Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector

Adenovirus (Ad) and adeno-associated virus(AAV) have attractive and complementary properties that canbe exploited for gene transfer purposes. Ad vectors are probablythe most efficient vehicles to deliver foreign genes both invitro and in vivo. AAV exhibits the unique ability to establishlatency by efficiently integrating at a specific locus of humanchromosome 19 (AAVS1). Two viral elements are necessaryfor the integration at AAVS1: Rep68y78 and the invertedterminal repeats (AAV-ITRs). In this study, we report thedevelopment of two helper-dependent adenoviral (HD) vectors,one carrying the Rep78 gene, the other an AAV-ITRflankedtransgene. Although Rep proteins have been demonstratedto interfere with Ad replication, HD Rep78 vector wassuccessfully amplified on serial passages in 293CRE4 cellswith a yield of 50–100 transducing units per cell. DNAintegration at the AAVS1 site also was demonstrated inhepatoma cells coinfected with the HD-expressing Rep78 andwith the second HD vector carrying a transgene flanked byAAV-ITRs. The high transduction efficiency, large cloningcapacity, and high titer of the HD, combined with the sitespecificintegration machinery provided by AAV-derived components,make the AdyAAV hybrid viruses a promising vehiclefor gene therap

Prediction of response to vemurafenib in BRAF V600E mutant cancers based on a network approach

Lung adenocarcinoma is the tumor with the highest number of switch genes (298) compared to its normal tissue, followed by thyroid (227) and colorectal (183) cancers. Switch genes codifying for kinases were 14,7 and 3 respectively.We looked for three homology sequences identified across vemurafenib targets and we found that thyroid cancer and lung adenocarcinoma have a similar number of putative targetable switch genes kinase (5-6); on the contrary, colorectal cancer has just one,with minor homology sequence

SVEP1 as a Genetic Modifier of TEK-Related Primary Congenital Glaucoma.

Purpose: Affecting children by age 3, primary congenital glaucoma (PCG) can cause debilitating vision loss by the developmental impairment of aqueous drainage resulting in high intraocular pressure (IOP), globe enlargement, and optic neuropathy. TEK haploinsufficiency accounts for 5% of PCG in diverse populations, with low penetrance explained by variable dysgenesis of Schlemm's canal (SC) in mice. We report eight families with TEK-related PCG, and provide evidence for SVEP1 as a disease modifier in family 8 with a higher penetrance and severity. Methods: Exome sequencing identified coding/splice site variants with an allele frequency less than 0.0001 (gnomAD). TEK variant effects were assayed in construct-transfected HEK293 cells via detection of autophosphorylated (active) TEK protein. An enucleated eye from an affected member of family 8 was examined via histology. SVEP1 expression in developing outflow tissues was detected by immunofluorescent staining of 7-day mouse anterior segments. SVEP1 stimulation of TEK expression in human umbilical vascular endothelial cells (HUVECs) was measured by TaqMan quantitative PCR. Results: Heterozygous TEK loss-of-function alleles were identified in eight PCG families, with parent-child disease transmission observed in two pedigrees. Family 8 exhibited greater disease penetrance and severity, histology revealed absence of SC in one eye, and SVEP1:p.R997C was identified in four of the five affected individuals. During SC development, SVEP1 is secreted by surrounding tissues. SVEP1:p.R997C abrogates stimulation of TEK expression by HUVECs. Conclusions: We provide further evidence for PCG caused by TEK haploinsufficiency, affirm autosomal dominant inheritance in two pedigrees, and propose SVEP1 as a modifier of TEK expression during SC development, affecting disease penetrance and severity

Experience with an online prospective database on adolescent idiopathic scoliosis: development and implementation

Considerable variability exists in the surgical treatment and outcomes of adolescent idiopathic scoliosis (AIS). This is due to the lack of evidence-based treatment guidelines and outcome measures. Although clinical trials have been extolled as the highest form of evidence for evaluating treatment efficacy, the disadvantage of cost, time, lack of feasibility, and ethical considerations indicate a need for a new paradigm for evidence based research in this spinal deformity. High quality clinical databases offer an alternative approach for evidence-based research in medicine. So, we developed and established Scolisoft, an international, multidimensional and relational database designed to be a repository of surgical cases for AIS, and an active vehicle for standardized surgical information in a format that would permit qualitative and quantitative research and analysis. Here, we describe and discuss the utility of Scolisoft as a new paradigm for evidence-based research on AIS. Scolisoft was developed using dot.net platform and SQL server from Microsoft. All data is deidentified to protect patient privacy. Scolisoft can be accessed at www.scolisoft.org. Collection of high quality data on surgical cases of AIS is a priority and processes continue to improve the database quality. The database currently has 67 registered users from 21 countries. To date, Scolisoft has 200 detailed surgical cases with pre, post, and follow up data. Scolisoft provides a structured process and practical information for surgeons to benchmark their treatment methods against other like treatments. Scolisoft is multifaceted and its use extends to education of health care providers in training, patients, ability to mine important data to stimulate research and quality improvement initiatives of healthcare organizations

Association of the Genomic Profile of Medullary Thyroid Carcinoma with Tumor Characteristics and Clinical Outcomes in an International Multicenter Study

