Variation of genotype and prevalence of Noro virus in U.S. students in Mexico with Traveler's Diarrhea, summer of 2004 and 2005

Noro virus, a positive single stranded RNA virus has been identified as a major etiologic agent in food borne gastroenteritis and diarrheal diseases. The emergence of this organism as a major non-bacterial cause in such outbreaks is partly due to the improved diagnostic tools like Reverse Transcription Polymerase chain reaction (RTPCR) that enable its detection. Noro virus accounts for nearly 96% of non-bacterial gastroenteritis outbreaks in US (1). Travelers' Diarrhea (TD) has remained a constant public health risk in the developed nations for decades and bacteria like Entero toxigenic Escherichia coli, Entero aggregative Escherichia coli have been described as the main etiologic agents for TD (2-4). A possible viral contribution to TD has been discovered in two studies (5, 6). The current study was designed to determine the prevalence of Noro virus in a population of 107 US students with TD acquired in Mexico in 2005 and to compare the prevalence to the prevalence of Noro virus in a similar study done in 2004. This study involved the testing of clinical stool specimens from 107 subjects in 2005 for the presence of Noro virus using RTPCR. The prevalence of Noro virus in 2004 used for comparison to 2005 data was obtained from published data (5). All subjects were recruited as TD subjects in a randomized, double-blinded clinical trial comparing a standard three day dosing of Rifaximin with and without an anti motility drug Loperamide. The prevalence of Noro virus geno group I was similar in both years, but geno group II prevalence differed across the two years (p = 0.003). This study finding suggests that the prevalence of Noro virus geno groups varies with time even within a specific geographic location. This study emphasizes the need for further systematic epidemiologic studies to determine the molecular epidemiology and the prevalence patterns of different geno groups of this virus. These are essential to planning and implementation of public health measures to lessen the burden of TD due to Noro virus infection among US travelers. ^

Microplankton abundance and biomass in the Eastern Pacific

Particles of detritus were counted by size-groups and microplankton cells in samples stained with acid fuchsin and acridine orange. Data were obtained for eutrophic and oligotrophic waters. Seston in the eutrophic layer of eutrophic waters consists of 22-65% phytoplankton, 3-18% microzooplankton, and 32-65% detritus; in oligotrophic waters - of 3-7% phytoplankton, 1-5% microzooplankton, and 92-97% detritus. Amount of detritus in seston increases with depth up to 4.4 µg C/l (sigma = 1.48) at 500-4000 m. Microplankton biomass in deep water contains mostly olive-green cells and bacteria; no microzooplankton <200 µm long was found below 200 m. Aggregates 10-50 µm in diameter and fragments of organisms 50-200 µm long were dominant by weight among detrital particles. No discernible associations of microorganisms with detrital particles were observed.

Elemental contents, non-carbonate, fatty acids and alcohols analysis from different Holes of IODP Expedition 310

During IODP Expedition 310 (Tahiti Sea Level), drowned Pleistocene-Holocene barrier-reef terraces were drilled on the slope of the volcanic island. The deglacial reef succession typically consists of a coral framework encrusted by coralline algae and later by microbialites; the latter make up < 80% of the rock volume. Lipid biomarkers were analyzed in order to identify organisms involved in reef-microbialite formation at Tahiti, as the genesis of deglacial microbialites and the conditions favoring their formation are not fully understood. Sterols plus saturated and monounsaturated short-chain fatty acids predominantly derived from both marine primary producers (algae) and bacteria comprise 44 wt% of all lipids on average, whereas long-chain fatty acids and long-chain alcohols derived from higher land plants represent an average of only 24 wt%. Bacterially derived mono-O-alkyl glycerol ethers (MAGEs) and branched fatty acids (10-Me-C16:0; iso- and anteiso-C15:0 and -C17:0) are exceptionally abundant in the microbial carbonates (average, 19 wt%) and represent biomarkers of intermediate-to-high specificity for sulfate-reducing bacteria. Both are relatively enriched in 13C compared to eukaryotic lipids. No lipid biomarkers indicative of cyanobacteria were preserved in the microbialites. The abundances of Al, Si, Fe, Mn, Ba, pyroxene, plagioclase, and magnetite reflect strong terrigenous influx with Tahitian basalt as the major source. Chemical weathering of the basalt most likely elevated nutrient levels in the reefs and this fertilization led to an increase in primary production and organic matter formation, boosting heterotrophic sulfate reduction. Based on the observed biomarker patterns, sulfate-reducing bacteria were apparently involved in the formation of microbialites in the coral reefs off Tahiti during the last deglaciation.

