Plant Root-Exudates Recruit Hyperparasitic Bacteria of Phytonematodes by Altered Cuticle Aging: Implications for Biological Control Strategies.

Phytonematodes are globally important functional components of the belowground ecology in both natural and agricultural soils; they are a diverse group of which some species are economically important pests, and environmentally benign control strategies are being sought to control them. Using eco-evolutionary theory, we test the hypothesis that root-exudates of host plants will increase the ability of a hyperparasitic bacteria, Pasteuria penetrans and other closely related bacteria, to infect their homologous pest nematodes, whereas non-host root exudates will not. Plant root-exudates from good hosts, poor hosts and non-hosts were characterized by gas chromatography-mass spectrometry (GC/MS) and we explore their interaction on the attachment of the hyperparasitic bacterial endospores to homologous and heterologous pest nematode cuticles. Although GC/MS did not identify any individual compounds as responsible for changes in cuticle susceptibility to endospore adhesion, standardized spore binding assays showed that Pasteuria endospore adhesion decreased with nematode age, and that infective juveniles pre-treated with homologous host root-exudates reduced the aging process and increased attachment of endospores to the nematode cuticle, whereas non-host root-exudates did not. We develop a working model in which plant root exudates manipulate the nematode cuticle aging process, and thereby, through increased bacterial endospore attachment, increase bacterial infection of pest nematodes. This we suggest would lead to a reduction of plant-parasitic nematode burden on the roots and increases plant fitness. Therefore, by the judicious manipulation of environmental factors produced by the plant root and by careful crop rotation this knowledge can help in the development of environmentally benign control strategies.

90-90-90 for everyone?: Access to HIV care and treatment for people with HIV who experience imprisonment in Ontario, Canada.

We examined HIV care and treatment in prison and after release for people with HIV in Ontario, Canada, and compared HIV care and treatment with the general population. We used administrative data to identify people with HIV released from provincial prison in 2010 and in the general population. We calculated the proportion of people with HIV who accessed HIV care in prison. We compared HIV care use between people with HIV on prison release and in the general population. We estimated the proportion of people with HIV on antiretroviral therapy in prison as the ratio of the average numbers of people prescribed antiretroviral therapy in prison in 2009/2010 and people with HIV in prison in January 2010. We compared the proportion of people with HIV on public drug benefits that filled an antiretroviral therapy prescription within 6 months for people postrelease and in the general population. Of 344 people with HIV on prison admission, 34.0% received HIV care in prison. Over 1 year, 63.6% of 330 people with HIV on prison release and 67.7% of 15,819 people with HIV in the general population accessed HIV care (p = 0.118), and 43.3% of people with HIV on prison release and 55.2% of people with HIV in the general population had 2 or more HIV care visits (p < 0.001). In prison, 52.4% of people with HIV (39.5/75.4) were on antiretroviral therapy. Of those accessing drug benefits, 60.1% of 226 people with HIV on prison release and 79.6% of 7458 people with HIV in the general population claimed an antiretroviral therapy prescription within 6 months (p < 0.001). Access to HIV care and treatment were suboptimal in prison, and sustained HIV care and treatment were worse for people post-release compared to the general population. Interventions are needed to support HIV care for this population.

Using concept mapping to inform the development of a transitional reintegration intervention program for formerly incarcerated people with HIV.

