The Synthesis and Characterization of New, Robust Titanium (IV) Scorpionate Complexes

Titanium complexes possessing sterically encumbered ligands have allowed for the preparation of reactive moieties (imido, alkylidene and alkylidyne species) relevant to reactions such as olefin polymerization and alkyne hydroamination. For this reason, we have targeted robust scorpionate ancillary ligands to support reactive titanium centers. Thus, a series of titanium complexes were synthesized using an achiral oxazoline-based scorpionate ligand, tris(4,4-dimethyl-2-oxazolinyl)phenyl borate [To^M^]^-^ as well as the related chiral ligand, tris(4-isopropyl-2-oxazolinyl)phenyl borate [To^P^]^-^. The complex [Ti(κ^3^- To^M^)Cl~3~] was prepared in moderate yield (43%) by the rapid (<1 min at room temperature) reaction of Li[To^M^] and TiCl~4~ in methylene chloride; this new compound was characterized by ^1^H NMR spectroscopy as the expected C~3v~-symmetric species. One route to Ti (IV) alkyls involves salt metathesis; accordingly, syntheses of [To^M^]Ti alkyl complexes by interaction of [Ti(κ^3^-To^M^)Cl~3~] and one or three equivalents of alkylating agents, such as benzyl potassium (KCH~2~C~6~H~5~), trimethylsilylmethyl
lithium (LiCH~2~Si(CH~3~) ~3~), or neopentyl lithium (LiCH~2~C(CH~3~)~3~) are currently under investigation. The complexes [Ti(=NBut) (κ~3~-To^M^)(Cl)(Bu^t^py)] (Bu^t^py=4 tert-butylpyridine) and [Ti(=NBu^t^) (κ~3~-To^P^)(Cl)(Bu^t^py)] were synthesized by reaction of the known Ti imido [Ti(=NBu^t^)(Cl)~2~(Bu^t^py)~2~] with Li[To^M^] or Li[To^P^], respectively, by stirring overnight in methylene chloride at ambient temperature. The complexes were identified using ^1^H NMR spectroscopy, ^1^H-^13^C HMQC and ^1^H-^15^N HMBC correlation experiments

Emergency vehicle traffic signal preemption system

An emergency vehicle traffic light preemption system for preemption of traffic lights at an intersection to allow safe passage of emergency vehicles. The system includes a real-time status monitor of an intersection which is relayed to a communications controller for transmission to emergency vehicles as well as to a central dispatch office. The system also provides for audio warnings at an intersection to protect pedestrians who may not be in a position to see visual warnings or for various reasons cannot hear the approach of emergency vehicles. A transponder mounted on an emergency vehicle provides autonomous control so the vehicle operator can attend to getting to an emergency and not be concerned with the operation of the system. Activation of a Code 3 situation provides communications with each intersection being approached by an emergency vehicle and indicates whether the intersection is preempted or if there is any conflict with other approaching emergency vehicles. On-board diagnostics handle various information including heading, speed, and acceleration sent to a communications controller which is transmitted to an intersection and which also simultaneously receives information regarding the status of an intersection

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

Which way up? Recognition of homologous DNA segments in parallel and antiparallel alignment

Homologous gene shuffling between DNA promotes genetic diversity and is an important pathway for DNA repair. For this to occur, homologous genes need to find and recognize each other. However, despite its central role in homologous recombination, the mechanism of homology recognition is still an unsolved puzzle. While specific proteins are known to play a role at later stages of recombination, an initial coarse grained recognition step has been proposed. This relies on the sequence dependence of the DNA structural parameters, such as twist and rise, mediated by intermolecular interactions, in particular electrostatic ones. In this proposed mechanism, sequences having the same base pair text, or are homologous, have lower interaction energy than those sequences with uncorrelated base pair texts; the difference termed the recognition energy. Here, we probe how the recognition energy changes when one DNA fragment slides past another, and consider, for the first time, homologous sequences in antiparallel alignment. This dependence on sliding was termed the recognition well. We find that there is recognition well for anti-parallel, homologous DNA tracts, but only a very shallow one, so that their interaction will differ little from the interaction between two nonhomologous tracts. This fact may be utilized in single molecule experiments specially targeted to test the theory. As well as this, we test previous theoretical approximations in calculating the recognition well for parallel molecules against MC simulations, and consider more rigorously the optimization of the orientations of the fragments about their long axes. The more rigorous treatment affects the recognition energy a little, when the molecules are considered rigid. However when torsional flexibility of the DNA molecules is introduced, we find excellent agreement between analytical approximation and simulation.Comment: Paper with supplemental material attached. 41 pages in all, 4 figures in main text, 3 figures in supplmental. To be submitted to Journa