Occurrence, abundance and biomass of photo- and heterotrophic plankton in Norwegian coastal waters

The purpose of the present study was to explore the composition and variation of the pico-, nano- and micro-plankton communities in Norwegian coastal waters and Skagerrak, and the co-occurrence of bacteria and viruses. Samples were collected along three cruise transects from Jaeren, Lista and Oksoy on the south coast of Norway and into the North Sea and Skagerrak. We also followed a drifting buoy for 55 h in Skagerrak in order to observe diel variations. Satellite ocean color images (SeaWiFS) of the chlorophyll a (chl a) distribution compared favorably to in situ measurements in open waters, while closer to the shore remote sensing chl a data was overestimated compared to the in situ data. Using light microscopy, we identified 49 micro- and 15 nanoplankton sized phototrophic forms as well as 40 micro- and 12 nanoplankton sized heterotrophic forms. The only picoeukaryote (0.2-2.0 µm) we identified was Resultor micron (Pedinophyceae). Along the transects a significant variation in the distribution and abundance of different plankton forms were observed, with Synechococcus spp and autotrophic picoeukaryotes as the most notable examples. There was no correlation between viruses and chl a, but between viruses and bacteria, and between viruses and some of the phytoplankton groups, especially the picoeukaryotes. Moreover, there was a negative correlation between nutrients and small viruses (Low Fluorescent Viruses) but a positive correlation between nutrients and large viruses (High Fluorescent Viruses). The abundance of autotrophic picoplankton, bacteria and viruses showed a diel variation in surface waters with higher values around noon and late at night and lower values in the evening. Synechococcus spp were found at 20 m depth 25-45 nautical miles from shore apparently forming a bloom that stretched out for more than 100 nautical miles from Skagerrak and up the south west coast of Norway. The different methods used for assessing abundance, distribution and diversity of microorganisms yielded complementary information about the plankton community. Flow cytometry enabled us to map the distribution of the smaller phytoplankton forms, bacteria and viruses in more detail than has been possible before but detection and quantification of specific forms (genus or species) still requires taxonomic skills, molecular analysis or both.

Results from field observations and in situ 13C feeding experiments in the Nazaré Canyon conducted during RRS James Cook 10 cruise Leg 2

Submarine canyon systems provide a heterogeneous habitat for deep-sea benthos in terms of topography, hydrography, and the quality and quantity of organic matter present. Enhanced meiofauna densities as found in organically enriched canyon sediments suggest that nematodes, as the dominant metazoan meiobenthic taxon, may play an important role in the benthic food web of these sediments. Very little is known about the natural diets and trophic biology of deep-sea nematodes, but enrichment experiments can shed light on nematode feeding selectivity and trophic position. An in-situ pulse-chase experiment (Feedex) was performed in the Nazaré Canyon on the Portuguese margin in summer 2007 to study nematode feeding behaviour. 13C-labelled diatoms and bacteria were added to sediment cores which were then sampled over a 14-day period. There was differential uptake by the nematode community of the food sources provided, indicating selective feeding processes. 13C isotope results revealed that selective feeding was less pronounced at the surface, compared to the sediment subsurface. This was supported by a higher trophic diversity in surface sediments compared to the subsurface, implying that more food items may be used by the nematode community at the sediment surface. Predatory and scavenging nematodes contributed relatively more to biomass than other feeding types and can be seen as key contributors to the nematode food web at the canyon site. Non-selective deposit feeding nematodes were the dominant trophic group in terms of abundance and contributed substantially to total nematode biomass. The high levels of 'fresh' (bioavailable) organic matter input and moderate hydrodynamic disturbance of the canyon environment lead to a more complex trophic structure in canyon nematode communities than that found on the open continental slope, and favours predator/scavengers and non-selective deposit feeders.