BACKGROUND: Accessing HIV-related care is challenging for formerly incarcerated people with HIV. Interventions informed by the perspectives of these individuals could facilitate engagement with care and address competing priorities that may act as barriers to this process. METHODS: We used concept mapping to identify and prioritize the main obstacles to engaging with HIV-related care following prison release. In brainstorming sessions, formerly incarcerated people with HIV generated responses to a focused prompt regarding the main barriers to reengaging with care. These were consolidated in 35 statements. Next, participants sorted the consolidated list of responses into groups and rated each from lowest to highest in terms of its importance and feasibility of being addressed. We used cluster analysis to generate concept maps that were interpreted with participants. RESULTS: Overall, 39 participants participated in brainstorming sessions, among whom 18 returned for rating and sorting. Following analysis, a seven-cluster map was generated, with participants rating the 'Practical Considerations' (e.g. lack of transportation from prison) and 'Survival Needs' (e.g. securing housing and food) clusters as most important. Although ratings were generally similar between women and men, women assigned greater importance to barriers related to reconnecting with children. CONCLUSIONS: Using concept mapping, we worked with formerly incarcerated people with HIV to identify and prioritize key challenges related to accessing health and social services following prison release. Transitional intervention programs should include programs and processes that address meeting basic subsistence needs and overcoming logistical barriers related to community re-entry.

Transitional care for formerly incarcerated persons with HIV: protocol for a realist review.

BACKGROUND: Little is known about the mechanisms that influence the success or failure of programs to facilitate re-engagement with health and social services for formerly incarcerated persons with HIV. This review aims to identify how interventions to address such transitions work, for whom and under what circumstances. METHODS: We will use realist review methodology to conduct our analysis. We will systematically search electronic databases and grey literature for English language qualitative and quantitative studies of interventions. Two investigators will independently screen citations and full-text articles, abstract data, appraise study quality and synthesize the literature. Data analysis will include identifying context-mechanism-outcome configurations, exploring and comparing patterns in these configurations, making comparisons across contexts and developing explanatory frameworks. DISCUSSION: This review will identify mechanisms that influence the success or failure of transition interventions for formerly incarcerated individuals with HIV. The findings will be integrated with those from complementary qualitative and quantitative studies to inform future interventions. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42016040054.

Global analysis of Chlorella variabilis NC64A mRNA profiles during the early phase of Paramecium bursaria chlorella virus-1 infection.

The PBCV-1/Chlorella variabilis NC64A system is a model for studies on interactions between viruses and algae. Here we present the first global analyses of algal host transcripts during the early stages of infection, prior to virus replication. During the course of the experiment stretching over 1 hour, about a third of the host genes displayed significant changes in normalized mRNA abundance that either increased or decreased compared to uninfected levels. The population of genes with significant transcriptional changes gradually increased until stabilizing at 40 minutes post infection. Functional categories including cytoplasmic ribosomal proteins, jasmonic acid biosynthesis and anaphase promoting complex/cyclosomes had a significant excess in upregulated genes, whereas spliceosomal snRNP complexes and the shikimate pathway had significantly more down-regulated genes, suggesting that these pathways were activated or shut-down in response to the virus infection. Lastly, we examined the expression of C. varibilis RNA polymerase subunits, as PBCV-1 transcription depends on host RNA polymerases. Two subunits were up-regulated, RPB10 and RPC34, suggesting that they may function to support virus transcription. These results highlight genes and pathways, as well as overall trends, for further refinement of our understanding of the changes that take place during the early stages of viral infection.

Deep RNA sequencing reveals hidden features and dynamics of early gene transcription in Paramecium bursaria chlorella virus 1.

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of the genus Chlorovirus (family Phycodnaviridae) that infects the unicellular, eukaryotic green alga Chlorella variabilis NC64A. The 331-kb PBCV-1 genome contains 416 major open reading frames. A mRNA-seq approach was used to analyze PBCV-1 transcriptomes at 6 progressive times during the first hour of infection. The alignment of 17 million reads to the PBCV-1 genome allowed the construction of single-base transcriptome maps. Significant transcription was detected for a subset of 50 viral genes as soon as 7 min after infection. By 20 min post infection (p.i.), transcripts were detected for most PBCV-1 genes and transcript levels continued to increase globally up to 60 min p.i., at which time 41% or the poly (A+)-containing RNAs in the infected cells mapped to the PBCV-1 genome. For some viral genes, the number of transcripts in the latter time points (20 to 60 min p.i.) was much higher than that of the most highly expressed host genes. RNA-seq data revealed putative polyadenylation signal sequences in PBCV-1 genes that were identical to the polyadenylation signal AAUAAA of green algae. Several transcripts have an RNA fragment excised. However, the frequency of excision and the resulting putative shortened protein products suggest that most of these excision events have no functional role but are probably the result of the activity of misled splicesomes.

