Limited Replication Research Articles

Limited Dengue Virus Replication in Field-Collected Aedes aegypti Mosquitoes Infected with Wolbachia

IntroductionDengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus.Methodology/Principal FindingsField collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes.Conclusions/SignificanceOur results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.

Recombinant Strain A/HK/Otar/6:2/2010 (H3N8) for Development of a Live Intranasal Equine Influenza Vaccine

Recombinant cold-adapted strain A/HK/Otar/6:2/2010 (H3N8) constructed by the method of classic genetic reassortment, inherited the hemagglutinin and neuraminidase genes from the epizootic A/equine/Otar/764/2007 (Н3N8) virus and genes of nonglycosylated proteins from attenuation donor A/Hong Kong/1/68/162/35 (H3N2). The resulted virus was of cold-adapted (efficient growth at 25°C) and temperature-sensitive (restricted replication at 39°C) phenotype. Like the parental virus of subtype H3N2, the resulted reassortant was attenuated, with limited replication in lungs of mice (1.75 lgEID50/mL) versus replication in turbinate (2.5 lgEID50/mL). Intranasal immunization of mice with A/HK/Otar/6:2/2010 (H3N8) reassortant did not induce animal weight reduction in contrast to epizootic A/equine/Otar/764/2007 (Н3N8) virus.

Antiviral Autophagy Restricts Rift Valley Fever Virus Infection and Is Conserved from Flies to Mammals

Autophagy has been implicated as a component of host defense, but the significance of antimicrobial autophagy invivo and the mechanism by which it is regulated during infection are poorly defined. Here we found that antiviral autophagy was conserved in flies and mammals during infection with Rift Valley fever virus (RVFV), a mosquito-borne virus that causes disease in humans and livestock. In Drosophila, Toll-7 limited RVFV replication and mortality through activation of autophagy. RVFV infection also elicited autophagy in mouse and human cells, and viral replication was increased in the absence of autophagy genes. The mammalian Toll-like receptor adaptor, MyD88, was required for anti-RVFV autophagy, revealing an evolutionarily conserved requirement for pattern-recognition receptors in antiviral autophagy. Pharmacologic activation of autophagy inhibited RVFV infection in mammalian cells, including primary hepatocytes and neurons. Thus, autophagy modulation might be an effective strategy for treating RVFV infection, which lacks approved vaccines and therapeutics.

Abstract P1-05-05: Epithelial cell reprogramming assay reveals inter-individual heterogeneity in stem/basal and progenitor cell profile of the normal breast

Abstract Objectives: The majority of our understanding of mammary epithelial cell hierarchy is from mouse models. In addition, due to limited replication potential, primary cells of either mouse or human origin have rarely been used for functional studies. In this study, we took advantage of the recently developed epithelial cell-reprogramming assay that utilizes irradiated fibroblasts and ROCK inhibitor to characterize breast epithelial cells from individuals with varying degree of breast cancer risk. Methods: Primary breast tissues were collected at the time of surgery and cultured either immediately or at convenient time after freezing in liquid nitrogen with ROCK inhibitor. Flow cytometry sorting was employed to fractionate and propagate stem-like/basal (CD49f+/EpCAM-), luminal progenitor (CD49f+/EpCAM+), and mature (CD49f-/EpCAM+) cell enriched fractions. Similar fractionation/characterization was performed using CD133, CD10, CD271, EGFR and ALDEFLUOR. Results: Inter-individual variability was observed in proportion of CD49f+/EpCAM+, CD49-/EpCAM+ and CD133+ cells. Epithelial cells from a patient considered high risk based on the history of hyperplasia as well as cells from BRCA1 and BRCA2 patients displayed unique CD49f and EpCAM staining pattern compared to cells isolated from adjoining “normal” tissue of two cancer patients. In addition, CD133+ population was higher in BRCA1 and BRCA2 mutant patients. Morphologically, cells isolated from BRCA1 patient displayed higher levels of cells with mesenchymal phenotype. Upon short-term culturing of cells under regular 2D culture without reprogramming conditions, cell surface marker profile remained unchanged suggesting that most of the cell surface marker profile is cell intrinsic. CD49f+/EpCAM-, CD49f+/EpCAMhigh, CD49f+/EpCAMmedium, CD49f-/EpCAM+, CD133+/EpCAM+, and CD10+/EpCAM+ cells were amicable for further propagation in culture. CD49f+/EpCAMmedium cells cultured for two weeks remained CD49f+/EpCAMmedium. Additional marker profiling including cancer stem cell markers is underway to further refine stem, progenitor, and mature cells. Conclusion: Ability to propagate distinct subpopulation of normal cells from patients with different breast cancer risk would allow investigation of the impact of cell type origin of cancer on the course of the disease including response to therapy and the development of in vitro assay for breast cancer risk assessment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-05-05.

