Methods for Scarless, Selection-Free Generation of Human Cells and Allele-Specific Functional Analysis of Disease-Associated SNPs and Variants of Uncertain Significance.

With the continued emergence of risk loci from Genome-Wide Association studies and variants of uncertain significance identified from patient sequencing, better methods are required to translate these human genetic findings into improvements in public health. Here we combine CRISPR/Cas9 gene editing with an innovative high-throughput genotyping pipeline utilizing KASP (Kompetitive Allele-Specific PCR) genotyping technology to create scarless isogenic cell models of cancer variants in ~1 month. We successfully modeled two novel variants previously identified by our lab in the PALB2 gene in HEK239 cells, resulting in isogenic cells representing all three genotypes for both variants. We also modeled a known functional risk SNP of colorectal cancer, rs6983267, in HCT-116 cells. Cells with extremely low levels of gene editing could still be identified and isolated using this approach. We also introduce a novel molecular assay, ChIPnQASO (Chromatin Immunoprecipitation and Quantitative Allele-Specific Occupation), which uses the same technology to reveal allele-specific function of these variants at the DNA-protein interaction level. We demonstrated preferential binding of the transcription factor TCF7L2 to the rs6983267 risk allele over the non-risk. Our pipeline provides a platform for functional variant discovery and validation that is accessible and broadly applicable for the progression of efforts towards precision medicine

Studies toward a prebiotic protometabolism

Understanding the origin of life is one of the foremost challenges for science. Despite the immense diversity displayed by biology on the macroscopic scale, cellular biochemistry is at its most fundamental level remarkably uniform. Recognising the minimal elements of biochemistry required for a living system represents one task within origins of life research, whereas the other is understanding the organic chemistry which could have led to their spontaneous organisation on the abiotic Earth. Significant attention has been paid to the prebiotic generation and assembly of the components of nucleic acids resulting in the demonstration of important prebiotic syntheses and in the uncovering of various key problems which have seeded much informative discussion within the prebiotic community. However, experimental investigations into the assembly of key sets of ubiquitous metabolites have been comparatively lacking and will be necessary to assess the relevance of central metabolic pathways to the earliest stages in the development of life. Presented herein are investigations into the prebiotic chemistry of a selection of small molecules central to one of the most fundamental and highly conserved metabolic pathways found in biology; triose glycolysis. By utilising a prebiotically relevant method of specifically generating aldehyde-2-phosphates from simple sugar precursors, it has been shown that several intermediates common to glycolysis can be generated in high yield under mild, aqueous conditions in a simple step-wise sequence which culminates in the generation (for the first time under prebiotically relevant conditions) of the highest-energy organophosphate utilised by nature; phosphoenolpyruvate. Based on the demonstrated transformations, a potentially prebiotic network of glycolytic reactions is proposed, sharing common precursors and reaction conditions with important existing work within the field concerning the generation of nucleotides, amino-acids and lipids. The predisposition of hydroxy-aldehydes to glycolytically important transformations and the relationship of the proposed prebiotic network to extant glycolysis is discussed

Shikimate pathway in apicomplexan parasites

No abstract available

Plasma Magnetohydrodynamics and Energy Conversion

Contains reports on five research projects.U. S. Air Force. Aeronautical Systems Division (Contract AF33(615)-1083)National Science Foundation (Grant GK-19

A simple scheme for expanding photonic cluster states for quantum information

We show how an entangled cluster state encoded in the polarization of single photons can be straightforwardly expanded by deterministically entangling additional qubits encoded in the path degree of freedom of the constituent photons. This can be achieved using a polarization--path controlled-phase gate. We experimentally demonstrate a practical and stable realization of this approach by using a Sagnac interferometer to entangle a path qubit and polarization qubit on a single photon. We demonstrate precise control over phase of the path qubit to change the measurement basis and experimentally demonstrate properties of measurement-based quantum computing using a 2 photon, 3 qubit cluster state

Australian security of payment legislation: impact of inconsistent case law

In Australia, a supreme court has a supervisory role over the statutory adjudication process that has been established within the security of payment legislation. In this role, the courts have quashed many adjudication determinations on the grounds of jurisdictional error in recent years. This is a problem as the courts’ involvement in statutory adjudication is contrary to the object of the legislation. When reviewing adjudication determinations, the courts have adopted different approaches with respect to determining the role of adjudicators and the essential jurisdictional facts that must exist in order for an adjudicator to have jurisdiction to hear a referred disputed matter. This diversification of judicial interpretation with respect to jurisdictional error is confusing, not only to construction professionals, but also to many lawyers. Via a desktop study– where the evidence is mainly garnered from case law, governmental reports and commentaries – this paper reviews the legal complexities involved in diagnosing jurisdictional errors. In doing so, the paper aims to answer the question as to why the adjudication process has become bogged down in the quagmire of judicial review. The paper concludes that the evolving inconsistency of case law in relation to statutory adjudication is a crucial factor contributing to the erosion of the object of the security of payment legislation in Australia. Moving forward, the paper argues that establishing a legislative review mechanism of jurisdictional challenges may be sufficient to address this problem

Examining the approaches to diminish judicial intervention in statutory adjudication in Australia

In Australia, statutory construction adjudication is a fast payment dispute resolution process designed to keep the cash flowing down the hierarchical contractual chain in construction projects. Its rapid, highly regulatory and temporarily binding nature have led to it being often described as a ‘quick and dirty’ process that delivers ‘rough and ready’ justice. Adjudicators often have to grapple with complex legal issues related to jurisdictional facts and interpretation of contract provisions, though the majority of them are not legally trained. This has often led to a poor quality of adjudication outcome for large and complex payment claims which has, in turn, led to a mounting dissatisfaction due to the many judicial challenges to adjudicators’ determinations seen in recent years. The evolving tension between the object of the security of payment legislation and excessive involvement of the courts has often been the subject of comment by the judiciary. This paper aims to examine the legislative and judicial approaches to support the object of the security of payment legislation to ease cash flow. The paper adopts a desktop study approach whereby evidence is gathered from three primary sources – judicial decisions, academic publications and governmental reports. The paper concludes that there is a need to adopt other measures which can provide more convenient relief to aggrieved parties to an adjudication process, such that the adjudication process is kept away from the courts as far as is possible. Specifically, it is proposed that a well-designed expanded legislative review scheme of allegedly flawed adjudication, based on that provided in the Western Australian legislation, might stand as a promising remedy to eliminate the evolving tension