Evaluation of higher plant virus resistance genes in the green alga, Chlorella variabilis NC64A, during the early phase of infection with Paramecium bursaria chlorella virus-1.

With growing industrial interest in algae plus their critical roles in aquatic systems, the need to understand the effects of algal pathogens is increasing. We examined a model algal host-virus system, Chlorella variabilis NC64A and virus, PBCV-1. C. variabilis encodes 375 homologs to genes involved in RNA silencing and in response to virus infection in higher plants. Illumina RNA-Seq data showed that 325 of these homologs were expressed in healthy and early PBCV-1 infected (≤60min) cells. For each of the RNA silencing genes to which homologs were found, mRNA transcripts were detected in healthy and infected cells. C. variabilis, like higher plants, may employ certain RNA silencing pathways to defend itself against virus infection. To our knowledge this is the first examination of RNA silencing genes in algae beyond core proteins, and the first analysis of their transcription during virus infection.

High abundances of cyanomyoviruses in marine ecosystems demonstrate ecological relevance.

The distribution of cyanomyoviruses was estimated using a quantitative PCR (qPCR) approach that targeted the g20 gene as a proxy for phage. Samples were collected spatially during a > 3000 km transect through the Sargasso Sea and temporally during a gyre-constrained phytoplankton bloom within the southern Pacific Ocean. Cyanomyovirus abundances were lower in the Sargasso Sea than in the southern Pacific Ocean, ranging from 2.75 × 10(3) to 5.15 × 10(4) mL(-1) and correlating with the abundance of their potential hosts (Prochlorococcus and Synechococcus). Cyanomyovirus abundance in the southern Pacific Ocean (east of New Zealand) followed Synechococcus host populations in the system: this included a decrease in g20 gene copies (from 4.3 × 10(5) to 9.6 × 10(3) mL(-1) ) following the demise of a Synechococcus bloom. When compared with direct counts of viruses, observations suggest that the cyanomyoviruses comprised 0.5 to >25% of the total virus community. We estimated daily lysis rates of 0.2-46% of the standing stock of Synechococcus in the Pacific Ocean compared with c. < 1.0% in the Sargasso Sea. In total, our observations confirm this family of viruses is abundant in marine systems and that they are an important source of cyanobacterial mortality.

Viral and bacterial abundance and production in the Western Pacific Ocean and the relation to other oceanic realms.

We completed a transect through the Western Pacific Warm Pool to examine how environmental variables may influence viral and bacterial abundance and production rates in this globally important oceanic region. Of the variables analyzed, viral abundance and production had the most significant relationship to bacterial cell abundance: viral parameters were not significantly correlated to the measured environmental variables, including temperature. Bacterial production rates were significantly correlated to temperature in open ocean waters, but not in waters close to land masses. Analyses of 16S rRNA gene by pyrosequencing indicated only minor changes in eubacterial community structure across the transect, with α-proteobacteria dominating all sampled populations. Diversity within the prokaryotic community did not correlate directly with viral abundance or activity. Comparisons to two other ocean-scale transects (> 8000 km of open ocean in total) in the Atlantic Ocean indicated that correlations between viral and bacterial abundance and production relative to environmental variables are regime dependent. In particular, correlations to temperature showed remarkable differences across the three transects. Collectively, our observations suggest that seemingly similar oceanic regions may have very different microbial community responses to environmental variables. Our observations and analyses demonstrate that ocean-scale generalizations may not apply in the case of viral ecology.

Pasteuria endospores from Heterodera cajani (Nematoda: Heteroderidae) exhibit inverted attachment and altered germination in cross-infection studies with Globodera pallida (Nematoda: Heteroderidae).