Isolation of Cancer Stem Like Cells from Human Adenosquamous Carcinoma of the Lung Supports a Monoclonal Origin from a Multipotential Tissue Stem Cell

There is increasing evidence that many solid tumors are hierarchically organized with the bulk tumor cells having limited replication potential, but are sustained by a stem-like cell that perpetuates the tumor. These cancer stem cells have been hypothesized to originate from transformation of adult tissue stem cells, or through re-acquisition of stem-like properties by progenitor cells. Adenosquamous carcinoma (ASC) is an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes. The origin of these mixtures is unclear as squamous carcinomas are thought to arise from basal cells in the upper respiratory tract while adenocarcinomas are believed to form from stem cells in the bronchial alveolar junction. We have isolated and characterized cancer stem-like populations from ASC through application of selective defined culture medium initially used to grow human lung stem cells. Homogeneous cells selected from ASC tumor specimens were stably expanded in vitro. Primary xenografts and metastatic lesions derived from these cells in NSG mice fully recapitulate both the adenocarcinoma and squamous features of the patient tumor. Interestingly, while the CSLC all co-expressed cytokeratins 5 and 7, most xenograft cells expressed either one, or neither, with <10% remaining double positive. We also demonstrated the potential of the CSLC to differentiate to multi-lineage structures with branching lung morphology expressing bronchial, alveolar and neuroendocrine markers in vitro. Taken together the properties of these ASC-derived CSLC suggests that ASC may arise from a primitive lung stem cell distinct from the bronchial-alveolar or basal stem cells.

Inhibiting AKT Phosphorylation Employing Non-Cytotoxic Anthraquinones Ameliorates TH2 Mediated Allergic Airways Disease and Rhinovirus Exacerbation

BackgroundSevere asthma is associated with T helper (TH) 2 and 17 cell activation, airway neutrophilia and phosphoinositide-3-kinase (PI3K) activation. Asthma exacerbations are commonly caused by rhinovirus (RV) and also associated with PI3K-driven inflammation. Anthraquinone derivatives have been shown to reduce PI3K-mediated AKT phosphorylation in-vitro.ObjectiveTo determine the anti-inflammatory potential of anthraquinones in-vivo. MethodsBALB/c mice were sensitized and challenged with crude house dust mite extract to induce allergic airways disease and treated with mitoxantrone and a novel non-cytotoxic anthraquinone derivative. Allergic mice were also infected with RV1B to induce an exacerbation.ResultsAnthraquinone treatment reduced AKT phosphorylation, hypoxia-inducible factor-1α and vascular endothelial growth factor expression, and ameliorated allergen- and RV-induced airways hyprereactivity, neutrophilic and eosinophilic inflammation, cytokine/chemokine expression, mucus hypersecretion, and expression of TH2 proteins in the airways. Anthraquinones also boosted type 1 interferon responses and limited RV replication in the lung.ConclusionNon-cytotoxic anthraquinone derivatives may be of therapeutic benefit for the treatment of severe and RV-induced asthma by blocking pro-inflammatory pathways regulated by PI3K/AKT.

Transcriptional coupling of DNA repair in sporulating Bacillus subtilis cells

In conditions of halted or limited genome replication, like those experienced in sporulating cells of Bacillus subtilis, a more immediate detriment caused by DNA damage is altering the transcriptional programme that drives this developmental process. Here, we report that mfd, which encodes a conserved bacterial protein that mediates transcription-coupled DNA repair (TCR), is expressed together with uvrA in both compartments of B. subtilis sporangia. The function of Mfd was found to be important for processing the genetic damage during B. subtilis sporulation. Disruption of mfd sensitized developing spores to mitomycin-C (M-C) treatment and UV-C irradiation. Interestingly, in non-growing sporulating cells, Mfd played an anti-mutagenic role as its absence promoted UV-induced mutagenesis through a pathway involving YqjH/YqjW-mediated translesion synthesis (TLS). Two observations supported the participation of Mfd-dependent TCR in spore morphogenesis: (i) disruption of mfd notoriously affected the efficiency of B. subtilis sporulation and (ii) in comparison with the wild-type strain, a significant proportion of Mfd-deficient sporangia that survived UV-C treatment developed an asporogenous phenotype. We propose that the Mfd-dependent repair pathway operates during B. subtilis sporulation and that its function is required to eliminate genetic damage from transcriptionally active genes.

