Efficient Gene Targeting Mediated by Adeno-Associated Virus and DNA Double-Strand Breaks

Gene targeting is the in situ manipulation of the sequence of an endogenous gene by the introduction of homologous exogenous DNA. Presently, the rate of gene targeting is too low for it to be broadly used in mammalian somatic cell genetics or to cure genetic diseases. Recently, it has been demonstrated that infection with recombinant adeno-associated virus (rAAV) vectors can mediate gene targeting in somatic cells, but the mechanism is unclear. This paper explores the balance between random integration and gene targeting with rAAV. Both random integration and spontaneous gene targeting are dependent on the multiplicity of infection (MOI) of rAAV. It has previously been shown that the introduction of a DNA double-stranded break (DSB) in a target gene can stimulate gene targeting by several-thousand-fold in somatic cells. Creation of a DSB stimulates the frequency of rAAV-mediated gene targeting by over 100-fold, suggesting that the mechanism of rAAV-mediated gene targeting involves, at least in part, the repair of DSBs by homologous recombination. Absolute gene targeting frequencies reach 0.8% with a dual vector system in which one rAAV vector provides a gene targeting substrate and a second vector expresses the nuclease that creates a DSB in the target gene. The frequencies of gene targeting that we achieved with relatively low MOIs suggest that combining rAAV vectors with DSBs is a promising strategy to broaden the application of gene targeting

Approximating the Nonlinear Newsvendor and Single-Item Stochastic Lot-Sizing Problems When Data Is Given by an Oracle

The single-item stochastic lot-sizing problem is to find an inventory replenishment policy in the presence of discrete stochastic demands under periodic review and finite time horizon. A closely related problem is the single-period newsvendor model. It is well known that the newsvendor problem admits a closed formula for the optimal order quantity whenever the revenue and salvage values are linear increasing functions and the procurement (ordering) cost is fixed plus linear. The optimal policy for the single-item lot-sizing model is also well known under similar assumptions. In this paper we show that the classical (single-period) newsvendor model with fixed plus linear ordering cost cannot be approximated to any degree of accuracy when either the demand distribution or the cost functions are given by an oracle. We provide a fully polynomial time approximation scheme for the nonlinear single-item stochastic lot-sizing problem, when demand distribution is given by an oracle, procurement costs are provided as nondecreasing oracles, holding/backlogging/disposal costs are linear, and lead time is positive. Similar results exist for the nonlinear newsvendor problem. These approximation schemes are designed by extending the technique of K-approximation sets and functions.National Science Foundation (U.S.) (Contract CMMI-0758069)United States. Office of Naval Research (Grant N000141110056

Receptor-mediated delivery of engineered nucleases for genome modification

Engineered nucleases, which incise the genome at predetermined sites, have a number of laboratory and clinical applications. There is, however, a need for better methods for controlled intracellular delivery of nucleases. Here, we demonstrate a method for ligand-mediated delivery of zinc finger nucleases (ZFN) proteins using transferrin receptor-mediated endocytosis. Uptake is rapid and efficient in established mammalian cell lines and in primary cells, including mouse and human hematopoietic stem-progenitor cell populations. In contrast to cDNA expression, ZFN protein levels decline rapidly following internalization, affording better temporal control of nuclease activity. We show that transferrin-mediated ZFN uptake leads to site-specific in situ cleavage of the target locus. Additionally, despite the much shorter duration of ZFN activity, the efficiency of gene correction approaches that seen with cDNA-mediated expression. The approach is flexible and general, with the potential for extension to other targeting ligands and nuclease architectures

Cluster Performance reconsidered: Structure, Linkages and Paths in the German Biotechnology Industry, 1996-2003

This paper addresses the evolution of biotechnology clusters in Germany between 1996 and 2003, paying particular attention to their respective composition in terms of venture capital, basic science institutions and biotechnology firms. Drawing upon the significance of co-location of "money and ideas", the literature stressing the importance of a cluster's openness and external linkages, and the path dependency debate, the paper aims to analyse how certain cluster characteristics correspond with its overall performance. After identifying different cluster types, we investigate their internal and external interconnectivity in comparative manner and draw on changes in cluster composition. Our results indicate that the structure, i.e. to which group the cluster belongs, and the openness towards external knowledge flows deliver merely unsystematic indications with regard to a cluster's overall success. Its ability to change composition towards a more balanced ratio of science and capital over time, on the other hand, turns out as a key explanatory factor. Hence, the dynamic perspective proves effective illuminating cluster growth and performance, where our explorative findings provide a promising avenue for further evolutionary research