A simple description of the states 0+0^+ and 2+2^+ in 168Er^{168}Er

A sixth-order quadrupole boson Hamiltonian is used to describe 26 states $0^+$ and 67 states $2^+$ which have been recently identified in $^{168}Er$. Two closed expressions are alternatively used for energy levels. One corresponds to a semi-classical approach while the other one represents the exact eigenvalue of the model Hamiltonian. The semi-classical expression involves four parameters, while the exact eigenvalue is determined by five parameters. In each of the two descriptions a least square fit procedure is adopted. Both expressions provide a surprisingly good agreement with the experimental data.Comment: 9 pages, 5 figure

State sum construction of two-dimensional open-closed Topological Quantum Field Theories

We present a state sum construction of two-dimensional extended Topological Quantum Field Theories (TQFTs), so-called open-closed TQFTs, which generalizes the state sum of Fukuma--Hosono--Kawai from triangulations of conventional two-dimensional cobordisms to those of open-closed cobordisms, i.e. smooth compact oriented 2-manifolds with corners that have a particular global structure. This construction reveals the topological interpretation of the associative algebra on which the state sum is based, as the vector space that the TQFT assigns to the unit interval. Extending the notion of a two-dimensional TQFT from cobordisms to suitable manifolds with corners therefore makes the relationship between the global description of the TQFT in terms of a functor into the category of vector spaces and the local description in terms of a state sum fully transparent. We also illustrate the state sum construction of an open-closed TQFT with a finite set of D-branes using the example of the groupoid algebra of a finite groupoid.Comment: 33 pages; LaTeX2e with xypic and pstricks macros; v2: typos correcte

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

Sequencing of idiopathic pulmonary fibrosis-related genes reveals independent single gene associations

BACKGROUND: Previous studies investigating a genetic basis for idiopathic pulmonary fibrosis (IPF) have focused on resequencing single genes in IPF kindreds or cohorts to determine the genetic contributions to IPF. None has investigated interactions among the candidate genes. OBJECTIVE: To compare the frequencies and interactions of mutations in six IPF-associated genes in a cohort of 132 individuals with IPF with those of a disease-control cohort of 192 individuals with chronic obstructive pulmonary disease (COPD) and the population represented in the Exome Variant Server. METHODS: We resequenced the genes encoding surfactant proteins A2 (SFTPA2), and C (SFTPC), the ATP binding cassette member A3 (ABCA3), telomerase (TERT), thyroid transcription factor (NKX2-1) and mucin 5B (MUC5B) and compared the collapsed frequencies of rare (minor allele frequency <1%), computationally predicted deleterious variants in each cohort. We also genotyped a common MUC5B promoter variant that is over-represented in individuals with IPF. RESULTS: We found 15 mutations in 14 individuals (11%) in the IPF cohort: (SFTPA2 (n=1), SFTPC (n=5), ABCA3 (n=4) and TERT (n=5)). No individual with IPF had two different mutations, but one individual with IPF was homozygous for p.E292V, the most common ABCA3 disease-causing variant. We did not detect an interaction between any of the mutations and the MUC5B promoter variant. CONCLUSIONS: Rare mutations in SFTPA2, SFTPC and TERT are collectively over-represented in individuals with IPF. Genetic analysis and counselling should be considered as part of the IPF evaluation