RON Receptor Tyrosine Kinase, a Negative Regulator of Inflammation, Is Decreased during Simian Immunodeficiency Virus–Associated Central Nervous System Disease

Expressed on tissue-resident macrophages, the receptor tyrosine kinase, recepteur d'orgine nantais (RON), functions to maintain inflammation homeostasis by activating genes that promote wound repair and resolve inflammation while repressing genes that perpetuate tissue damage and cell death. Chronic HIV-1 infection is associated with dysregulated inflammation, and we hypothesize that diminished RON expression contributes to the development of end organ diseases such as HIV-1-associated CNS disease. To explore RON function in vivo, we used CNS tissue from a well-characterized SIV macaque model and examined the temporal regulation of RON in the brain during the course of infection. Following prolonged SIV infection, RON expression was inversely correlated with the development of CNS disease; RON was maintained in animals that did not develop CNS lesions and was reduced in SIV-infected macaques that demonstrated moderate to severe inflammatory lesions. Arginase-1 expression was reduced in the brain during late infection, whereas expression of the inflammatory genes, IL-12p40 and TNF-α, was elevated. To validate a role for RON in regulating HIV-1 in primary cells, we used human tissue-resident macrophages isolated from tonsil as a tractable cell model. RON signaling in tissue-resident macrophages, both ligand dependent and independent, limited HIV-1 replication. Furthermore, prolonged HIV-1 infection in vitro resulted in downregulation of RON. We propose a model in which, following chronic HIV-1 infection in the brain, RON expression is decreased, genes that quell inflammation are repressed, and inflammatory mediators are induced to promote tissue inflammation.

Genetic analysis of poliovirus strains isolated from sewage in Poland

The study describes genetic characterization of poliovirus (PV) strains isolated from sewage samples in Poland. The analyses were performed for the detection of any putative polio revertants and recombinants in three genomic regions by sequencing analysis. Thirty-six strains were analyzed. The analyzed strains were identified by neutralization assay as 7 strains of serotype P1, 10 strains of serotype P2, and 19 strains of serotype P3. Sewage isolates were sequenced in 5'UTR, VP1, and 3D genomic regions. All detected PVs were classified as vaccine strains on the basis of VP1 sequence. Mutational differences in the VP1 sequences of isolated viruses ranged from 0.0% to 0.4%, indicating a limited replication period. The genetic analysis of the 3D region showed that some strains have recombinant genomes. Nine strains were found as dipartite recombinants (seven strains--S3/S2, one strain--S2/S1, one strain--S3/S1), while one strain was found as tripartite recombinant (S3/S2/S1). No recombinants with non-PV enteroviruses were identified. None of wild-type PVs or vaccine-derived polioviruses (VDPVs) were detected. This study showed the absence of wild or VDPV circulation in the country and demonstrated the usefulness of environmental surveillance in addition to acute flaccid paralysis (AFP) surveillance in support of polio eradication initiatives.

Human H7N9 Influenza A Viruses Replicate in Swine Respiratory Tissue Explants

Recently, novel H7N9 influenza viruses have caused an unprecedented outbreak in humans. Pigs are an important intermediate host for influenza; thus, we assessed the replication ability of three human H7N9 viruses (A/Anhui/1/2013, A/Shanghai/1/2013, A/Shanghai/2/2013) in swine tissue explants. All viruses tested replicated efficiently in explants from tracheas and bronchi, with limited replication in alveolar cells. Swine respiratory tissue explants can serve as an efficient model for screening replication potential of newly emerging H7N9 viruses.

Preliminary Characterization of Physical Properties of Cross-Laminated-Timber (CLT) Panels for Hygrothermal Modelling

