AAV Serotype Influences Gene Transfer in Corneal Stroma \u3cem\u3ein vivo\u3c/em\u3e

This study evaluated the cellular tropism and relative transduction efficiency of three AAV serotypes, AAV6, AAV8 and AAV9, for corneal gene delivery using mouse cornea in vivo and donor human cornea ex vivo. The AAV6, AAV8 and AAV9 serotypes having AAV2 plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Viral vectors (109 vg/μl) were topically applied onto mouse cornea in vivo and human cornea ex vivo after removing the epithelium. Human corneas were processed for transgene delivery at day 5 after viral vector application. Mouse corneas were harvested at 4, 14 and 30 days after vector application for AP staining. Transduction efficiency was calculated by quantifying pixels of AP-stained area using Image J software and also confirmed by functional AP enzyme activity in the corneal lysates. Cellular toxicity of the three AAV serotypes was tested with TUNEL assay. Inflammatory response was detected by immunostaining for CD11b and F4/80. All three AAV serotypes successfully transduced mouse and human corneas. The order of transduction efficiency was AAV9\u3eAAV8\u3eAAV6. The transduction efficiency of AAV9 was 1.1–1.4 fold higher (p\u3e0.05) as compared to AAV8 and 3.5–5.5 fold higher (p\u3c0.01) as compared to AAV6. The level of transgene expression for all the three serotypes was greater at 14 days compared to 4 days and this high level of transgene expression was maintained up to the tested time point of 30 days. Corneas exposed to any of the three AAV serotypes did not show significant TUNEL positive cells or any inflammatory response as tested by CD11b or F4/80 staining suggesting that tested AAV serotypes do not induce cell death or inflammation and are safe for corneal gene therapy

Role of Transforming Growth Factor Beta in Corneal Function, Biology and Pathology

Transforming growth factor-beta (TGFβ) is a pleiotropic multifunctional cytokine that regulates several essential cellular processes in many parts of the body including the cornea. Three isoforms of TGFβ are known in mammals and the human cornea expresses all of them. TGFβ1 has been shown to play a central role in scar formation in adult corneas whereas TGFβ2 and TGFβ3 have been implicated to play a critical role in corneal development and scarless wound healing during embryogenesis. The biological effects of TGFβ in the cornea have been shown to follow SMAD dependent as well as SMAD-independent signaling pathways depending upon cellular responses and microenvironment. Corneal TGFβ expression is necessary for maintaining corneal integrity and corneal wound healing. On the other hand, TGFβ is perhaps the most important cytokine in the pathogenesis of fibrotic disease in the cornea. Although the transformation of keratocytes to myofibroblasts induced by TGFβ is largely believed to cause corneal fibrosis or scarring, the precise molecular mechanism(s) involved in this process is still unknown. Currently no drugs are available to treat corneal scarring effectively without causing significant side effects. Many approaches to treat TGFβ-mediated corneal scarring are under investigation. These include blocking of TGFβ, TGFβ receptor, TGFβ function and/or TGFβ maturation. Other strategies such as modulating keratocyte proliferation, apoptosis, transcription and DNA condensation are also being investigated. The potential of gene therapy to neutralize the pathologic effects of TGFβ has also been demonstrated recently

Targeted Decorin Gene Therapy Delivered with Adeno-Associated Virus Effectively Retards Corneal Neovascularization \u3cem\u3ein vivo\u3c/em\u3e

