Development of a novel polyamide-based agent to inhibit EVI1 function

The EVI1 gene at chromosome 3q26 is associated with acute myeloid leukemogenesis, due to both chromosomal rearrangement and to overexpression in the absence of rearrangement. Some rearrangements such as t(3;3) and inv(3) result in overexpression of EVI1 protein, while translocation t(3;21) yields an AML1-MDS1-EVI1 (AME) fusion protein. EVI1 possesses two zinc finger domains, an N-terminal domain with fingers 1–7, which binds to GACAAGATA, and a C-terminal domain (fingers 8–10) which binds GAAGATGAG. Inhibition of EVI1 function with a small molecule compound may provide a targeted therapy for EVI1-expressing leukemias. As a first step towards inhibiting the leukemogenic function of EVI1, we performed structure-function studies on both EVI1 and AME protein to determine what domains are critical for malignant transformation activity. Assays were Rat1 fibroblasts in a soft agar colony forming assay for EVI1; primary bone marrow cells in a serial replating assay for AME. Both assays revealed that mutation of arginine 205 in zinc finger 6 of EVI1, which completely abrogates sequencespecific DNA binding via the N-terminal zinc finger domain, resulted in complete loss of transforming activity; mutations in other domains, such as the C-terminal zinc finger domain, CtBP binding domain, and the domains of AML1 had less of an effect or no effect on transforming activity. In an effort to inhibit EVI1 leukemogenic function, we developed a polyamide, DH-IV-298, designed to block zinc fingers 1–7 binding to the GACAAGATA motif. DNAseI footprinting revealed a specific interaction between DH-IV-298 and the GACAAGATA motif; no significant interaction was observed elsewhere; a mismatch polyamide failed to footprint at equivalent concentrations; and DH-IV-298 failed to bind to a control DNA lacking the GACAAGATA motif. Electromobility shift assay showed that, at a 1:1 polyamide:DNA ratio, DH-IV-298 lowered EVI1:DNA affinity by over 98%, while mismatch was significantly less effective (74% reduction). To assess the effect of DH-IV-298 on EVI1 binding to DNA in vivo, we performed CAT reporter assays in a NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as a EVI1-responsive CAT reporter. Removal of tetracycline resulted in a four-fold increase in CAT activity that was completely blocked by DH-IV-298. The mismatch polyamide was significantly less effective than DH-IV-298. Further studies are being performed to assess the effect on endogenous gene expression, and on growth of leukemic cells that express EVI1. These studies provide evidence that a cell permeable small molecule compound may effectively block the activity of a leukemogenic transcription factor

Chelator free gallium-68 radiolabelling of silica coated iron oxide nanorods via surface interactions

The commercial availability of combined magnetic resonance imaging (MRI)/positron emission tomography (PET) scanners for clinical use has increased demand for easily prepared agents which offer signal or contrast in both modalities. Herein we describe a new class of silica coated iron–oxide nanorods (NRs) coated with polyethylene glycol (PEG) and/or a tetraazamacrocyclic chelator (DO3A). Studies of the coated NRs validate their composition and confirm their properties as in vivo T₂ MRI contrast agents. Radiolabelling studies with the positron emitting radioisotope gallium-68 (t1/2 = 68 min) demonstrate that, in the presence of the silica coating, the macrocyclic chelator was not required for preparation of highly stable radiometal-NR constructs. In vivo PET-CT and MR imaging studies show the expected high liver uptake of gallium-68 radiolabelled nanorods with no significant release of gallium-68 metal ions, validating our innovation to provide a novel simple method for labelling of iron oxide NRs with a radiometal in the absence of a chelating unit that can be used for high sensitivity liver imaging

The receptor locus for Escherichia coli F4ab/F4ac in the pig maps distal to the MUC4 - LMLN region

Enterotoxigenic Escherichia coli (ETEC) with fimbriae of the F4 family are one of the major causes of diarrhea and death among neonatal and young piglets. Bacteria use the F4 fimbriae to adhere to specific receptors expressed on the surface of the enterocytes. F4 fimbriae exist in three different antigenic variants, F4ab, F4ac, and F4ad, of which F4ac is the most common. Resistance to ETEC F4ab/F4ac adhesion in pigs has been shown to be inherited as an autosomal recessive trait. In previous studies the ETEC F4ab/F4ac receptor locus (F4bcR) was mapped to the q41 region on pig chromosome 13. A polymorphism within an intron of the mucin 4 (MUC4) gene, which is one of the possible candidate genes located in this region, was shown earlier to cosegregate with the F4bcR alleles. Recently, we discovered a Large White boar from a Swiss experimental herd with a recombination between F4bcR and MUC4. A three-generation pedigree including 45 offspring was generated with the aim to use this recombination event to refine the localization of the F4bcR locus. All pigs were phenotyped using the microscopic adhesion test and genotyped for a total of 59 markers. The recombination event was mapped to a 220-kb region between a newly detected SNP in the leishmanolysin-like gene (LMLN g.15920) and SNP ALGA0072075. In this study the six SNPs ALGA0072075, ALGA0106330, MUC13-226, MUC13-813, DIA0000584, and MARC0006918 were in complete linkage disequilibrium with F4bcR. Based on this finding and earlier investigations, we suggest that the locus for F4bcR is located between the LMLN locus and microsatellite S028

The nature of freedom

https://stars.library.ucf.edu/prism/1771/thumbnail.jp

Computation with Advice

Computation with advice is suggested as generalization of both computation with discrete advice and Type-2 Nondeterminism. Several embodiments of the generic concept are discussed, and the close connection to Weihrauch reducibility is pointed out. As a novel concept, computability with random advice is studied; which corresponds to correct solutions being guessable with positive probability. In the framework of computation with advice, it is possible to define computational complexity for certain concepts of hypercomputation. Finally, some examples are given which illuminate the interplay of uniform and non-uniform techniques in order to investigate both computability with advice and the Weihrauch lattice

Comparability & reimbursement for the translation of scalable, automated stem cell cultures

The research in this thesis focuses primarily on two critical challenges that inhibit the late stage translation of cell-based therapies and Regenerative Medicines (RMs). These include product comparability after a change in manufacturing process or site; and the reimbursement of RMs, in particular those which target multiple simultaneous indications, or Multimorbidity . The automation and standardisation of stem cell cultures also represent key themes of this thesis, which may facilitate the development of scalable, reproducible manufacturing processes for cell-based therapies. Furthermore, given the current uncertainty regarding the characterisation and potency of Human Mesenchymal Stromal (or Stem) Cells (hMSCs) that has inhibited the successful clinical translation of hMSC-based products, understanding the characterisation and putative modes of action of these cells was also a priority throughout this research. Also, due to the increasing number of Human Embryonic Stem Cell (hESC) derived therapies progressing towards market, and the industry-wide shift towards Human Induced Pluripotent Stem Cells (hiPSC) as an alternative to hESCs, the measurement of the growth and characterisation of these cells types represents an important method of demonstrating product comparability after alternative manufacturing process steps in the present thesis. Finally, due to the potential of hiPSCs as a source of large numbers of hMSCs, the culture conditions required to direct the differentiation of hiPSCs to hMSCs are explored