Theoretical analysis of the electronic structure of the stable and metastable c(2x2) phases of Na on Al(001): Comparison with angle-resolved ultra-violet photoemission spectra

Using Kohn-Sham wave functions and their energy levels obtained by density-functional-theory total-energy calculations, the electronic structure of the two c(2x2) phases of Na on Al(001) are analysed; namely, the metastable hollow-site structure formed when adsorption takes place at low temperature, and the stable substitutional structure appearing when the substrate is heated thereafter above ca. 180K or when adsorption takes place at room temperature from the beginning. The experimentally obtained two-dimensional band structures of the surface states or resonances are well reproduced by the calculations. With the help of charge density maps it is found that in both phases, two pronounced bands appear as the result of a characteristic coupling between the valence-state band of a free c(2x2)-Na monolayer and the surface-state/resonance band of the Al surfaces; that is, the clean (001) surface for the metastable phase and the unstable, reconstructed "vacancy" structure for the stable phase. The higher-lying band, being Na-derived, remains metallic for the unstable phase, whereas it lies completely above the Fermi level for the stable phase, leading to the formation of a surface-state/resonance band-structure resembling the bulk band-structure of an ionic crystal.Comment: 11 pages, 11 postscript figures, published in Phys. Rev. B 57, 15251 (1998). Other related publications can be found at http://www.rz-berlin.mpg.de/th/paper.htm

A hierarchical model for the cash transfer system design problem

This paper presents a hierarchical model that incorporates strategic, tactical, and operational decisions of cash transfer management system of a bank. The aim of the model is to decide on the location of cash management centers, the number and routes of vehicles, and the cash inventory management policies to minimize the cost of owning and operating a cash transfer system while maintaining a pre-defined service level. Owing to the difficulty of finding optimal decisions in such integrated models, an iterative solution approach is proposed in which strategic, tactical, and operational problems are solved separately via a feedback mechanism. Numerical results show that such an approach is quite effective in reaching at greatly improved solutions with just a few iterations, making it a very promising approach for similar models

Sourcing Flexibility, Spot Trading, and Procurement Contract Structure

We analyze the structure and pricing of option contracts for an industrial good in the presence of spot trading. We combine the analysis of spot trading and buyers' disparate private valuations for different suppliers' products, and we jointly endogenize the determination of three major dimensions in contract design: (i) sales contracts versus options contracts, (ii) flat-price versus volume-dependent contracts, and (iii) volume discounts versus volume premia. We build a model in which a supplier of an industrial good transacts with a manufacturer who uses the supplier's product to produce an end good with an uncertain demand. We show that, consistent with industry observations, volume-dependent optimal sales contracts always demonstrate volume discounts (i.e., involve concave pricing). However, options are more complex agreements, and optimal option contracts can involve both volume discounts and volume premia. Three major contract structures commonly emerge in optimality. First, if the seller has a high discount rate relative to the buyer and the seller's production costs or the production capacity is low, the optimal contracts tend to be flat-price sales contracts. Second, when the seller has a relatively high discount rate compared to the buyer but production costs or production capacity are high, the optimal contracts are sales contracts with volume discounts. Third, if the buyer's discount rate is high relative to the seller's, then the optimal contracts tend to be volume-dependent options contracts and can involve both volume discounts and volume premia. However, when the seller's production capacity is sufficiently low, it is possible to observe flat-price option contracts. Furthermore, we provide links between production and spot market characteristics, contract design, and efficiency.National Science Foundation (U.S.) (contract CMMI-0758069)National Science Foundation (U.S.) (contract DMI-0245352

Stem Cell-Specific Mechanisms Ensure Genomic Fidelity within HSCs and upon Aging of HSCs

SummaryWhether aged hematopoietic stem and progenitor cells (HSPCs) have impaired DNA damage repair is controversial. Using a combination of DNA mutation indicator assays, we observe a 2- to 3-fold increase in the number of DNA mutations in the hematopoietic system upon aging. Young and aged hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) do not show an increase in mutation upon irradiation-induced DNA damage repair, and young and aged HSPCs respond very similarly to DNA damage with respect to cell-cycle checkpoint activation and apoptosis. Both young and aged HSPCs show impaired activation of the DNA-damage-induced G1-S checkpoint. Induction of chronic DNA double-strand breaks by zinc-finger nucleases suggests that HSPCs undergo apoptosis rather than faulty repair. These data reveal a protective mechanism in both the young and aged hematopoietic system against accumulation of mutations in response to DNA damage