The Effect of Fish Introductions on the Diatom and Cladoceran Communities of Lake Opeongo,Ontario, Canada

Fish introductions are one of the most widespread anthropogenic perturbations to aquatic ecosystems. Paradoxically, the effects of these introductions on aquatic ecosystems are typically poorly documented. This project studied the effect of fish introductions on Lake Opeongo, an oligotrophic lake in Algonquin Provincial Park, Ontario, Canada (45° 42′ N, 78° 22′ W), using the remains of algae (diatoms) and zooplankton (cladocerans) preserved in the sediments. It was hypothesized that the introduction of cisco or lake herring (Coregonus artedii Lesueur) in 1948, which filled the underutilized pelagic forage fish niche, should have altered nutrient availability for phytoplankton. Prior to cisco introduction, the diatom community of Lake Opeongo reflected a relatively stable oligotrophic state established before European settlement, and consisted of the Cyclotella stelligera complex with subdominants Tabellaria flocculosa IIIp and the Aulacoseira distans complex. No marked changes occurred until ca. 1962 when the diatom community shifted to an assemblage with increased total phosphorus preferences, consisting of Asterionella formosa and lesser amounts of Cyclotella bodanica var lemanica, the C. stelligera complex, Fragilaria crotonensis and T. flocculosa IIIp. The dominant cladoceran Bosmina longirostris increased significantly in relative abundance since the introduction of cisco. The most likely cause of this shift was increased nutrient recycling and/or trophic level changes caused by human manipulation of the fish community of the lake.     

Expression of Flp Protein in a Baculovirus/Insect Cell System for Biotechnological Applications

The Saccharomyces cerevisiae Flp protein is a site-specific recombinase that recognizes and binds to the Flp recognition target (FRT) site, a specific sequence comprised of at least two inverted repeats separated by a spacer. Binding of four monomers of Flp is required to mediate recombination between two FRT sites. Because of its site-specific cleavage characteristics, Flp has been established as a genome engineering tool. Amongst others, Flp is used to direct insertion of genes of interest into eukaryotic cells based on single and double FRT sites. A Flp-encoding plasmid is thereby typically cotransfected with an FRT-harboring donor plasmid. Moreover, Flp can be used to excise DNA sequences that are flanked by FRT sites. Therefore, the aim of this study was to determine whether Flp protein and its step-arrest mutant, FlpH305L, recombinantly expressed in insect cells, can be used for biotechnological applications. Using a baculovirus system, the proteins were expressed as C-terminally 3?×?FLAG-tagged proteins and were purified by anti-FLAG affinity selection. As demonstrated by electrophoretic mobility shift assays (EMSAs), purified Flp and FlpH305L bind to FRT-containing DNA. Furthermore, using a cell assay, purified Flp was shown to be active in recombination and to mediate efficient insertion of a donor plasmid into the genome of target cells. Thus, these proteins can be used for applications such as DNA-binding assays, in vitro recombination, or genome engineering.     

Lipopolysaccharide can modify differentiation and immunomodulatory potential of periodontal ligament stem cells via ERK1,2 signaling

Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast‐like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF‐β, fibronectin (FN), α‐SMA, and NG2. LPS also increased protein and gene expression levels of anti‐inflammatory COX‐2 and pro‐inflammatory IL‐6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS‐treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen‐stimulated proliferation of CD4⁺ and the ratio of CD4⁺CD25^high/CD4⁺CD25^low lymphocytes. LPS‐treated PDLSCs did not change the frequency of CD34⁺ and CD45⁺ cells, but decreased the frequency of CD33⁺ and CD14⁺ myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU‐GM number. The results indicated that LPS‐activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features.