Inducible gene modification in the gastric epithelium of Tff1‐CreERT2, Tff2‐rtTA,Tff3‐luc mice

Temporal and spatial regulation of genes mediated by tissue‐specific promoters and conditional gene expression systems provide a powerful tool to study gene function in health, disease, and during development. Although transgenic mice expressing the Cre recombinase in the gastric epithelium have been reported, there is a lack of models that allow inducible and reversible gene modification in the stomach. Here, we exploited the gastrointestinal epithelium‐specific expression pattern of the three trefoil factor (Tff) genes and bacterial artificial chromosome transgenesis to generate a novel mouse strain that expresses the CreERT2 recombinase and the reverse tetracycline transactivator (rtTA). The Tg(Tff1‐CreERT2;Tff2‐rtTA;Tff3‐Luc) strain confers tamoxifen‐inducible irreversible somatic recombination and allows simultaneous doxycycline‐dependent reversible gene activation in the gastric epithelium of developing and adult mice. This strain also confers luciferase activity to the intestinal epithelium to enable in vivo bioluminescence imaging. Using fluorescent reporters as conditional alleles, we show Tff1‐CreERT2 and Tff2‐rtTA transgene activity in a partially overlapping subset of long‐term regenerating gastric stem/progenitor cells. Therefore, the Tg(Tff1‐CreERT2;Tff2‐rtTA;Tff3‐Luc) strain can confer intermittent transgene expression to gastric epithelial cells that have undergone previous gene modification, and may be suitable to genetically model therapeutic intervention during development, tumorigenesis, and other genetically tractable diseases. Birth Defects Research (Part A) 106:626–635, 2016. © 2016 Wiley Periodicals, Inc.     

Long-term survival of haptophyte and prasinophyte resting stages in marine sediment

Resting stages of marine phytoplankton have been shown to have potential for long-term survival and to remain viable in marine sediments for up to about a century. This study documents for the first time long-term survival in haptophytes and prasinophytes, by germination of resting stages of Isochrysis galbana and Mantoniella squamata from up to 40-year-old sediment layers. Germination was induced by setting up sediment slurries in L1 medium at 15°C. Cyst formation was induced in culture strains acquired from the germinations by keeping mixtures of strains in the light or dark at salinities of 20 or 30. The identity of the two species was confirmed by light and electron microscopy as well as LSU and SSU rDNA-based phylogenetic analyses.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Salt‐enhanced cultivation as a morphology engineering tool for filamentous actinomycetes: Increased production of labyrinthopeptin A1 in Actinomadura namibiensis

Salt‐enhanced cultivation as a morphology engineering tool for the filamentous actinomycete Actinomadura namibiensis was evaluated in 500‐mL shaking flasks (working volume 100 mL) with the aim of increasing the concentration of the pharmaceutically interesting peptide labyrinthopeptin A1. Among the inorganic salts added to a complex production medium, the addition of (NH₄)₂SO₄ led to the highest amount of labyrinthopeptin A1 production. By using 50 mM (NH₄)₂SO₄, the labyrinthopeptin A1 concentration increased up to sevenfold compared to the non‐supplemented control, resulting in 325 mg L^?1 labyrinthopeptin A1 after 10 days of cultivation. The performance of other ammonium‐ and sulfate‐containing salts (e.g., NH₄Cl, K₂SO₄) was much lower than the performance of (NH₄)₂SO₄. A positive correlation between the uptake of glycerol as one of the main carbon sources and nongrowth‐associated labyrinthopeptin productivity was found. The change in the cell morphology of A. namibiensis in conjunction with increased osmolality by the addition of 50 mM (NH₄)₂SO₄, was quantified by image analysis. A. namibiensis always developed a heterogeneous morphology with pellets and loose mycelia present simultaneously. In contrast to the non‐supplemented control, the morphology of (NH₄)₂SO₄‐supplemented cultures was characterized by smaller and circular pellets that were more stable against disintegration in the stationary production phase.     

Satellite tracking of gulls and genomic characterization of faecal bacteria reveals environmentally mediated acquisition and dispersal of antimicrobial‐resistant Escherichia coli on the Kenai Peninsula,Alaska

Gulls (Larus spp.) have frequently been reported to carry Escherichia coli exhibiting antimicrobial resistance (AMR E. coli); however, the pathways governing the acquisition and dispersal of such bacteria are not well described. We equipped 17 landfill‐foraging gulls with satellite transmitters and collected gull faecal samples longitudinally from four locations on the Kenai Peninsula, Alaska to assess: (a) gull attendance and transitions between sites, (b) spatiotemporal prevalence of faecally shed AMR E. coli, and (c) genomic relatedness of AMR E. coli isolates among sites. We also sampled Pacific salmon (Oncorhynchus spp.) harvested as part of personal‐use dipnet fisheries at two sites to assess potential contamination with AMR E. coli. Among our study sites, marked gulls most commonly occupied the lower Kenai River (61% of site locations) followed by the Soldotna landfill (11%), lower Kasilof River (5%) and upper Kenai River (<1%). Gulls primarily moved between the Soldotna landfill and the lower Kenai River (94% of transitions among sites), which were also the two locations with the highest prevalence of AMR E. coli. There was relatively high spatial and temporal variability in AMR E. coli prevalence in gull faeces and there was no evidence of contamination on salmon harvested in personal‐use fisheries. We identified E. coli sequence types and AMR genes of clinical importance, with some isolates possessing genes associated with resistance to as many as eight antibiotic classes. Our findings suggest that gulls acquire AMR E. coli at habitats with anthropogenic inputs and subsequent movements may represent pathways through which AMR is dispersed.