Pyrosequencing-based characterization of gastrointestinal bacteria of Atlantic salmon (Salmo salar L.) within a commercial mariculture system.

AIMS: The relationship of Atlantic salmon gastrointestinal (GI) tract bacteria to environmental factors, in particular water temperature within a commercial mariculture system, was investigated. METHODS AND RESULTS: Salmon GI tract bacterial communities commercially farmed in south-eastern Tasmania were analysed, over a 13-month period across a standard commercial production farm cycle, using 454 16S rRNA-based pyrosequencing. Faecal bacterial communities were highly dynamic but largely similar between randomly selected fish. In postsmolt, the faecal bacteria population was dominated by Gram-positive fermentative bacteria; however, by midsummer, members of the family Vibrionaceae predominated. As fish progressed towards harvest, a range of different bacterial genera became more prominent corresponding to a decline in Vibrionaceae. The sampled fish were fed two different commercial diet series with slightly different protein, lipid and digestible energy level; however, the effect of these differences was minimal. CONCLUSIONS: The overall data demonstrated dynamic hind gut communities in salmon that were related to season and fish growth phases but were less influenced by differences in commercial diets used routinely within the farm system studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides understanding of farmed salmon GI bacterial communities and describes the relative impact of diet, environmental and farm factors.

Niche differentiation of ammonia-oxidising archaea (AOA) and bacteria (AOB) in response to paper and pulp mill effluent.

Sediment organic loading has been shown to affect estuarine nitrification and denitrification, resulting in changes to sediment biogeochemistry and nutrient fluxes detrimental to estuarine health. This study examined the effects of organic loading on nutrient fluxes and microbial communities in sediments receiving effluent from a paper and pulp mill (PPM) by applying microcosm studies and molecular microbial ecology techniques. Three sites near the PPM outfall were compared to three control sites, one upstream and two downstream of the outfall. The control sites showed coupled nitrification-denitrification with minimal ammonia release from the sediment. In contrast, the impacted sites were characterised by nitrate uptake and substantial ammonia efflux from the sediments, consistent with a decoupling of nitrification and denitrification. Analysis of gene diversity demonstrated that the composition of nitrifier communities was not significantly different at the impacted sites compared to the control sites; however, analysis of gene abundance indicated that whilst there was no difference in total bacteria, total archaea or ammonia-oxidising archaea (AOA) abundance between the control and impacted sites, there was a significant reduction in ammonia-oxidising bacteria (AOB) at the impacted sites. The results of this study demonstrate an effect of organic loading on estuarine sediment biogeochemistry and highlight an apparent niche differentiation between AOA and AOB.

Colonization and community dynamics of class Flavobacteria on diatom detritus in experimental mesocosms based on Southern Ocean seawater.

In order to better understand the ecology of microorganisms responsible for secondary production in the Southern Ocean the activity of Flavobacteria communities on diatom detritus in seawater mesocosms was investigated. Seawater was collected from different parts of the Southern Ocean including the Polar Front Zone (PFZ), ice-edge area of the Antarctic Zone (AZ), and a site in the AZ ice pack. Detritus from the cosmopolitan marine diatom Nitzschia closterium Ehrenberg was resuspended in mesocosms containing seawater filtered to remove particulate organic matter, including particle-associated bacteria and most eukaryotes, but retaining native planktonic bacterial assemblages. Mesocosms were incubated at 2 degrees C and samples analysed for changes in community composition using denaturing gradient gel electrophoresis (DGGE), real-time PCR and fluorescent in-situ hybridization (FISH). DGGE banding patterns and FISH images demonstrated rapid bacterial colonization of the detritus, dominated by members of class gamma-Proteobacteria, alpha-Proteobacteria and Flavobacteria. Real-time PCR data indicated members of class Flavobacteria were involved in initial colonization of detrital aggregate, however relative abundance stayed at similar levels found for the original native particle-associated populations. 16S rRNA gene DGGE banding patterns and sequence analysis demonstrated significant variation in Flavobacteria community structure occurred in the first 20 days of the experiment before community stabilization occurred. The community structures between the three mesocosms also markedly differed and major colonizers were primarily derived from detectable members of the initial particle-associated Flavobacteria community, however the abundant uncultured Flavobacteria agg58 clone-related and DE cluster 2 clades, previously identified in Southern Ocean seawater were not observed to colonize the detritus.

