time, in many string-based models, the axion coupling parameter F_a is several orders of magnitude higher than the standard cosmological bounds. We re-examine this problem in a variety of models, showing that F_a is close to the GUT scale or above in many models that have GUT-like phenomenology, as well as some that do not. On the other hand, in some models with Standard Model gauge fields supported on vanishing cycles, it is possible for F_a to be well below the GUT scale.Comment: 62 pages, v2; references, acknowledgements and minor corrections adde

Antibiotic use during pregnancy increases offspring asthma severity in a dose‐dependent manner

Background: The use of antibiotics during pregnancy is associated with increased allergic asthma risk in the offspring, and given that approximately 25% of pregnant women are prescribed antibiotics, it is important to understand the mechanisms contributing to this phenomenon. Currently, there are no studies that directly test this association experimentally. Our objective was to develop a mouse model in which antibiotic treatment during pregnancy results in increased offspring asthma susceptibility. Methods: Pregnant mice were treated daily from gestation day 8-17 with an oral solution of the antibiotic vancomycin, and three concentrations were tested. At weaning, offspring were subjected to an adjuvant-free experimental asthma protocol using ovalbumin as an allergen. The composition of the gut microbiome was determined in mothers and offspring with samples collected from five different time points; shortchain fatty acids were also analyzed in allergic offspring. Results: We found that maternal antibiotic treatment during pregnancy was associated with increased offspring asthma severity in a dose-dependent manner. Furthermore, maternal vancomycin treatment during pregnancy caused marked changes in the gut microbiome composition in both mothers and pups at several different time points. The increased asthma severity and intestinal microbiome changes in pups were also associated with significantly decreased cecal short-chain fatty acid concentrations. Conclusion: Consistent with the "Developmental Origins Hypothesis," our results confirm that exposure to antibiotics during pregnancy shapes the neonatal intestinal environment and increases offspring allergic lung inflammation

Chasing Brane Inflation in String-Theory

We investigate the embedding of brane anti-brane inflation into a concrete type IIB string theory compactification with all moduli fixed. Specifically, we are considering a D3-brane, whose position represents the inflaton $\phi$, in a warped conifold throat in the presence of supersymmetrically embedded D7-branes and an anti D3-brane localized at the tip of the warped conifold cone. After presenting the moduli stabilization analysis for a general D7-brane embedding, we concentrate on two explicit models, the Ouyang and the Kuperstein embeddings. We analyze whether the forces, induced by moduli stabilization and acting on the D3-brane, might cancel by fine-tuning such as to leave us with the original Coulomb attraction of the anti D3-brane as the driving force for inflation. For a large class of D7-brane embeddings we obtain a negative result. Cancelations are possible only for very small intervals of $\phi$ around an inflection point but not globally. For the most part of its motion the inflaton then feels a steep, non slow-roll potential. We study the inflationary dynamics induced by this potential.Comment: 34 pages, 4 figures. Final version published in JCA

Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy

Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 1012 vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD−/− mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD−/− mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD−/− mice maintained euglycemia, whereas untreated VLCAD−/− mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans

F-Theory GUT Vacua on Compact Calabi-Yau Fourfolds

We present compact three-generation F-theory GUT models meeting in particular the constraints of D3-tadpole cancellation and D-term supersymmetry. To this end we explicitly construct elliptically fibered Calabi-Yau fourfolds as complete intersections in a toric ambient space. Toric methods enable us to control the singular geometry of the SU(5) GUT model. The GUT brane wraps a non-generic del Pezzo surface admitting GUT symmetry breaking via hypercharge flux. It is contractible to a curve and we demonstrate the existence of a consistent decoupling limit. We compute the Euler characteristic of the singular Calabi-Yau fourfold to show that our three-generation flux solutions obtained via the spectral cover construction are consistent with D3-tadpole cancellation.Comment: 22+12 pages; v2: minor clarifications on decoupling limi

Improved In vivo Assessment of Pulmonary Fibrosis in Mice using X-Ray Dark-Field Radiography

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with a median life expectancy of 4-5 years after initial diagnosis. Early diagnosis and accurate monitoring of IPF are limited by a lack of sensitive imaging techniques that are able to visualize early fibrotic changes at the epithelial-mesenchymal interface. Here, we report a new x-ray imaging approach that directly visualizes the air-tissue interfaces in mice in vivo. This imaging method is based on the detection of small-angle x-ray scattering that occurs at the air-tissue interfaces in the lung. Small-angle scattering is detected with a Talbot-Lau interferometer, which provides the so-called x-ray dark-field signal. Using this imaging modality, we demonstrate-for the first time-the quantification of early pathogenic changes and their correlation with histological changes, as assessed by stereological morphometry. The presented radiography method is significantly more sensitive in detecting morphological changes compared with conventional x-ray imaging, and exhibits a significantly lower radiation dose than conventional x-ray CT. As a result of the improved imaging sensitivity, this new imaging modality could be used in future to reduce the number of animals required for pulmonary research studies