Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology

A type of chromosome-free cell called SimCells (simple cells) has been generated from Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. The removal of the native chromosomes of these bacteria was achieved by double-stranded breaks made by heterologous I-CeuI endonuclease and the degradation activity of endogenous nucleases. We have shown that the cellular machinery remained functional in these chromosome-free SimCells and was able to process various genetic circuits. This includes the glycolysis pathway (composed of 10 genes) and inducible genetic circuits. It was found that the glycolysis pathway significantly extended longevity of SimCells due to its ability to regenerate ATP and NADH/NADPH. The SimCells were able to continuously express synthetic genetic circuits for 10 d after chromosome removal. As a proof of principle, we demonstrated that SimCells can be used as a safe agent (as they cannot replicate) for bacterial therapy. SimCells were used to synthesize catechol (a potent anticancer drug) from salicylic acid to inhibit lung, brain, and soft-tissue cancer cells. SimCells represent a simplified synthetic biology chassis that can be programmed to manufacture and deliver products safely without interference from the host genome

Repetitive noninvasive monitoring of hsvl-tk-expressing t cells intravenously infused into nonhuman primates using positron emission tomography and computed tomography with 18F-FEAU

Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) has been successfully used to treat patients with different types of cancer. However, the long-term spatial-temporal dynamics of the distribution of systemically infused CTLs rlargely unknown. Noninvasive imaging of adoptively transferred CTLs using molecular-genetic reporter imaging with positron emission tomography and computed tomography (PET-CT) represents an innovative approach to understanding the long-term migratory patterns and therapeutic potential of adoptively transferred T cells. Here we report the application of repetitive PET-CT imaging with [18F]fluoro-5-ethyl-1-beta-D- arabinofuranosyluracil (18F-FEAU) in two nonhuman primates demonstrating that autologous polyclonal macaque T lymphocytes activated and transduced with a retroviral vector encoding for the sr39 mutant herpes simplex virus 1 thymidine kinase (sr39HSV1-tk) reporter gene can be detected after intravenous infusion in discrete lymphoid organs and in sites of inflammation. This study represents a proof of principle and supports the application of 18F-FEAU PET-CT imaging for monitoring the distribution of intravenously administered sr39HSV1-tk gene-transduced CTLs in humans

Ex vivo fucosylation improves human cord blood engraftment in NOD-SCID IL-2Rγ null mice

Delayed engraftment remains a major hurdle after cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the bone marrow microenvironment. Because fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the bone marrow microvasculature can occur, a simple 30-minute ex vivo incubation of CB hematopoietic progenitor cells with fucosyltransferase-VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB hematopoietic progenitor cell hypofucosylation was investigated in vivo in NOD-SCID interleukin (IL)-2Rγ null (NSG) mice. By isolating fucosylated and nonfucosylated CD34 + cells from CB, we showed that only fucosylated CD34 + cells are responsible for engraftment in NSG mice. In addition, because the proportion of CD34 + cells that are fucosylated in CB is significantly less than in bone marrow and peripheral blood, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed after CB transplantation. Because engraftment appears to be correlated with the fucosylation of CD34 + cells, we hypothesized that increasing the proportion of CD34 + cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase-VI significantly increases the levels of CD34 + fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells or pattern of long-term, multilineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting

Retarded long-range interaction in split-ring-resonator square arrays

We systematically investigate the optical extinction spectra of planar gold split-ring-resonator square arrays operating at ∼200-THz frequency versus the lattice constant and versus angle of incidence. We find a strong dependence of the resonance dampi

Phosphorylation of the Stat1 transactivating domain is required for the response to type I interferons

Stat1 (signal transducer and activator of transcription 1) regulates transcription in response to the type I interferons IFN-α and IFN-β, either in its dimerized form or as a subunit of the interferon-stimulated gene factor 3 (Isgf3) complex (consisting of Stat1, Stat2 and interferon-regulating factor 9). Full-length Stat1-α and the splice variant Stat1-β, which lacks the carboxyl terminus and the Ser727 phosphorylation site, are found in all cell types. IFN-induced phosphorylation of Stat1-α on Ser727 occurs in the absence of the candidate kinase, protein kinase C-δ. When expressed in Stat1-deficient cells, Stat1-β and a Stat1-S727A mutant both restored the formation of Stat1 dimers and of the Isgf3 complex on treatment with IFN-β. By contrast, only Stat1-α restored the ability of IFN-β to induce high levels of transcription from target genes of Stat1 dimers and Isgf3 and to induce an antiviral state. Our data suggest an important contribution of the Stat1 C terminus and its phosphorylation at Ser727 to the transcriptional activities of the Stat1 dimer and the Isgf3 complex