Purpose: The prognostic importance of RET and RAS mutations and their relationship to clinicopathologic parameters and outcomes in medullary thyroid carcinoma (MTC) need to be clarified. Experimental Design: A multicenter retrospective cohort study was performed utilizing data from 290 patients with MTC. The molecular profile was determined and associations were examined with clinicopathologic data and outcomes. Results: RET germ line mutations were detected in 40 patients (16.3%). Somatic RET and RAS mutations occurred in 135 (46.9%) and 57 (19.8%) patients, respectively. RETM918T was the most common somatic RET mutation (n = 75). RET somatic mutations were associated with male sex, larger tumor size, advanced American Joint Committee Cancer (AJCC) stage, vascular invasion, and high International Medullary Thyroid Carcinoma Grading System (IMTCGS) grade. When compared with other RET somatic mutations, RETM918T was associated with younger age, AJCC (eighth edition) IV, vascular invasion, extrathyroidal extension, and positive margins. RET somatic or germ line mutations were significantly associated with reduced distant metastasis-free survival on univariate analysis, but there were no significant independent associations on multivariable analysis, after adjusting for tumor grade and stage. There were no significant differences in outcomes between RET somatic and RET germ line mutations, or between RETM918T and other RET mutations. Other recurrent molecular alterations included TP53 (4.2%), ARID2 (2.9%), SETD2 (2.9%), KMT2A (2.9%), and KMT2C (2.9%). Among them, TP53 mutations were associated with decreased overall survival (OS) and disease-specific survival (DSS), independently of tumor grade and AJCC stage. Conclusions: RET somatic mutations were associated with high-grade, aggressive primary tumor characteristics, and decreased distant metastatic-free survival but this relationship was not significant after accounting for tumor grade and disease stage. RETM918T was associated with aggressive primary tumors but was not independently associated with clinical outcomes. TP53 mutation may represent an adverse molecular event associated with decreased OS and DSS in MTC, but its prognostic value needs to be confirmed in future studies

DNA Structure Modulates the Oligomerization Properties of the AAV Initiator Protein Rep68

Rep68 is a multifunctional protein of the adeno-associated virus (AAV), a parvovirus that is mostly known for its promise as a gene therapy vector. In addition to its role as initiator in viral DNA replication, Rep68 is essential for site-specific integration of the AAV genome into human chromosome 19. Rep68 is a member of the superfamily 3 (SF3) helicases, along with the well-studied initiator proteins simian virus 40 large T antigen (SV40-LTag) and bovine papillomavirus (BPV) E1. Structurally, SF3 helicases share two domains, a DNA origin interaction domain (OID) and an AAA+ motor domain. The AAA+ motor domain is also a structural feature of cellular initiators and it functions as a platform for initiator oligomerization. Here, we studied Rep68 oligomerization in vitro in the presence of different DNA substrates using a variety of biophysical techniques and cryo-EM. We found that a dsDNA region of the AAV origin promotes the formation of a complex containing five Rep68 subunits. Interestingly, non-specific ssDNA promotes the formation of a double-ring Rep68, a known structure formed by the LTag and E1 initiator proteins. The Rep68 ring symmetry is 8-fold, thus differing from the hexameric rings formed by the other SF3 helicases. However, similiar to LTag and E1, Rep68 rings are oriented head-to-head, suggesting that DNA unwinding by the complex proceeds bidirectionally. This novel Rep68 quaternary structure requires both the DNA binding and AAA+ domains, indicating cooperativity between these regions during oligomerization in vitro. Our study clearly demonstrates that Rep68 can oligomerize through two distinct oligomerization pathways, which depend on both the DNA structure and cooperativity of Rep68 domains. These findings provide insight into the dynamics and oligomeric adaptability of Rep68 and serve as a step towards understanding the role of this multifunctional protein during AAV DNA replication and site-specific integration

Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus

Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at oriT was similar to TrwC. Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily.This work was supported by the Medical Research Council (MRC) grant MR/N022890/1 to EH and grant 1001764 to RML; National Institutes of Health (NIH) grant RO1-GM09285 to CRE; Spanish Ministry of Economy and competitiveness (MINECO) grant BIO2013-46414-P to ML and AFM is supported by a Doc.Mobility fellowship from the Swiss National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Fiscal Shocks and Policy Regimes in Some OECD Countries

The objective of this paper is twofold. First, for some OECD countries we econometrically select the fiscal policy regimes, i.e. a "set of rules'' for the conduct of fiscal policy. Second, we identify different types of fiscal policy shocks related to expenditure and taxation, and simulate their effects on GDP and the price level. Estimation and simulation are obtained within the structural VAR framework. We found that France and Italy are characterized by fiscal regimes that target either the government expenditure on salaries and transfers, or the residual spending. For Germany and the US instead the selected fiscal regimes exclude any (contemporaneous) influence of innovations in government wages and transfers on both taxation and residual spending, i.e. decisions on government wages and transfers do not precede other fiscal-policy decisions. Except for France, government expenditure on salaries and transfers has the strongest procyclical effect on output, but lower than expected: a one percent decrease in public spending on wages and transfers (as a ratio of GDP) lowers output at most by 0.1 percent. Innovations on taxes that decrease fiscal pressure have the standard positive effect on output, but not in the long run