Mutational analysis of the GB virus B internal ribosome entry site.

GB virus B (GBV-B) is a recently discovered hepatotropic flavivirus that is distantly related to hepatitis C virus (HCV). We show here that translation of its polyprotein is initiated by internal entry of ribosomes on GBV-B RNA. We analyzed the translational activity of dicistronic RNA transcripts containing wild-type or mutated 5' nontranslated GBV-B RNA (5'NTR) segments, placed between the coding sequences of two reporter proteins, in vitro in rabbit reticulocyte lysate and in vivo in transfected BT7-H cells. We related these results to a previously proposed model of the secondary structure of the GBV-B 5'NTR (M. Honda, et al. RNA 2:955-968, 1996). We identified an internal ribosome entry site (IRES) bounded at its 5' end by structural domain II, a location analogous to the 5' limit of the IRES in both the HCV and pestivirus 5'NTRs. Mutational analysis confirmed the structure proposed for domain II of GBV-B RNA, and demonstrated that optimal IRES-mediated translation is dependent on each of the putative RNA hairpins in this domain, including two stem-loops not present in the HCV or pestivirus structures. IRES activity was also absolutely dependent on (i) phylogenetically conserved, adenosine-containing bulge loops in domain III and (ii) the primary nucleotide sequence of stem-loop IIIe. IRES-directed translation was inhibited by a series of point mutations predicted to stabilize stem-loop IV, which contains the initiator AUG codon in its loop segment. A reporter gene was translated most efficiently when fused directly to the initiator AUG codon, with no intervening downstream GBV-B sequence. This finding indicates that the 3' limit of the GBV-B IRES is at the initiator AUG and that it does not require downstream polyprotein-coding sequence as suggested for the HCV IRES. These results show that the GBV-B IRES, while sharing a common general structure, differs both structurally and functionally from other flavivirus IRES elements.

Cell cycle regulation of hepatitis C virus internal ribosomal entry site-directed translation.

BACKGROUND & AIMS: Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located within the 5' nontranslated segment of the viral RNA. This RNA segment is known to form binary complexes with isolated 40S ribosome subunits in vitro, but there is little understanding of how the process of virus translation is regulated in vivo. METHODS: We established 2 stably transformed cell lines from Huh-7 cells that constitutively express dicistronic RNA transcripts containing sequences encoding 2 reporter proteins (Renilla luciferase and firefly luciferase) separated by a functional HCV IRES. The translation of the upstream Renilla luciferase reading frame is initiated in these cells by the usual cellular cap-dependent mechanism, whereas translation of the downstream firefly luciferase reading frame is initiated by the IRES. RESULTS: Compared with cap-dependent translation, the activity of the IRES was greatest in actively growing cells and relatively reduced in resting cells. In synchronized cultures of these stably transformed cells, the IRES activity varied with the cell cycle and was greatest during the mitotic (M) phases and lowest during the quiescent (G(0)) phases. CONCLUSIONS: These findings suggest that HCV translation is regulated by cellular proteins that vary in abundance during the cell cycle and that viral translation may be enhanced by factors that stimulate the regeneration of hepatocytes in patients with chronic hepatitis C.

Proton dosimetry intercomparison based on the ICRU report 59 protocol.

