Live cell imaging of X chromosome reactivation during somatic cell reprogramming

Generation of induced pluripotent stem cells (iPSCs) with naive pluripotency is important for their applications in regenerative medicine. In female iPSCs, acquisition of naive pluripotency is coupled to X chromosome reactivation (XCR) during somatic cell reprogramming, and live cell monitoring of XCR is potentially useful for analyzing how iPSCs acquire naive pluripotency. Here we generated female mouse embryonic stem cells (ESCs) that carry the enhanced green fluorescent protein (EGFP) and humanized Kusabira-Orange (hKO) genes inserted into an intergenic site near either the Syap1 or Taf1 gene on both X chromosomes. The ESC clones, which initially expressed both EGFP and hKO, inactivated one of the fluorescent protein genes upon differentiation, indicating that the EGFP and hKO genes are subject to X chromosome inactivation (XCI). When the derived somatic cells carrying the EGFP gene on the inactive X chromosome (Xi) were reprogrammed into iPSCs, the EGFP gene on the Xi was reactivated when pluripotency marker genes were induced. Thus, the fluorescent protein genes inserted into an intergenic locus on both X chromosomes enable live cell monitoring of XCI during ESC differentiation and XCR during reprogramming. This is the first study that succeeded live cell imaging of XCR during reprogramming.     

Characterization of Fusarium oxysporum β-1,6-Galactanase, an Enzyme That Hydrolyzes Larch Wood Arabinogalactan

A type II arabinogalactan-degrading enzyme (FoGal1) was purified from Fusarium oxysporum 12S, and the corresponding cDNA was isolated. FoGal1 had high similarity to enzymes of glycoside hydrolase family 5. Treatment of larch wood arabinogalactan with the recombinant enzyme indicated that FoGal1 is a β-1,6-galactanase that preferentially debranches β-1,6-galactobiose from the substrate.     

Deoxyribonucleic Acid Polymerase Activities in Normal and Leukovirus-Infected Chicken Embryo Cells

Chicken embryo cells normally contain, in addition to deoxyribonucleic acid (DNA)-dependent DNA (D-DNA) polymerases, a novel "R-DNA-polymerase" which specifically copies polyriboadenylic acid strands. This R-DNA polymerase cannot copy natural ribonucleic acid or polyribocytidylic acid strands to a significant extent. Infection of cells with the leukovirus RAV-2 leads to the intracellular formation of large amounts of the viral RNA-dependent DNA polymerase whose properties differ from the cell R-DNA polymerase. Chicken cells transformed by a Rous sarcoma virus mutant which produce noninfectious alpha-type Rous sarcoma virus (f), a leukovirus known to be deficient in the viral RNA-dependent DNA polymerase, do not contain detectable viral RNA-dependent DNA polymerase, whereas the cellular R-DNA polymerase is found in normal amounts. There seems to be no relationship between the cellular R-DNA polymerase and the RNA-dependent DNA polymerase of the avian leukoviruses.     

Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants

Key message

The first Good Manufacturing Practices production of a purification-free rice-based oral cholera vaccine (MucoRice-CTB) from transgenic plants in a closed cultivation system yielded a product meeting regulatory requirements.

Abstract

Despite our knowledge of their advantages, plant-based vaccines remain unavailable for human use in both developing and industrialized countries. A leading, practical obstacle to their widespread use is producing plant-based vaccines that meet governmental regulatory requirements. Here, we report the first production according to current Good Manufacturing Practices of a rice-based vaccine, the cholera vaccine MucoRice-CTB, at an academic institution. To this end, we established specifications and methods for the master seed bank (MSB) of MucoRice-CTB, which was previously generated as a selection-marker-free line, evaluated its propagation, and given that the stored seeds must be renewed periodically. The production of MucoRice-CTB incorporated a closed hydroponic system for cultivating the transgenic plants, to minimize variations in expression and quality during vaccine manufacture. This type of molecular farming factory can be operated year-round, generating three harvests annually, and is cost- and production-effective. Rice was polished to a ratio of 95 % and then powdered to produce the MucoRice-CTB drug substance, and the identity, potency, and safety of the MucoRice-CTB product met pre-established release requirements. The formulation of MucoRice-CTB made by fine-powdering of drug substance and packaged in an aluminum pouch is being evaluated in a physician-initiated phase I study.

     

Replication of hepatitis B virus which carries foreign DNA in vitro.

Targeting a specific DNA sequence to the desired tissues is an important step in gene therapy. The hepatitis B virus (HBV) is the only DNA virus that has hepatocyte specificity. We attempted to construct an HBV-based vector for targeting the liver. We observed the replication and secretion of virus particles in an HBV construct that lacks X gene and carries an extra 63 bp DNA fragment in vitro. Replication was observed in the cell line HuH-7 but not HepG2. From this construct, we designed an HBV-based vector that could carry foreign DNA. HBV based vectors provide for the possibilities of generating therapeutic agents for individual patients. Our host vector system may be used to clear out the HBV from the HBV carrier or chronic hepatitis B patients by introducing a genetically engineered HBV into these patients.     

Strain improvement of Lentzea sp. 7887 for higher yield per unit volume on hydroxylation of cyclosporine derivative FR901459

A Gram-positive bacterium Lentzea sp. 7887 hydroxylates a cyclosporine derivative FR901459 into AS1837812 (9-hydroxide), which is an important intermediate of candidate drugs that target the hepatitis C virus. We screened a UV-induced mutant, named M-1, which showed about 1.2-fold higher conversion yields, 2-fold higher substrate concentrations (3.69 mM), and 2.5-fold higher yield per unit volume than the wild-type strain.     

Gene duplications and the evolution of c-type lysozyme during adaptive radiation of East African cichlid fish

Given the reported degraded nature of DOC in the Rio Negro, and low oxygen, pH, and bacterial riverine levels, we hypothesized: (1) DOC would have strong humic and fulvic acid fluorescence signals with high aromaticity and large mean molecular weight; and (2) photo-oxidation rates would be slow, and reactive oxygen species (ROS) concentrations low, producing no oxidative stress in biota. We surveyed the environment and properties of DOC and explored DOC photo-oxidation and fish sensitivity to DOC products. DOC properties were investigated using absorption and fluorescence indices and parallel factor analysis (PARAFAC) of excitation–emission matrices. ROS concentrations were measured spectrophotometrically. A native fish, Hemigrammus levis, was exposed to photo-oxidizing DOC and its tissues (brain, gill, liver) assayed for changes in antioxidant and biotransformation enzymes. With respect to our hypotheses, (1) DOC was highly terrigenous, with high SAC₃₄₀ values (aromaticity), high capacity to produce ROS, and high tryptophan-like fluorescence (bacterial, autochthonous signal); (2) photo-oxidation rates were appreciable, while products were related to mean UV-radiation levels (total radiation was constant). ROS levels were often higher than freshwater averages, yet fish experienced no oxidative stress. Results suggest photo-oxidation influences patterns in C-cycling, bacterial production and community dynamics between wet and dry seasons.