A simplified in vitro ligation approach to clone an E1B55k-deleted double-targeted conditionally-replicative adenovirus

<p>Abstract</p> <p>Background</p> <p>Construction of conditionally-replicative Adenovirus (CRAd) is complex and time-consuming. While homologous recombination (HR) using a two-plasmid system in bacteria is commonly used to generate CRAds, alternative methods may be required when HR fails. Previously, <it>in vitro </it>ligation has been suggested to facilitate construction of E1/E3-deleted, replication-incompetent Ad vectors. However, <it>in vitro </it>ligation has only rarely been used to generate CRAds and may be a complex procedure for molecular biologists who are not experts in the field.</p> <p>Methods and Results</p> <p>A modified <it>in vitro </it>ligation approach was developed to construct a double-targeted, E1B55k-deleted CRAd. The method allowed the incorporation of a tumor-specific promoter, e.g. the heat-shock protein 70 (hsp70) promoter, upstream of <it>E1a</it>, deletion of the <it>E1B55k </it>gene, and HR-free cloning of the recombined <it>E1Δ55k </it>gene into the Ad genome. The genetic structure of the CRAd was confirmed using restriction analysis and PCR. The replication rate of the hsp70E1Δ55k CRAd was 1.5–2% of Ad without E1Δ55k deletion.</p> <p>Conclusion</p> <p>A 3-step cloning approach can generate a double-targeted, <it>E1B55k</it>-deleted CRAd using a straight-forward, modified <it>in vitro </it>ligation procedure.</p

Moral decision-making in real life: <br /> factors affecting moral orientation and behaviour justification

The study addresses two separate but related issues in connection with people"s real-life moral decisions and judgements. First, the notion of moral orientation is examined in terms of its consistency across varying contexts, its relation to gender and to gender role. Secondly, a new aspect of moral reasoning is explored—the influence on moral decision-making of considering the consequences of an action. Fifty-eight undergraduate students were asked to discuss two personal and two impersonal real-life moral dilemmas. The results reveal a significant interaction between gender role and type of dilemma. However, moral orientation was not consistent across various dilemmas and gender was not related to any particular orientation. Also the results indicate a significant difference between the reasoning of consequences of personal-antisocial conflicts and impersonal-antisocial conflicts. These findings suggest that different moral orientations may be embedded in life experience and connect with an individual"s sense of his or her moral identity in real-life situations

A PCP Characterization of AM

We introduce a 2-round stochastic constraint-satisfaction problem, and show that its approximation version is complete for (the promise version of) the complexity class AM. This gives a `PCP characterization' of AM analogous to the PCP Theorem for NP. Similar characterizations have been given for higher levels of the Polynomial Hierarchy, and for PSPACE; however, we suggest that the result for AM might be of particular significance for attempts to derandomize this class. To test this notion, we pose some `Randomized Optimization Hypotheses' related to our stochastic CSPs that (in light of our result) would imply collapse results for AM. Unfortunately, the hypotheses appear over-strong, and we present evidence against them. In the process we show that, if some language in NP is hard-on-average against circuits of size 2^{Omega(n)}, then there exist hard-on-average optimization problems of a particularly elegant form. All our proofs use a powerful form of PCPs known as Probabilistically Checkable Proofs of Proximity, and demonstrate their versatility. We also use known results on randomness-efficient soundness- and hardness-amplification. In particular, we make essential use of the Impagliazzo-Wigderson generator; our analysis relies on a recent Chernoff-type theorem for expander walks.Comment: 18 page

Moral decision-making in real life: &lt;br /&gt; factors affecting moral orientation and behaviour justification

The study addresses two separate but related issues in connection with people"s real-life moral decisions and judgements. First, the notion of moral orientation is examined in terms of its consistency across varying contexts, its relation to gender and to gender role. Secondly, a new aspect of moral reasoning is explored—the influence on moral decision-making of considering the consequences of an action. Fifty-eight undergraduate students were asked to discuss two personal and two impersonal real-life moral dilemmas. The results reveal a significant interaction between gender role and type of dilemma. However, moral orientation was not consistent across various dilemmas and gender was not related to any particular orientation. Also the results indicate a significant difference between the reasoning of consequences of personal-antisocial conflicts and impersonal-antisocial conflicts. These findings suggest that different moral orientations may be embedded in life experience and connect with an individual"s sense of his or her moral identity in real-life situations