BACKGROUND AND PURPOSE: A new protocol for calibration of proton beams was established by the ICRU in report 59 on proton dosimetry. In this paper we report the results of an international proton dosimetry intercomparison, which was held at Loma Linda University Medical Center. The goals of the intercomparison were, first, to estimate the level of consistency in absorbed dose delivered to patients if proton beams at various clinics were calibrated with the new ICRU protocol, and second, to evaluate the differences in absorbed dose determination due to differences in 60Co-based ionization chamber calibration factors. MATERIALS AND METHODS: Eleven institutions participated in the intercomparison. Measurements were performed in a polystyrene phantom at a depth of 10.27 cm water equivalent thickness in a 6-cm modulated proton beam with an accelerator energy of 155 MeV and an incident energy of approximately 135 MeV. Most participants used ionization chambers calibrated in terms of exposure or air kerma. Four ionization chambers had 60Co-based calibration in terms of absorbed dose-to-water. Two chambers were calibrated in a 60Co beam at the NIST both in terms of air kerma and absorbed dose-to-water to provide a comparison of ionization chambers with different calibrations. RESULTS: The intercomparison showed that use of the ICRU report 59 protocol would result in absorbed doses being delivered to patients at their participating institutions to within +/-0.9% (one standard deviation). The maximum difference between doses determined by the participants was found to be 2.9%. Differences between proton doses derived from the measurements with ionization chambers with N(K)-, or N(W) - calibration type depended on chamber type. CONCLUSIONS: Using ionization chambers with 60Co calibration factors traceable to standard laboratories and the ICRU report 59 protocol, a distribution of stated proton absorbed dose is achieved with a difference less than 3%. The ICRU protocol should be adopted for clinical proton beam calibration. A comparison of proton doses derived from measurements with different chambers indicates that the difference in results cannot be explained only by differences in 60Co calibration factors.

An infectious molecular clone of a Japanese genotype 1b hepatitis C virus.

We describe an infectious molecular clone of a Japanese genotype 1b strain of hepatitis C virus (HCV-N). The molecularly cloned sequence of HCV-N was compared with alignments of other HCV sequences, leading to the identification of 15 unique, nonconservative amino acid substitutions within the HCV-N open reading frame (ORF). These were repaired to the consensus genotype 1b residue, and the infectivity of RNA transcribed from the repaired clone was assessed by intrahepatic inoculation of a chimpanzee. Viral RNA was first detected in the serum of this chimpanzee 3 weeks following inoculation, and was intermittently present over the next 14 weeks. A strong and persistent anti-HCV serological response developed 13 weeks following inoculation, with seroconversion in the recombinant immunoblot assay (RIBA). A weaker, transient serological response, characterized by seroconversion in a third-generation enzyme-linked immunosorbent assay (ELISA) but not RIBA, occurred between weeks 1 and 5. This may have represented an anamnestic response to HCV antigens translated directly from the intrahepatically inoculated RNA, because the animal previously had undergone 2 unsuccessful attempts at rescue of HCV by intrahepatic RNA inoculation. There was neither biochemical nor histological evidence of liver disease. Although this is within the range of expected outcomes in an HCV-naive chimpanzee, prior immunologic priming may have modified the infection in this animal. The HCV-N clone is the first infectious molecular clone of HCV that is comprised entirely of genotype 1b sequence, and it contains an ORF sequence that is significantly divergent from that of a previously described genotype 1a/1b chimera.

Natural variation in translational activities of the 5' nontranslated RNAs of hepatitis C virus genotypes 1a and 1b: evidence for a long-range RNA-RNA interaction outside of the internal ribosomal entry site.