Concentration of dissolved phosphates and total phosphorus in seawater near the Cape Utrish, Black Sea

Dynamics of growth of natural phytoplankton and bacterioplankton in deep seawater upwelled to the upper sea layer were studied. Seawater from the lower part of the aerobic zone of the Black Sea was shown to have high bio-productive potential and can be used as an environment for algae and bacteria cultivation.

Ocean acidification weakens the immune response of blood clam through hampering the NF-kappa beta and toll-like receptor pathways

The impact of pCO2 driven ocean acidification on marine bivalve immunity remains poorly understood. To date, this impact has only been investigated in a few bivalve species and the underlying molecular mechanism remains unknown. In the present study, the effects of the realistic future ocean pCO2 levels (pH at 8.1, 7.8, and 7.4) on the total number of haemocyte cells (THC), phagocytosis status, blood cell types composition, and expression levels of twelve genes from the NF-kappa beta signaling and toll-like receptor pathways of a typical bottom burrowing bivalve, blood clam (Tegillarca granosa), were investigated. The results obtained showed that while both THC number and phagocytosis frequency were significantly reduced, the percentage of red and basophil granulocytes were significantly decreased and increased, respectively, upon exposure to elevated pCO2. In addition, exposure to pCO2 acidified seawater generally led to a significant down-regulation in the inducer and key response genes of NF-kappa beta signaling and toll-like receptor pathways. The results of the present study revealed that ocean acidification may hamper immune responses of the bivalve T. granosa which subsequently render individuals more susceptible to pathogens attacks such as those from virus and bacteria.

Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms

Protease inhibitors from plants have been involved in defence mechanisms against pests and pathogens. Phytocystatins and trypsin/α-amylase inhibitors are two of the best characterized protease inhibitor families in plants. In barley, thirteen cystatins (HvCPI-1 to 13) and the BTI-CMe trypsin inhibitor have been previously studied. Their capacity to inhibit pest digestive proteases, and the negative in vivo effect caused by plants expressing these inhibitors on pests support the defence function of these proteins. Barley cystatins are also able to inhibit in vitro fungal growth. However, the antifungal effect of these inhibitors in vivo had not been previously tested. Moreover, their in vitro and in vivo effect on plant pathogenous bacteria is still unknown. In order to obtain new insights on this feature, in vitro assays were made against different bacterial and fungal pathogens of plants using the trypsin inhibitor BTI-CMe and the thirteen barley cystatins. Most barley cystatins and the BTI-CMe inhibitor were able to inhibit mycelial growth but no bacterial growth. Transgenic Arabidopsis plants independently expressing the BTI-CMe inhibitor and the cystatin HvCPI-6 were tested against the same bacterial and fungal pathogens. Neither the HvCPI-6 expressing transgenic plants nor the BTI-CMe ones were more resistant to plant pathogen fungi and bacteria than control Arabidopsis plants. The differences observed between the in vitro and in planta assays against phytopathogenic fungi are discussed

Identification and characterization of a single-stranded DNA-binding protein from the archaeon Methanococcus jannaschii

Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria and eukarya. We report here the identification and characterization of the SSB of an archaeon, Methanococcus jannaschii. The M. jannaschii SSB (mjaSSB) has significant amino acid sequence similarity to the eukaryotic SSB, replication protein A (RPA), and contains four tandem repeats of the core single-stranded DNA (ssDNA) binding domain originally defined by structural studies of RPA. Homologous SSBs are encoded by the genomes of other archaeal species, including Methanobacterium thermoautotrophicum and Archaeoglobus fulgidus. The purified mjaSSB binds to ssDNA with high affinity and selectivity. The apparent association constant for binding to ssDNA is similar to that of RPA under comparable experimental conditions, and the affinity for ssDNA exceeds that for double-stranded DNA by at least two orders of magnitude. The binding site size for mjaSSB is ≈20 nucleotides. Given that RPA is related to mjaSSB at the sequence level and to Escherichia coli SSB at the structural level, we conclude that the SSBs of archaea, eukarya, and bacteria share a common core ssDNA-binding domain. This ssDNA-binding domain was presumably present in the common ancestor to all three major branches of life.

Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification

Macromolecular transport systems in bacteria currently are classified by function and sequence comparisons into five basic types. In this classification system, type II and type IV secretion systems both possess members of a superfamily of genes for putative NTP hydrolase (NTPase) proteins that are strikingly similar in structure, function, and sequence. These include VirB11, TrbB, TraG, GspE, PilB, PilT, and ComG1. The predicted protein product of tadA, a recently discovered gene required for tenacious adherence of Actinobacillus actinomycetemcomitans, also has significant sequence similarity to members of this superfamily and to several unclassified and uncharacterized gene products of both Archaea and Bacteria. To understand the relationship of tadA and tadA-like genes to those encoding the putative NTPases of type II/IV secretion, we used a phylogenetic approach to obtain a genealogy of 148 NTPase genes and reconstruct a scenario of gene superfamily evolution. In this phylogeny, clear distinctions can be made between type II and type IV families and their constituent subfamilies. In addition, the subgroup containing tadA constitutes a novel and extremely widespread subfamily of the family encompassing all putative NTPases of type IV secretion systems. We report diagnostic amino acid residue positions for each major monophyletic family and subfamily in the phylogenetic tree, and we propose an easy method for precisely classifying and naming putative NTPase genes based on phylogeny. This molecular key-based method can be applied to other gene superfamilies and represents a valuable tool for genome analysis.

Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene.

With global heavy metal contamination increasing, plants that can process heavy metals might provide efficient and ecologically sound approaches to sequestration and removal. Mercuric ion reductase, MerA, converts toxic Hg2+ to the less toxic, relatively inert metallic mercury (Hg0) The bacterial merA sequence is rich in CpG dinucleotides and has a highly skewed codon usage, both of which are particularly unfavorable to efficient expression in plants. We constructed a mutagenized merA sequence, merApe9, modifying the flanking region and 9% of the coding region and placing this sequence under control of plant regulatory elements. Transgenic Arabidopsis thaliana seeds expressing merApe9 germinated, and these seedlings grew, flowered, and set seed on medium containing HgCl2 concentrations of 25-100 microM (5-20 ppm), levels toxic to several controls. Transgenic merApe9 seedlings evolved considerable amounts of Hg0 relative to control plants. The rate of mercury evolution and the level of resistance were proportional to the steady-state mRNA level, confirming that resistance was due to expression of the MerApe9 enzyme. Plants and bacteria expressing merApe9 were also resistant to toxic levels of Au3+. These and other data suggest that there are potentially viable molecular genetic approaches to the phytoremediation of metal ion pollution.

Interactions of maize bushy stunt phytoplasma with the leafhopper vector, Dalbulus maidis (Delong and Wolcott) (Hemiptera: Cicadellidae) and associated microbiota