Abstract Cross-laminated-timber (CLT) panels are a type of relatively new wood-based structural panel, typically manufactured by laminating three or more layers of lumber together, with each layer rotated 90° relative to the neighbouring layers. This study explored preliminary assessment of the physical properties of a range of specimens for the purpose of initiating generating material property data for hygrothermal simulation of CLT building enclosure assemblies. Three types of five-layer CLT panels (nominally 130 mm thick) were made with Canadian softwood species, including spruce-pine-fir (SPF) from Eastern Canada and British Columbia, and hem fir from British Columbia, all glued with emulsion polymer isocyanate (EPI) as the adhesive applied between neighbouring layers. One type of three-layer CLT (nominally 90 mm thick) commercially manufactured in Europe using European spruce, with polyurethane adhesive, was also tested. Physical properties including density, thermal conductivity, liquid-water absorption, water-vapor permeability, sorption (moisture storage function), and air permeability were measured, mostly based on established international standards. To assess the impact of the adhesive on the hygrothermal properties of CLTs, test specimens used for most tests included at least one layer of adhesive, except those used for sorption tests, which used very small specimens. The properties were expected to mostly reflect the properties of the lumber used for CLT manufacturing. Variations in properties were found among these different CLT specimens; however, it was generally concluded that the differences caused by wood species and manufacturing methods would not be large enough to cause considerable differences in the hygrothermal properties or significantly impact the outcomes of hygrothermal simulation. With small-scale testing and very limited replication this study showed that the water-vapor permeability of CLT generally increased with an increase in relative humidity (RH), and the air permeance of the CLT specimens without visible gaps and checks was negligible.

Limited change in the diversity and structure of subtidal communities over four decades

A unique archive of photographs from 1969 to 1974 permitted a test of the hypothesis that the diversity and composition of contemporary epilithic communities on subtidal rock walls in the San Juan Islands, WA, USA, has changed over 40 years. Notably, the richness and diversity of sessile taxa was significantly higher in 2008–2011. Furthermore, the multivariate community structure of sessile and mobile taxa differed between the historic and modern eras. Historic communities were characterized by a high percent cover of bare rock and non-calcified algal crusts, consistent with the effects of grazing by chitons and urchins. The rate of sessile community turnover, an index less susceptible to spatial sampling artifacts than richness or diversity, was not significantly different between the two eras. Together with the naturally high spatial variability in these epilithic communities, and the limited replication of historic quadrats, we interpret cautiously the data as evidence of limited change despite a clear shift in temperature and predator (fish) guild composition. The lack of substantial change in rock wall communities may be due in part to their vertical topography, which limits physical disturbance and the preemption of space by weedy algae, two processes that are often associated with “phase-shifts” in other marine ecosystems.

Enhancement of systemic tumor immunity for squamous cell carcinoma cells by an oncolytic herpes simplex virus

RH2 is a neurovirulent γ134.5 gene-deficient herpes simplex virus type 1 (HSV-1) with a lytic ability in human squamous cell carcinoma (SCC) cells; it is related to spontaneously occurring HSV-1 mutant HF10. The effect of RH2 on SCC was examined using a syngeneic C3H mouse model. After infection of mouse SCCVII cells with RH2, cell viability was decreased at first, but recovered by prolonged culture, indicating the limited replication of RH2. The antitumor ability of RH2 was examined using a bilateral SCCVII tumor model. The growth of the RH2-injected tumors was suppressed compared with that of phosphate-buffered saline-injected tumors. Moreover, the growth of contralateral tumor of RH2-treated mice was also suppressed significantly. The splenocytes of C3H mice treated with RH2 lysed more SCCVII cells than NFSaY83 cells and YAC-1 cells. The cytotoxicity of the splenocytes on SCCVII cells was significantly greater than that of splenocytes from tumor-bearing mice. Removal of CD8(+) T cells from splenocytes decreased their cell killing activity remarkably. The antitumor effect of RH2 on SCCVII xenografts in nude mice was not demonstrated. These results indicate that RH2 exhibited a suppressive effect on mouse SCC, even if the replication of RH2 was limited. This is ascribed to the ability of RH2 to enhance existing tumor-specific cytotoxic T lymphocyte activity.

An M2e-based synthetic peptide vaccine for influenza A virus confers heterosubtypic protection from lethal virus challenge

BackgroundVaccination is considered as the most effective preventive method to control influenza. The hallmark of influenza virus is the remarkable variability of its major surface glycoproteins, HA and NA, which allows the virus to evade existing anti-influenza immunity in the target population. So it is necessary to develop a novel vaccine to control animal influenza virus. Also we know that the ectodomain of influenza matrix protein 2 (M2e) is highly conserved in animal influenza A viruses, so a vaccine based on the M2e could avoid several drawbacks of the traditional vaccines. In this study we designed a novel tetra-branched multiple antigenic peptide (MAP) based vaccine, which was constructed by fusing four copies of M2e to one copy of foreign T helper (Th) cell epitope, and then investigated its immune responses.ResultsOur results show that the M2e-MAP induced strong M2e-specific IgG antibody,which responses following 2 doses immunization in the presence of Freunds’ adjuvant. M2e-MAP vaccination limited viral replication substantially. Also it could attenuate histopathological damage in the lungs of challenged mice and counteracted weight loss. M2e-MAP-based vaccine protected immunized mice against the lethal challenge with PR8 virus.ConclusionsBased on these findings, M2e-MAP-based vaccine seemed to provide useful information for the research of M2e-based influenza vaccine. Also it show huge potential to study vaccines for other similarly viruses.