The Pasteuria group of Gram-positive, endospore-forming bacteria are parasites of invertebrates and exhibit differences in host specificity. We describe a cross-infection study between an isolate of Pasteuria from pigeon pea cyst nematode, Heterodera cajani, which also infects the potato cyst nematode, Globodera pallida, from the United Kingdom. A proportion of the attached endospores, 13% on H. cajani and 22% on G. pallida adhere to the cuticle in an inverted orientation. Inverted and conventionally attached endospores germinated and produced bacillus-like rods that completed their life cycle in < 15 weeks within females of G. pallida. This is the first example in which the life cycle of a Pasteuria population was systematically followed in two different nematode genera. A 1430-base pair fragment of the 16S rRNA gene sequence of the Pasteuria isolate from H. cajani revealed 98.6% similarity to the orthologous gene in Pasteuria nishizawae. Additionally, their respective endospore sizes were not significantly different, in contrast their host ranges are. Potential reasons for this remain unclear and are discussed.

The mitochondrial genome of the soybean cyst nematode, Heterodera glycines.

We sequenced the entire coding region of the mitochondrial genome of Heterodera glycines. The sequence obtained comprised 14.9 kb, with PCR evidence indicating that the entire genome comprised a single, circular molecule of approximately 21-22 kb. The genome is the most T-rich nematode mitochondrial genome reported to date, with T representing over half of all nucleotides on the coding strand. The genome also contains the highest number of poly(T) tracts so far reported (to our knowledge), with 60 poly(T) tracts ≥ 12 Ts. All genes are transcribed from the same mitochondrial strand. The organization of the mitochondrial genome of H. glycines shows a number of similarities compared with Radopholus similis, but fewer similarities when compared with Meloidogyne javanica. Very few gene boundaries are shared with Globodera pallida or Globodera rostochiensis. Partial mitochondrial genome sequences were also obtained for Heterodera cardiolata (5.3 kb) and Punctodera chalcoensis (6.8 kb), and these had identical organizations compared with H. glycines. We found PCR evidence of a minicircular mitochondrial genome in P. chalcoensis, but at low levels and lacking a noncoding region. Such circularised genome fragments may be present at low levels in a range of nematodes, with multipartite mitochondrial genomes representing a shift to a condition in which these subgenomic circles predominate.

Application of the major capsid protein as a marker of the phylogenetic diversity of Emiliania huxleyi viruses.

Studies of the Phycodnaviridae have traditionally relied on the DNA polymerase (pol) gene as a biomarker. However, recent investigations have suggested that the major capsid protein (MCP) gene may be a reliable phylogenetic biomarker. We used MCP gene amplicons gathered across the North Atlantic to assess the diversity of Emiliania huxleyi-infecting Phycodnaviridae. Nucleotide sequences were examined across >6000 km of open ocean, with comparisons between concentrates of the virus-size fraction of seawater and of lysates generated by exposing host strains to these same virus concentrates. Analyses revealed that many sequences were only sampled once, while several were over-represented. Analyses also revealed nucleotide sequences distinct from previous coastal isolates. Examination of lysed cultures revealed a new richness in phylogeny, as MCP sequences previously unrepresented within the existing collection of E. huxleyi viruses (EhV) were associated with viruses lysing cultures. Sequences were compared with previously described EhV MCP sequences from the North Sea and a Norwegian Fjord, as well as from the Gulf of Maine. Principal component analysis indicates that location-specific distinctions exist despite the presence of sequences common across these environments. Overall, this investigation provides new sequence data and an assessment on the use of the MCP gene.

Poly(T) variation in heteroderid nematode mitochondrial genomes is predominantly an artefact of amplification.

