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.     

Rapid MALDI biotyper-based identification and cluster analysis of <Emphasis Type="Italic">Streptococcus iniae</Emphasis>

Streptococcus iniae causes severe mortalities among cultured marine species, especially in the olive flounder (Paralichthys olivaceus), which is economically important in Korea and Japan. Recently, there has been growing concern regarding the emergence of S. iniae as a zoonotic pathogen. Here, 89 S. iniae isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of S. iniae data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either “no reliable identification” or were incorrectly identified as Streptococcus pyogenes at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as S. iniae at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between Streptococcus species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying S. iniae. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen.     

BRONCHIAL ASTHMA—Patterns of Morbidity and Mortality in the United States, 1951-1959

On a nationwide basis, bronchial asthma occurs at the rate of 23 cases per 1,000 population. Young males develop bronchial asthma more readily and more severely than young females. Males dying from asthma outnumber females 2 to 1.Eight per cent of the asthmatic persons in the United States have not sought medical attention for this condition. Repeated attacks of severe bronchial asthma increase the likelihood of premature death.Approximately 6,000 deaths due to asthma occur annually in the United States, with a seasonal increase during the winter months. The estimated fatality rate of asthma in the general population is 1.5 deaths per 1,000 asthmatics.     

Complete Genomic and Lysis-Cassette Characterization of the Novel Phage,KBNP1315, which Infects Avian Pathogenic Escherichia coli (APEC)

Avian pathogenic Escherichia coli (APEC) is a major pathogen that causes avian colibacillosis and is associated with severe economic losses in the chicken-farming industry. Here, bacteriophage KBNP1315, infecting APEC strain KBP1315, was genomically and functionally characterized. The evolutionary relationships of KBNP1315 were analyzed at the genomic level using gene (protein)-sharing networks, the Markov clustering (MCL) algorithm, and comparative genomics. Our network analysis showed that KBNP1315 was connected to 30 members of the Autographivirinae subfamily, which comprises the SP6-, T7-, P60-, phiKMV-, GAP227- and KP34-related groups. Network decomposition suggested that KBNP1315 belongs to the SP6-like phages, but our comparison of putative encoded proteins revealed that key proteins of KBNP1315, including the tail spike protein and endolysin, had relative low levels of amino acid sequence similarity with other members of the SP6-like phages. Thus KBNP1315 may only be distantly related to the SP6-like phages, and (based on the difference in endolysin) its lysis mechanism may differ from theirs. To characterize the lytic functions of the holin and endolysin proteins from KBNP1315, we expressed these proteins individually or simultaneously in E. coli BL21 (DE3) competent cell. Interestingly, the expressed endolysin was secreted into the periplasm and caused a high degree of host cell lysis that was dose-dependently delayed/blocked by NaN₃-mediated inhibition of the SecA pathway. The expressed holin triggered only a moderate inhibition of cell growth, whereas coexpression of holin and endolysin enhanced the lytic effect of endolysin. Together, these results revealed that KBNP1315 appears to use a pin-holin/signal-arrest-release (SAR) endolysin pathway to trigger host cell lysis.     

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.