Analysis of adenoviral attachment to human platelets

<p>Abstract</p> <p>Background</p> <p>Systemic adenoviral (Ad) vector administration is associated with thrombocytopenia. Recently, Ad interaction with mouse platelets emerged as a key player determining liver uptake and platelet clearance. However, whether Ad can activate platelets is controversial. Thus, <it>in vitro </it>analysis of Ad attachment to platelets is of interest.</p> <p>Methods</p> <p>We developed a direct flow cytometry assay to specifically detect Ad particles adherent to human platelets. The method was pre-validated in nucleated cells. Blocking assays were employed to specifically inhibit Ad attachment to platelets. Platelet activation was analyzed using annexin v flow cytometry.</p> <p>Results</p> <p>We found <it>in vitro </it>that Ad binding to human platelets is synergistically enhanced by the combination of platelet activation by thrombin and MnCl2 supplementation. Of note, Ad binding could activate human platelets. Platelets bound Ad displaying an RGD ligand in the fiber knob more efficiently than unmodified Ad. In contrast to a previous report, CAR expression was not detected on human platelets. Integrins appear to mediate Ad binding to platelets, at least partially. Finally, αIIbβ3-deficient platelets from a patient with Glanzmann thrombasthenia could bind Ad 5-fold more efficiently than normal platelets.</p> <p>Conclusion</p> <p>The flow cytometry methodology developed herein allows the quantitative measurement of Ad attachment to platelets and may provide a useful <it>in vitro </it>approach to investigate Ad interaction with platelets.</p

Future directions for the management of pain in osteoarthritis.

Osteoarthritis (OA) is the predominant form of arthritis worldwide, resulting in a high degree of functional impairment and reduced quality of life owing to chronic pain. To date, there are no treatments that are known to modify disease progression of OA in the long term. Current treatments are largely based on the modulation of pain, including NSAIDs, opiates and, more recently, centrally acting pharmacotherapies to avert pain. This review will focus on the rationale for new avenues in pain modulation, including inhibition with anti-NGF antibodies and centrally acting analgesics. The authors also consider the potential for structure modification in cartilage/bone using growth factors and stem cell therapies. The possible mismatch between structural change and pain perception will also be discussed, introducing recent techniques that may assist in improved patient phenotyping of pain subsets in OA. Such developments could help further stratify subgroups and treatments for people with OA in future

Circumventing antivector immunity: potential use of nonhuman adenoviral vectors

Adenoviruses are efficient gene delivery vectors based on their ability to transduce a wide variety of cell types and drive high-level transient transgene expression. While there have been advances in modifying human adenoviral (HAdV) vectors to increase their safety profile, there are still pitfalls that need to be further addressed. Preexisting humoral and cellular immunity against common HAdV serotypes limits the efficacy of gene transfer and duration of transgene expression. As an alternative, nonhuman AdV (NHAdV) vectors can circumvent neutralizing antibodies against HAdVs in immunized mice and monkeys and in human sera, suggesting that NHAdV vectors could circumvent preexisting humoral immunity against HAdVs in a clinical setting. Consequently, there has been an increased interest in developing NHAdV vectors for gene delivery in humans. In this review, we outline the recent advances and limitations of HAdV vectors for gene therapy and describe examples of NHAdV vectors focusing on their immunogenicity, tropism, and potential as effective gene therapy vehicles

The problem of equilibration and the computation of correlation functions on a quantum computer

We address the question of how a quantum computer can be used to simulate experiments on quantum systems in thermal equilibrium. We present two approaches for the preparation of the equilibrium state on a quantum computer. For both approaches, we show that the output state of the algorithm, after long enough time, is the desired equilibrium. We present a numerical analysis of one of these approaches for small systems. We show how equilibrium (time)-correlation functions can be efficiently estimated on a quantum computer, given a preparation of the equilibrium state. The quantum algorithms that we present are hard to simulate on a classical computer. This indicates that they could provide an exponential speedup over what can be achieved with a classical device.Comment: 25 pages LaTex + 8 figures; various additional comments, results and correction