Phytoplasmas are bacteria with a persistent propagative transmission by insect vectors that generates direct and indirect interactions among them. In order to understand these interactions for maize bushy stunt phytoplasma (MBSP) and the leafhopper vector Dalbulus maidis (Hemiptera: Cicadellidae), two research lines were addressed. The first one aimed to determine the indirect effects of maize infection by MBSP on some biological and behavioral parameters of the vector, whereas a second line investigated direct interactions of the phytoplasma with D. maidis during its movement through the vector body following acquisition from plants, and associated microbiota. Indirect effects were investigated in choice experiments in which alighting and oviposition preferences by D. maidis were compared on healthy vs. MBSP-infected plants with variable incubation time (diseased plants with early and advanced symptoms, or still asymptomatic). Likewise, indirect effect of MBSP on the D. maidis biology was determined in two life table experiments in which the vector was reared on healthy vs. MBSP-infected plants expressing advanced disease symptoms or still asymptomatic. Choice experiments showed that alighting and oviposition preferences of D. maidis on MBSP-infected plants compared to healthy plants depend on the pathogen incubation period in the plant. The leafhopper preferred MBSP-infected plants over healthy ones during the asymptomatic phase of the disease, but rejected infected plants with advanced symptoms. The vector was able to acquire MBSP from asymptomatic infected plants shortly (3 days) after inoculation, but transmission efficiency increased when acquisition occurred at later stages of the pathogen incubation period (≥14 days) in the source plants and the test plants showed disease symptoms faster. These results suggest that MBSP modulates D. maidis preference for asymptomatic infected plants in the early stages of the crop, allowing rapid spread of this pathogen. Maize infection by the phytoplasma had a neutral effect on most life table parameters of D. maidis; a lower net reproductivity rate (Ro) was observed in the cohort reared on MBSP-infected plants with advanced symptoms, which was compensated to some extent by a higher sexual ratio. MBSP acquisition by all vector nymphal stadia was confirmed by PCR, and the pathogen as detected in both male and female reproductive organs. Concerning direct MBSP-vector interactions, transmission electron microscopy analyses showed phytoplasma-like cells in the midgut lumen, microvilli and epithelial cells, suggesting that MBSP enters the epithelium midgut through the microvilli wall. Within the epithelial cells, mitochondria and bacteria-like cells (possibly endosymbionts) were observed together with masses of phythoplasma-like cells. In the hemocoel, phytoplasma-like cells grouped into a matrix were also observed in association with bacteria-like cells similar to those observed in the midgut epithelium. Similar associations were found in the salivary gland. Interestingly, in-situ hybridization (FISH) technique revealed a variation in diversity and abundance of the microbiota in intestine and salivary glands of D. maidis adults over time after MBSP acquisition from plants. Sulcia sp., Cardinium sp. and eubacteria increased their abundance over time, whereas Rickettsia sp. decreased. The frequent association of the vector microbiota with the phytoplasma in some tissues of D. maidis suggests that endosymbiotic bacteria may play some role in MBSP-vector interactions.

Fluorescent Biological Aerosol Particles in Coastal Canada and the Link to Atmospheric Ice Nuclei

Bioaerosols are a subgroup of atmospheric aerosols and are often linked to the spread of human, animal and plant diseases. Bioaerosols also may play an indirect effect on environmental processes, including the formation of precipitation and alteration of the global climate through their role as nuclei for cloud droplet formation. Several types of biological organisms (e.g., fungi and bacteria) have been shown to be effective ice nuclei (IN) and cloud condensation nuclei (CCN). During 21 days in August 2013 we participated in a collaborative international campaign at a rural, coastal site near the village of Ucluelet on the west coast of Vancouver Island, British Columbia, Canada. The experiments were conducted as part of the NETCARE project (the NETwork on Climate and Aerosols: Addressing Key Uncertainties in Remote Canadian Environments), in part to examine cloud nuclei properties of marine aerosol. The study was conducted from a mobile trailer located approximately 100 m from the coast. A suite of aerosol instrumentation was operated for approximately one month. Key instruments utilized as a part of this thesis include the wideband integrated bioaerosol sensor (WIBS-4A) and the multiple orifice uniform deposition impactor (MOUDI) coupled with an off-line droplet freezing technique (DFT) for the measurement of ice nucleation activity of particles in immersion mode. The WIBS measures the concentration and properties of individual fluorescent particles suspended in the air, which can serve as a proxy for airborne biological particle content. Particles shown to be fluorescent by the WIBS instrument were divided into seven categories based on the pattern of fluorescence each particle exhibited in the three fluorescent channels. Results of the WIBS analysis show that the fluorescent particle concentration in the region correlated well with IN number. The fluorescent particle concentration correlated well with the number of particles shown to be ice active as a function of both particle size and freezing temperature. Correlations involving marine aerosols and marine biological activity indicate that the majority of IN measured at the coastal site likely are not from have marine sources.

Highly accurate RNA and DNA sequencing: Application to longstanding questions in aging and cancer

Thesis (Ph.D.)--University of Washington, 2016-06