Increased S-Nitrosylation and Proteasomal Degradation of Caspase-3 during Infection Contribute to the Persistence of Adherent Invasive Escherichia coli (AIEC) in Immune Cells

Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn’s disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients.

The facultative intracellular pathogen Cryptococcus neoformans is susceptible to in vivo clearance by alveolar macrophages (P4211)

Abstract Cryptococcus is a yeast commonly found in soil, decaying wood and bird excreta. Normally characterized as an opportunistic human infection, cryptococcosis is a leading cause of meningitis and death in HIV/AIDS patients in Sub-Saharan Africa. Recent Cryptococcus gattii outbreaks within healthy, immunocompetent populations in the Pacific Northwest are calling this characterization into question. Recent studies support the ability of Cryptococcus to replicate intracellularly, particularly in macrophages, leading to classification as a facultative intracellular pathogen. However, in vivo analysis of this capacity for intracellular replication in the context of a disease model for pulmonary infection has been lacking. Here we employ a GFP expressing strain of Cryptococcus neoformans and present novel dye labeling strategies to assess whether intracellular replication is productive in a mouse model of pulmonary cryptococcosis. We show that, while limited intracellular replication occurs, cryptococcal clearance by alveolar macrophages within the first forty-eight hours of pulmonary infection predominates. Cryptococcus may correctly be characterized as a facultative intracellular pathogen; however, alveolar macrophages possess the capacity to clear this pathogen, keeping early infection in check. Our finding, showing the importance of the alveolar macrophage in early cryptococcal clearance, opens the possibility of novel treatment strategies for a clinically important human pathogen.

Targeted Overexpression of CKI-Insensitive Cyclin-Dependent Kinase 4 Increases Functional β-Cell Number Through Enhanced Self-Replication in Zebrafish

β-Cells of the islet of Langerhans produce insulin to maintain glucose homeostasis. Self-replication of β-cells is the predominant mode of postnatal β-cell production in mammals, with about 20% of rodent β cells dividing in a 24-hour period. However, replicating β-cells are rare in adults. Induction of self-replication of existing β-cells is a potential treatment for diabetes. In zebrafish larvae, β-cells rarely self-replicate, even under conditions that favor β-cell genesis such overnutrition and β-cell ablation. It is not clear why larval β-cells are refractory to replication. In this study, we tested the hypothesis that insufficient activity of cyclin-dependent kinase 4 may be responsible for the low replication rate by ectopically expressing in β-cells a mutant CDK4 (CDK4(R24C)) that is insensitive to inhibition by cyclin-dependent kinase inhibitors. Our data show that expression of CDK4(R24C) in β-cells enhanced β-cell replication. CDK4(R24C) also dampened compensatory β-cell neogenesis in larvae and improved glucose tolerance in adult zebrafish. Our data indicate that CDK4 inhibition contributes to the limited β-cell replication in larval zebrafish. To our knowledge, this is the first example of genetically induced β-cell replication in zebrafish.

The HC-Pro and P3 Cistrons of an Avirulent Soybean mosaic virus Are Recognized by Different Resistance Genes at the Complex Rsv1 Locus

The complex Rsv1 locus in soybean plant introduction (PI) 'PI96983' confers extreme resistance (ER) against Soybean mosaic virus (SMV) strain N but not SMV-G7 and SMV-G7d. Both the SMV helper-component proteinase (HC-Pro) and P3 cistrons can serve as avirulence factors recognized by Rsv1. To understand the genetics underlying recognition of the two cistrons, we have utilized two soybean lines (L800 and L943) derived from crosses between PI96983 (Rsv1) and Lee68 (rsv1) with distinct recombination events within the Rsv1 locus. L800 contains a single PI96983-derived member (3gG2) of an Rsv1-associated subfamily of nucleotide-binding leucine-rich repeat (NB-LRR) genes. In contrast, although L943 lacks 3gG2, it contains a suite of five other NB-LRR genes belonging to the same family. L800 confers ER against SMV-N whereas L943 allows limited replication at the inoculation site. SMV-N-derived chimeras containing HC-Pro from SMV-G7 or SMV-G7d gained virulence on L943 but not on L800 whereas those with P3 replacement gained virulence on L800 but not on L943. In reciprocal experiments, SMV-G7- and SMV-G7d-derived chimeras with HC-Pro replacement from SMV-N lost virulence on L943 but retained virulence on L800 whereas those with P3 replacement lost virulence on L800 while remaining virulent on L943. These data demonstrate that distinct resistance genes at the Rsv1 locus, likely belonging to the NB-LRR class, mediate recognition of HC-Pro and P3.