We assessed the rate of in vitro polymerase errors at polythymidine [poly(T)] tracts in the mitochondrial DNA (mtDNA) of a heteroderid nematode (Heterodera cajani). The mtDNA of these nematodes contain unusually high numbers of poly(T) tracts, and have previously been suggested to contain biological poly(T) length variation. However, using a cloned molecule, we observed that poly(T) variation was generated in vitro at regions containing more than six consecutive Ts. This artefactual error rate was estimated at 7.3 × 10(-5) indels/poly(T) tract >6 Ts/cycle. This rate was then compared to the rate of poly(T) variation detected after the amplification of a biological sample, in order to estimate the 'biological + artefactual' rate of poly(T) variation. There was no significant difference between the artefactual and the artefactual + biological rates, suggesting that the majority of poly(T) variation in the biological sample was artefactual. We then examined the generation of poly(T) variation in a range of templates with tracts up to 16 Ts long, utilizing a range of Heteroderidae species. We observed that T deletions occurred five times more frequently than insertions, and a trend towards increasing error rates with increasing poly(T) tract length. These findings have significant implications for studies involving genomes with many homopolymer tracts.

ISOLATION OF A NON-PHAGE-LIKE LYTIC VIRUS INFECTING AUREOCOCCUS ANOPHAGEFFERENS(1).

We have been working to characterize viruses that infect the HAB-forming pelagophyte Aureococcus anophagefferens Hargraves et Sieburth. Field samples were collected during brown-tide events in 2002 and tested for the presence of lytic agents. Here, we describe a recently isolated, lytic virus-like particle (VLP) that is morphologically similar to particles observed in thin sections of infected A. anophagefferens cells from natural samples. TEM and SEM have revealed VLPs consistent with the morphological characteristics of previously described Phycodnaviridae. Large icosahedral particles (∼140 nm) of similar shape and morphology dominate cell lysates and are accompanied by smaller phage-like particles and heterotrophic prokaryotes that appear to be incurable from our cultures. To determine which of these particles interacts with the Aureococcus cells, we preserved cultures during the early stage of infection so that SEM could be used to visualize those particles that attach to the surface of naïve cultures. SEM revealed that 63% of the large icosahedral-shaped particles attached to A. anophagefferens cells after only 30 min of exposure, while no significant frequency of attachment to the alga was observed for the phage-like particles. The results of these observations are in contrast to previous studies, where phage-like particles were reported to infect cells. When considered in conjunction with field observations, the results suggest that this newly isolated virus represents the dominant virus-morphotype associated with bloom collapse and termination.

Nitric oxide production by decidual endothelial cells is not reduced in preeclampsia.

OBJECTIVE: To investigate whether decidual endothelial cells (DEC) contribute to the pathogenesis of preeclampsia through abnormal nitric oxide production. Decidual endothelial cells from normal (NDEC) and preeclamptic (PEDEC) pregnancies, and also human umbilical vein endothelial cells (HUVEC), were examined. METHODS: HUVEC, NDEC, and PEDEC were incubated for 45 min in serum-free media with the addition of potential stimulators [calcium ionophore (A23187), sepiapterin, and a combination of cytokines (TNF-alpha, gamma-IFN and LPS)], and the competitive inhibitor, NG-monomethyl-L-arginine (L-NMMA). These were added alone or in combination. Supernatants were measured for nitrate/nitrite (NOx) levels and the cells acid-extracted for measurement of cyclic guanosine monophosphate (cGMP). The effect of 30 min of shear stress (approximately 20 dynes/cm2) on NO and cGMP production by NDEC and PEDEC and on production of prostacyclin and thromboxane A2, was assessed. RESULTS: PEDEC and HUVEC both produced more NO than NDEC under all conditions examined. Cell-associated cGMP levels, however, were not different among the cell groups but were increased by A23187 and inhibited by L-NMMA. In control conditions, shear stress stimulated cGMP levels 5-fold (p<0.01) in both NDEC and PEDEC, and PGI2 production 2-fold (p<0.05). CONCLUSIONS: DEC from preeclamptic women do not have reduced NO production and respond normally to shear stress by increasing cGMP and PGI2 production. Our results are consistent with other reports of equal or higher NO levels in preeclampsia and indicate that reduced NO production by endothelial cells is not the explanation for the vasoconstriction of uterine vessels.