Decorin, small leucine-rich proteoglycan, has been shown to modulate angiogenesis in nonocular tissues. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the rabbit stroma with adeno-associated virus serotype 5 (AAV5) impedes corneal neovascularization (CNV) in vivo without significant side effects. An established rabbit CNV model was used. Targeted decorin gene therapy in the rabbit stroma was delivered with a single topical AAV5 titer (100 μl; 5x10^12 vg/ml) application onto the stroma for two minutes after removing corneal epithelium. The levels of CNV were examined with stereomicroscopy, H&E staining, lectin, collagen type IV, CD31 immunocytochemistry and CD31 immunoblotting. Real-time PCR quantified mRNA expression of pro- and anti-angiogenic genes. Corneal health in live animals was monitored with clinical, slit-lamp and optical coherence tomography biomicroscopic examinations. Selective decorin delivery into stroma showed significant 52% (p\u3c0.05), 66% (p\u3c0.001), and 63% (p\u3c0.01) reduction at early (day 5), mid (day 10), and late (day 14) stages of CNV in decorin-delivered rabbit corneas compared to control (no decorin delivered) corneas in morphometric analysis. The H&E staining, lectin, collagen type IV, CD31 immunostaining (57–65, p\u3c0.5), and CD31 immunoblotting (62–67%, p\u3c0.05) supported morphometric findings. Quantitative PCR studies demonstrated decorin gene therapy down-regulated expression of VEGF, MCP1 and angiopoietin (pro-angiogenic) and up-regulated PEDF (anti-angiogenic) genes. The clinical, biomicroscopy and transmission electron microscopy studies revealed that AAV5– mediated decorin gene therapy is safe for the cornea. Tissue-targeted AAV5-mediated decorin gene therapy decreases CNV with no major side effects, and could potentially be used for treating patients

Supersymmetric particle mass measurement with the boost-corrected contransverse mass

A modification to the contransverse mass (MCT) technique for measuring the masses of pair-produced semi-invisibly decaying heavy particles is proposed in which MCT is corrected for non-zero boosts of the centre-of-momentum (CoM) frame of the heavy states in the laboratory transverse plane. Lack of knowledge of the mass of the CoM frame prevents exact correction for this boost, however it is shown that a conservative correction can nevertheless be derived which always generates an MCT value which is less than or equal to the true value of MCT in the CoM frame. The new technique is demonstrated with case studies of mass measurement with fully leptonic ttbar events and with SUSY events possessing a similar final state.Comment: 33 pages, 33 .eps figures, JHEP3 styl

Minimal unsatisfiable formulas with bounded clause-variable difference are fixed-parameter tractable

Recognition of minimal unsatisfiable CNF formulas (unsatisfiable CNF formulas which become satisfiable if any clause is removed) is a classical DP-complete problem. It was shown recently that minimal unsatisfiable formulas with n variables and n+k clauses can be recognized in time . We improve this result and present an algorithm with time complexity ; hence the problem turns out to be fixed-parameter tractable (FTP) in the sense of Downey and Fellows (Parameterized Complexity, 1999). Our algorithm gives rise to a fixed-parameter tractable parameterization of the satisfiability problem: If for a given set of clauses F, the number of clauses in each of its subsets exceeds the number of variables occurring in the subset at most by k, then we can decide in time whether F is satisfiable; k is called the maximum deficiency of F and can be efficiently computed by means of graph matching algorithms. Known parameters for fixed-parameter tractable satisfiability decision are tree-width or related to tree-width. Tree-width and maximum deficiency are incomparable in the sense that we can find formulas with constant maximum deficiency and arbitrarily high tree-width, and formulas where the converse prevails

Using Subsystem MT2 for Complete Mass Determinations in Decay Chains with Missing Energy at Hadron Colliders

We propose to use the MT2 concept to measure the masses of all particles in SUSY-like events with two unobservable, identical particles. To this end we generalize the usual notion of MT2 and define a new MT2(n,p,c) variable, which can be applied to various subsystem topologies, as well as the full event topology. We derive analytic formulas for its endpoint MT2{max}(n,p,c) as a function of the unknown test mass Mc of the final particle in the subchain and the transverse momentum pT due to radiation from the initial state. We show that the endpoint functions MT2{max}(n,p,c)(Mc,pT) may exhibit three different types of kinks and discuss the origin of each type. We prove that the subsystem MT2(n,p,c) variables by themselves already yield a sufficient number of measurements for a complete determination of the mass spectrum (including the overall mass scale). As an illustration, we consider the simple case of a decay chain with up to three heavy particles, X2 -> X1 -> X0, which is rather problematic for all other mass measurement methods. We propose three different MT2-based methods, each of which allows a complete determination of the masses of particles X0, X1 and X2. The first method only uses MT2(n,p,c) endpoint measurements at a single fixed value of the test mass Mc. In the second method the unknown mass spectrum is fitted to one or more endpoint functions MT2{max}(n,p,c)(Mc,pT) exhibiting a kink. The third method is hybrid, combining MT2 endpoints with measurements of kinematic edges in invariant mass distributions. As a practical application of our methods, we show that the dilepton W+W- and tt-bar samples at the Tevatron can be used for an independent determination of the masses of the top quark, the W boson and the neutrino, without any prior assumptions.Comment: 47 pages, 9 figures. revised version, published in JHEP. Major addition: a new appendix with the complete set of formulas for the MT2 endpoints as functions of the upstream transverse momentum pT and test mass M