Cold front induced changes on the Florida panhandle shelf during October 2008

A significant step transition between seasonally stratified and destratified hydrographic conditions occurred during an October 2008 cruise to the Florida Panhandle Shelf along a cross-shelf transect that was sampled before and after a cold front passed through the area. Meteorological measurements from nearby ocean and land-based stations characterized the event. Cross-shelf continuous Acrobat profiles and discrete CTD stations characterized water column hydrographic patterns, while mid-shelf multicorer and box corer samples characterized sediment texture and nutrients. Water samples collected from selected depths biased toward the sediment interface were analyzed for nutrient content and phytoplankton community composition. Pre-front, the cross-shelf water column exhibited vertical stratification with complex temperature and salinity patterns. A prominent near-bottom chlorophyll a maximum of ∼1.5μgL−1 between the 25–35m isobaths occurred with the 1% light level at ∼18m depth and a near-bottom nitrate+nitrite (NO3−+NO2−) maximum >3μM between the 30–40m isobaths. HPLC-determined phytoplankton community composition in the near-bottom chlorophyll a maximum consisted of gyroxanthin-containing dinoflagellates (Karenia brevis) and less abundant diatoms, both verified by FlowCAM analysis, mixed with detectable cryptophytes and chlorophytes. Sediment trends based on limited core replicates suggested the sediments were a potential source of nutrients to near-bottom populations of K. brevis and that shell hash could provide abundant pore space for K. brevis incursions. Between the 40–50m isobaths, diatoms, cryptophytes and chlorophytes dominated near-bottom, gyroxanthin-containing dinoflagellates and prasinophytes occurred throughout the water column, and cyanophytes dominated near-surface. Post-front, the cross-shelf water column exhibited destratification with temperature and salinity increasing offshore. A chlorophyll a maximum of ∼0.75μg Chl aL−1 left the sediment between 25–35m isobaths and extended offshore especially in the lower water column with the 1% light level at ∼15m depth and NO3−+NO2− concentrations ∼2μM to the 60m isobath. HPLC-determined phytoplankton community composition of the offshore plume retained the signature of gyroxanthin-containing dinoflagellates and chlorophytes. Between the 30–50m isobaths, prasinophytes increased in the lower water column, while cyanophytes increased at all depths across the shelf. The observed step transition from stratification to destratification on the Florida Panhandle Shelf contributed to altered phytoplankton community patterns in response to predominant downwelling favorable winds. Pre-front, K. brevis cells were broadly distributed cross-shelf, but concentrated near-bottom between the 25–35m isobaths and staged for prolific bloom seeding in response to the upwelling favorable west winds more typical of spring-summer. Post-front, K. brevis cells were mixed throughout the mid-shelf water column and were staged for diffuse bloom seeding in response to either the downwelling or upwelling favorable winds occurring fall-winter. Cyanophytes located predominantly near-surface offshore pre-front, were ubiquitous cross-shelf and more closely associated with K. brevis post-front.

Spatial arrangement of legionella colonies in intact biofilms from a model cooling water system.

There is disagreement among microbiologists about whether Legionella requires a protozoan host in order to replicate. This research sought to determine where in biofilm Legionellae are found and whether all biofilm associated Legionella would be located within protozoan hosts. While it is accepted that Legionella colonizes biofilm, its life cycle and nutritional fastidiousness suggest that Legionella employs multiple survival strategies to persist within microbial systems. Fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) demonstrated an undulating biofilm surface architecture and a roughly homogenous distribution of heterotrophic bacteria with clusters of protozoa. Legionella displayed 3 distinct spatial arrangements either contained within or directly associated with protozoa, or dispersed in loosely associated clusters or in tightly packed aggregations of cells forming dense colonial clusters. The formation of discreet clusters of tightly packed Legionella suggests that colony formation is influenced by specific environmental conditions allowing for limited extracellular replication. This work represents the first time that an environmentally representative, multispecies biofilm containing Legionella has been fluorescently tagged and Legionella colony morphology noted within a complex microbial system.