The 5' nontranslated RNA (5'NTR) of a genotype 1b hepatitis C virus (HCV-N) directs cap-independent translation of the HCV-N polyprotein with about twofold less efficiency than the 5'NTR of a genotype 1a virus under physiologic conditions (Hutchinson strain, or HCV-H) (M. Honda et al., Virology 222:31-42, 1996). Here, we show by mutational analysis that substitution of the AG dinucleotide sequence at nucleotides (nt) 34 and 35 of HCV-N with GA (present in HCV-H) restores the translational activity to that of the HCV-H 5'NTR both in vitro and in vivo. These nucleotides are located upstream of the minimal essential internal ribosome entry site (IRES), as a 6-nt deletion spanning nt 32 to 37 also increased the translational activity of the HCV-N 5'NTR to that of HCV-H. Thus, the upstream AG dinucleotide sequence has an inhibitory effect on IRES-directed translation. Surprisingly, however, this inhibitory effect was observed only when the translated, downstream RNA sequence contained nt 408 to 929 of HCV (capsid-coding RNA). Further analysis of RNA transcripts containing frameshift mutations demonstrated that the nucleotide sequence of the transcript, and not the amino acid sequence of the expressed capsid protein, determines this difference in translation efficiency. The difference between the translational activities of the HCV-N and HCV-H transcripts was increased when translation was carried out in reticulocyte lysates containing high K+ concentrations, with a sevenfold difference evident at 130 to 150 mM K+. These results suggest that there is an RNA-RNA interaction involving 5'NTR and capsid-coding sequences flanking the IRES and that this is responsible for the reduced IRES activity of the genotype 1b virus, HCV-N.

Effects of proton and gamma radiation on lymphocyte populations and acute response to antigen.

BACKGROUND: The clinical use of proton radiation in the management of cancer, as well as benign disorders, is rapidly increasing. The major goal of this study was to compare the effects of proton and gamma (60Co) radiation on cell-mediated and humoral immunological parameters. MATERIALS AND METHODS: C57BL/6 mice were exposed to a single dose of 3 Gray (Gy) protons or gamma-rays and intraperitoneally injected 1 day later with sheep red blood cells (sRBC). On 4, 10, 15, and 29 days after exposure, subsets from each group were euthanised; nonirradiated controls (with and without sRBC injection) were included. Body and relative spleen weights, leukocyte counts, spontaneous blastogenesis, lymphocyte populations, and anti-sRBC titers were evaluated. RESULTS: The data showed significant depression (p < 0.05) in nearly all assays on days 4 and 10 after irradiation. B lymphocytes (CD19+) were the most radiosensitive, although reconstitution back to normal levels was observed by day 15. T cell (CD3+) and T helper cell (CD4+) recovery was evident by day 29, whereas the T cytotoxic cell (CD8+) count remained significantly below normal. Natural killer cells (NK1.1+) were relatively radioresistant. Anti-sRBC antibody production was slow and low titers were obtained after irradiation. No significant differences were noted between the two types of radiation. CONCLUSIONS: Taken together, the data show that whole-body irradiation with protons or gamma-rays, at the dose employed, results in marked, but transient, immunosuppression. However, at the time points of testing and with the assays used, little or no differences were found between the two forms of radiation.

Dose uniformity from a computerized three-dimensional tissue compensating system.

A verification of dose uniformity of the Huestis Compu-Former, a three dimensional megavoltage tissue compensator, is presented. Tissue compensators were built for three different anthropomorphic phantoms: a head and neck, a mantle, and a breast. Film densitometry was used to evaluate the effectiveness of the tissue compensators by comparing isodose curves generated for a compensated and an uncompensated field. Evaluation of the isodose distributions for the three regions confirmed the use of the Compu-Former as a reliable tissue compensating system.

Human births and the phase of the moon.

PIP: Published studies on the frequency of births as related to the lunar cycle are inconsistent with each other. The distribution of all births during 51 lunar cycles, from March 17, 1974, to April 30, 1978, was analyzed by the authors at the University of California, Los Angeles, Hospital. There were 11.691 live births, of which 8142 were natural, 141 multiple, and 168 stillbirths. In none of the 4 samples was the mean number of births occurring on the date of the full moon above average, showing that the birthrate during the period surveyed did not in any way correlate with the cycle of lunar phases.^ieng