A Combined Enrichment and Aptamer Pulldown Assay for Francisella tularensis Detection in Food and Environmental Matrices

Francisella tularensis, a Gram-negative bacterium and causative agent of tularemia, is categorized as a Class A select agent by the Centers for Disease Control and Prevention due to its ease of dissemination and ability to cause disease. Oropharyngeal and gastrointestinal tularemia may occur due to ingestion of contaminated food and water. Despite the concern to public health, little research is focused on F. tularensis detection in food and environmental matrices. Current diagnostics rely on host responses and amplification of F. tularensis genetic elements via Polymerase Chain Reaction; however, both tools are limited by development of an antibody response and limit of detection, respectively. During our investigation to develop an improved culture medium to aid F. tularensis diagnostics, we found enhanced F. tularensis growth using the spent culture filtrate. Addition of the spent culture filtrate allowed for increased detection of F. tularensis in mixed cultures of food and environmental matrices. Ultraperformance liquid chromatography (UPLC)/MS analysis identified several unique chemicals within the spent culture supernatant of which carnosine had a matching m/z ratio. Addition of 0.625 mg/mL of carnosine to conventional F. tularensis medium increased the growth of F. tularensis at low inoculums. In order to further enrich F. tularensis cells, we developed a DNA aptamer cocktail to physically separate F. tularensis from other bacteria present in food and environmental matrices. The combined enrichment steps resulted in a detection range of 1–10⁶ CFU/mL (starting inoculums) in both soil and lettuce backgrounds. We propose that the two-step enrichment process may be utilized for easy field diagnostics and subtyping of suspected F. tularensis contamination as well as a tool to aid in basic research of F. tularensis ecology.     

Marek's disease virus (MDV) ICP4, pp38, and meq genes are involved in the maintenance of transformation of MDCC-MSB1 MDV-transformed lymphoblastoid cells.

An antisense strategy has been used to identify genes important for the maintenance of transformation of MDCC-MSB1 (MSB1) Marek's disease virus-transformed lymphoblastoid cells. Oligodeoxynucleotides antisense to the predicted translation initiation regions of ICP4 and pp38 mRNAs inhibited proliferation of MSB1 cells but not MDCC-CU91 (CU91) reticuloendotheliosis virus-transformed cells. Control oligodeoxynucleotides having the same base composition but a different sequence did not inhibit MSB1 cell proliferation. In addition, ICP4 and pp38 antisense oligodeoxynucleotides resulted in 77- and 100-fold reductions in colony formation by MSB1 cells in soft agar, respectively. To extend and corroborate these results, a novel system based on efficiently regulated expression of eukaryotic genes by a chimeric mammalian transactivator, LAP267 (S. B. Baim, M. A. Labow, A. J. Levine, and T. Shenk, Proc. Natl. Acad. Sci. USA 88:5072-5076, 1991), was used. MSB1-derived stably transfected cell lines in which RNA antisense to Marek's disease virus ICP4, pp38, or meq could be induced by treatment of the cells with isopropyl-beta-D-thiogalactopyranoside (IPTG) were constructed. Control cell lines in which expression of ICP4 sense or pUC19 sequences could be induced by IPTG were also constructed. Induction of the cell lines indicated that ICP4 antisense RNA, but not ICP4 sense RNA or pUC19 RNA, inhibited proliferation of MSB1 cells. Induction of ICP4, meq, or pp38 antisense RNAs, but not ICP4 sense or pUC19 RNAs, had a dramatic effect on relative colony formation by MSB1 cells in soft agar. These results indicate that ICP4, pp38, and Meq are all involved in the maintenance of transformation of MSB1 cells.     

Interconversion of replication and recombination structures: implications for terminal repeats and concatemers

Replication and recombination structures can be interconverted by branch-migration. Using this simple concept a novel mechanism is proposed for generating concatemers through an initial single-strand DNA invasion into a duplex. Only DNAs with terminal repeats can form concatemers, and Herpes Simplex Virus DNA replication is considered in detail. The model is more parsimonious than other models such as Watson's for concatemer formation.     

Using remote underwater video to estimate freshwater fish species richness

Species richness records from replicated deployments of baited remote underwater video stations (BRUVS) and unbaited remote underwater video stations (UBRUVS) in shallow (<1 m) and deep (>1 m) water were compared with those obtained from using fyke nets, gillnets and beach seines. Maximum species richness (14 species) was achieved through a combination of conventional netting and camera‐based techniques. Chanos chanos was the only species not recorded on camera, whereas Lutjanus argentimaculatus, Selenotoca multifasciata and Gerres filamentosus were recorded on camera in all three waterholes but were not detected by netting. BRUVSs and UBRUVSs provided versatile techniques that were effective at a range of depths and microhabitats. It is concluded that cameras warrant application in aquatic areas of high conservation value with high visibility. Non‐extractive video methods are particularly desirable where threatened species are a focus of monitoring or might be encountered as by‐catch in net meshes.     

The Focal Adhesion: A Regulated Component of Aortic Stiffness

Increased aortic stiffness is an acknowledged predictor and cause of cardiovascular disease. The sources and mechanisms of vascular stiffness are not well understood, although the extracellular matrix (ECM) has been assumed to be a major component. We tested here the hypothesis that the focal adhesions (FAs) connecting the cortical cytoskeleton of vascular smooth muscle cells (VSMCs) to the matrix in the aortic wall are a component of aortic stiffness and that this component is dynamically regulated. First, we examined a model system in which magnetic tweezers could be used to monitor cellular cortical stiffness, serum-starved A7r5 aortic smooth muscle cells. Lysophosphatidic acid (LPA), an activator of myosin that increases cell contractility, increased cortical stiffness. A small molecule inhibitor of Src-dependent FA recycling, PP2, was found to significantly inhibit LPA-induced increases in cortical stiffness, as well as tension-induced increases in FA size. To directly test the applicability of these results to force and stiffness development at the level of vascular tissue, we monitored mouse aorta ring stiffness with small sinusoidal length oscillations during agonist-induced contraction. The alpha-agonist phenylephrine, which also increases myosin activation and contractility, increased tissue stress and stiffness in a PP2- and FAK inhibitor 14-attenuated manner. Subsequent phosphotyrosine screening and follow-up with phosphosite-specific antibodies confirmed that the effects of PP2 and FAK inhibitor 14 in vascular tissue involve FA proteins, including FAK, CAS, and paxillin. Thus, in the present study we identify, for the first time, the FA of the VSMC, in particular the FAK-Src signaling complex, as a significant subcellular regulator of aortic stiffness and stress.