Collider signatures of goldstini in gauge mediation

We investigate the collider signatures of the multiple goldstini scenario in the framework of gauge mediation. This class of models is characterized by a visible sector (e.g. the MSSM or any extension) coupled by gauge interactions to more than one SUSY breaking sector. The spectrum consists of a light gravitino LSP, behaving as a goldstino, and a number of neutral fermions (the pseudo-goldstini) with a mass between that of the LSP and that of the lightest particle of the observable sector (LOSP). We consider the two situations where the LOSP is either a gaugino-like neutralino or a stau and we assume only one pseudo-goldstino of a mass of O(100) GeV. The coupling of the LOSP to the pseudo-goldstino can be enhanced with respect to those of the gravitino giving rise to characteristic signatures. We show that the decay modes of the LOSP into a SM particle and a pseudo-goldstino can be significant. For both LOSP scenarios we analyze (pseudo)-goldstini production at colliders. Compared to standard gauge mediation the final state spectrum is softer and more structured.Comment: v2: analysis of the stau LOSP scenario added, sections rearranged, and Introduction and Conclusions rewritten to include the added scenario. Version to appear in JHE

Interpreting a 1 fb^-1 ATLAS Search in the Minimal Anomaly Mediated Supersymmetry Breaking Model

Recent LHC data significantly extend the exclusion limits for supersymmetric particles, particularly in the jets plus missing transverse momentum channels. The most recent such data have so far been interpreted by the experiment in only two different supersymmetry breaking models: the constrained minimal supersymmetric standard model (CMSSM) and a simplified model with only squarks and gluinos and massless neutralinos. We compare kinematical distributions of supersymmetric signal events predicted by the CMSSM and anomaly mediated supersymmetry breaking (mAMSB) before calculating exclusion limits in mAMSB. We obtain a lower limit of 900 GeV on squark and gluino masses at the 95% confidence level for the equal mass limit, tan(beta)=10 and mu>0.Comment: 18 pages, 11 figure

Conceptualizing the adventure-sports coach

As a comparatively recent development, the adventure-sports coach struggles for a clear and distinct identity. The generic term ‘instructor’ no longer characterizes the role and function of this subgroup of outdoor professionals. Indeed, although the fields of adventure/outdoor education and leadership are comparatively well researched, the arrival of this ‘new kid on the block’ appears to challenge both the adventure-sports old guard and traditional views of sports coaching. In an attempt to offer clarity and stimulate debate, this paper attempts to conceptualize the adventure-sports coach in the context of the existing roles in the field and current motivations for activity in the outdoors. We identify issues that are specific to the adventure-sports coach while also recognizing those skills and competencies shared with other professionals, both in the adventure sports profession and traditional sports coaching fields. Based on this review, we offer a conceptual model which may be used to focus debate, stimulate research and, at a possible later stage, to underpin accreditation, training and professional development

Reducing combinatorial uncertainties: A new technique based on MT2 variables

We propose a new method to resolve combinatorial ambiguities in hadron collider events involving two invisible particles in the final state. This method is based on the kinematic variable MT2 and on the MT2-assisted-on-shell reconstruction of invisible momenta, that are reformulated as `test' variables Ti of the correct combination against the incorrect ones. We show how the efficiency of the single Ti in providing the correct answer can be systematically improved by combining the different Ti and/or by introducing cuts on suitable, combination-insensitive kinematic variables. We illustrate our whole approach in the specific example of top anti-top production, followed by a leptonic decay of the W on both sides. However, by construction, our method is also directly applicable to many topologies of interest for new physics, in particular events producing a pair of undetected particles, that are potential dark-matter candidates. We finally emphasize that our method is apt to several generalizations, that we outline in the last sections of the paper.Comment: 1+23 pages, 8 figures. Main changes in v3: (1) discussion at the end of sec. 2 improved; (2) added sec. 4.2 about the method's dependence on mass information. Matches journal versio