The Big Dam on the Little San Diego

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Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms

Site Selective Antibody-Oligonucleotide Conjugation via Microbial Transglutaminase.

Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to siRNAs and ASOs has shown clinical efficacy in targeting diseases driven by liver hepatocytes. However, targeting non-hepatic diseases with oligonucleotide therapeutics has remained problematic for several reasons, including targeting specific cell types and endosomal escape. Monoclonal antibody (mAb) targeting of siRNAs and ASOs has the potential to deliver these drugs to a variety of specific cell and tissue types. However, most conjugation strategies rely on random chemical conjugation through lysine or cysteine residues resulting in conjugate heterogeneity and a distribution of Drug:Antibody Ratios (DAR). To produce homogeneous DAR-2 conjugates with two siRNAs per mAb, we developed a novel two-step conjugation procedure involving microbial transglutaminase (MTGase) tagging of the antibody C-terminus with an azide-functionalized linker peptide that can be subsequently conjugated to dibenzylcyclooctyne (DBCO) bearing oligonucleotides through azide-alkyne cycloaddition. Antibody-siRNA (and ASO) conjugates (ARCs) produced using this strategy are soluble, chemically defined targeted oligonucleotide therapeutics that have the potential to greatly increase the number of targetable cell types

Black holes admitting a Freudenthal dual

The quantised charges x of four dimensional stringy black holes may be assigned to elements of an integral Freudenthal triple system whose automorphism group is the corresponding U-duality and whose U-invariant quartic norm Delta(x) determines the lowest order entropy. Here we introduce a Freudenthal duality x -> \tilde{x}, for which \tilde{\tilde{x}}=-x. Although distinct from U-duality it nevertheless leaves Delta(x) invariant. However, the requirement that \tilde{x} be integer restricts us to the subset of black holes for which Delta(x) is necessarily a perfect square. The issue of higher-order corrections remains open as some, but not all, of the discrete U-duality invariants are Freudenthal invariant. Similarly, the quantised charges A of five dimensional black holes and strings may be assigned to elements of an integral Jordan algebra, whose cubic norm N(A) determines the lowest order entropy. We introduce an analogous Jordan dual A*, with N(A) necessarily a perfect cube, for which A**=A and which leaves N(A) invariant. The two dualities are related by a 4D/5D lift.Comment: 32 pages revtex, 10 tables; minor corrections, references adde

On spherical twisted conjugacy classes

Let G be a simple algebraic group over an algebraically closed field of good odd characteristic, and let theta be an automorphism of G arising from an involution of its Dynkin diagram. We show that the spherical theta-twisted conjugacy classes are precisely those intersecting only Bruhat cells corresponding to twisted involutions in the Weyl group. We show how the analogue of this statement fails in the triality case. We generalize to good odd characteristic J-H. Lu's dimension formula for spherical twisted conjugacy classes.Comment: proof of Lemma 6.4 polished. The journal version is available at http://www.springerlink.com/content/k573l88256753640

Jamaican bioethanol: an environmental and economic life cycle assessment

E10 is a blend of 10% bioethanol and 90% gasoline that can be used in the engines of most cars without causing damage. Currently for the E10 blend, Jamaica imports gasoline from Trinidad & Tobago and bioethanol from Brazil because the bioethanol production in Jamaica is at an early stage. However, the country has great potential for bioethanol production. In order to assess the environmental and economic feasibility of bioethanol in Jamaica, this paper presents an economic and environmental life cycle assessment for a case study in Jamaica in two different scenarios. The Baseline Scenario represents the use of E10 in the current conditions in which bioethanol comes from Brazil and gasoline from Trinidad & Tobago. Scenario I represents the use of E10 with bioethanol from Jamaica and gasoline from Trinidad & Tobago. The comparative environmental life cycle assessment revealed that the Baseline Scenario had better results than Scenario I in ten environmental categories. The economic assessment results in Scenario I were 7% higher than in the Baseline Scenario. Hence, the current context (Baseline Scenario) was identified as the scenario with the best economic performance. Therefore, the current situation in Jamaica (Baseline Scenario) scored better results than Scenario I from an environmental and an economical point of views. It is recommended to increase the bagasse cogeneration of Scenario I to lower the environmental impacts. To improve their productivity, it is necessary to improve the Jamaican sugar infrastructure by combining molasses and cane juice to produce bioethanol.Publicad

Sustainability assessment of electricity cogeneration from sugarcane bagasse in Jamaica

Cogeneration from sugarcane bagasse in Jamaica represents a significant opportunity to reduce CO2 emissions and its dependence on a fossil fuel-based energy matrix. Generation of electricity through cogeneration is a huge opportunity in countries where the sugarcane industry is in decline. This article draws on the findings of a case-study on electricity generation through cogeneration in Jamaica to provide some key messages that may be useful for policy-makers and the private sector to make electricity generation by cogeneration a more competitive option the for investors. To this end, this article analyses two scenarios: the first is a Baseline Scenario that assesses the impact of cogeneration technology already installed in a Jamaican sugarcane company where the cogeneration stage produces 2,2 MW; the second one considers that the cogeneration technology is changed to a new biomass based power plant upgrading the cogeneration stage in order to produce 5 MW of power from bagasse. The assessment was carried out by using a complete Life Cycle Assessment, Life Cycle Costing and Social Life Cycle Assessment. The results revealed that generation of electricity from cogeneration derived from bagasse is a suitable alternative adding economic, environmental and social value.Publicad

An elementary stringy estimate of transport coefficients of large temperature QCD

Modeling QCD at large temperature with a simple holographic five dimensional theory encoding minimal breaking of conformality, allows for the calculation of all the transport coefficients, up to second order, in terms of a single parameter. In particular, the shear and bulk relaxation times are provided. The result follows by deforming the AdS background with a scalar dual to a marginally relevant operator, at leading order in the deformation parameter.Comment: 11 pages; v2: comments and references adde