A Sample-to-Sequence Protocol for Genus Targeted Transcriptomic Profiling: Application to Marine Synechococcus.

Recent studies using whole community metagenomic and metatranscriptomic approaches are revealing important new insights into the functional potential and activity of natural marine microbial communities. Here, we complement these approaches by describing a complete ocean sample-to-sequence protocol, specifically designed to target a single bacterial genus for purposes of both DNA and RNA profiling using fluorescence activated cell sorting (FACS). The importance of defining and understanding the effects of a sampling protocol are critical if we are to gain meaningful data from environmental surveys. Rigorous pipeline trials with a cultured isolate, Synechococcus sp. BL107 demonstrate that water filtration has a well-defined but limited impact on the transcriptomic profile of this organism, whilst sample storage and multiple rounds of cell sorting have almost no effect on the resulting RNA sequence profile. Attractively, the means to replicate the sampling strategy is within the budget and expertise of most researchers

A new family of “megaphages” abundant in the marine environment

AbstractMegaphages, bacteriophages harbouring extremely large genomes, have recently been found to be ubiquitous, being described from a variety of microbiomes ranging from the animal gut to soil and freshwater systems. However, no complete marine megaphage has been identified to date. Here, using both short and long read sequencing, we assembled &gt;900 high-quality draft viral genomes from water in the English Channel. One of these genomes included a novel megaphage, Mar_Mega_1 at &gt;650 Kb, making it one of the largest phage genomes assembled to date. Utilising phylogenetic and network approaches, we found this phage represents a new family of megaphages. Genomic analysis showed Mar_Mega_1 shares relatively few homologues with its closest relatives, but, as with other megaphages Mar_Mega_1 contained a variety of auxiliary metabolic genes responsible for carbon metabolism and nucleotide biosynthesis, including a NADP-dependent isocitrate dehydrogenase [Idh] and nicotinamide-nucleotide amidohydrolase [PncC], which have not previously been identified in megaphages. Mar_Mega_1 was abundant in a marine virome sample and related phages are widely prevalent in the oceans.</jats:p

Shared Skeletal Support in a Coral-Hydroid Symbiosis

Hydroids form symbiotic relationships with a range of invertebrate hosts. Where they live with colonial invertebrates such as corals or bryozoans the hydroids may benefit from the physical support and protection of their host's hard exoskeleton, but how they interact with them is unknown. Electron microscopy was used to investigate the physical interactions between the colonial hydroid Zanclea margaritae and its reef-building coral host Acropora muricata. The hydroid tissues extend below the coral tissue surface sitting in direct contact with the host's skeleton. Although this arrangement provides the hydroid with protective support, it also presents problems of potential interference with the coral's growth processes and exposes the hydroid to overgrowth and smothering. Desmocytes located within the epidermal layer of the hydroid's perisarc-free hydrorhizae fasten it to the coral skeleton. The large apical surface area of the desmocyte and high bifurcation of the distal end within the mesoglea, as well as the clustering of desmocytes suggests that a very strong attachment between the hydroid and the coral skeleton. This is the first study to provide a detailed description of how symbiotic hydroids attach to their host's skeleton, utilising it for physical support. Results suggest that the loss of perisarc, a characteristic commonly associated with symbiosis, allows the hydroid to utilise desmocytes for attachment. The use of these anchoring structures provides a dynamic method of attachment, facilitating detachment from the coral skeleton during extension, thereby avoiding overgrowth and smothering enabling the hydroid to remain within the host colony for prolonged periods of time

Evaluation of the role of glutathione in the lead-induced toxicity in Saccharomyces cerevisiae

The effect of intracellular reduced glutathione (GSH) in the lead stress response of Saccharomyces cerevisiae was investigated. Yeast cells exposed to Pb, for 3 h, lost the cell proliferation capacity (viability) and decreased intracellular GSH level. The Pb-induced loss of cell viability was compared among yeast cells deficient in GSH1 (∆gsh1) or GSH2 (∆gsh2) genes and wild-type (WT) cells. When exposed to Pb, ∆gsh1 and ∆gsh2 cells did not display an increased loss of viability, compared with WT cells. However, the depletion of cellular thiols, including GSH, by treatment of WT cells with iodoacetamide (an alkylating agent, which binds covalently to thiol group), increased the loss of viability in Pb-treated cells. In contrast, GSH enrichment, due to the incubation of WT cells with amino acids mixture constituting GSH (l-glutamic acid, l-cysteine and glycine), reduced the Pb-induced loss of proliferation capacity. The obtained results suggest that intracellular GSH is involved in the defence against the Pb-induced toxicity; however, at physiological concentration, GSH seems not to be sufficient to prevent the Pb-induced loss of cell viability

U.S. Physicians’ Views on Financing Options to Expand Health Insurance Coverage: A National Survey

Background: Physician opinion can influence the prospects for health care reform, yet there are few recent data on physician views on reform proposals or access to medical care in the United States. Objective: To assess physician views on financing options for expanding health care coverage and on access to health care. Design and Participants: Nationally representative mail survey conducted between March 2007 and October 2007 of U.S. physicians engaged in direct patient care. Measurements: Rated support for reform options including financial incentives to induce individuals to purchase health insurance and single-payer national health insurance; rated views of several dimensions of access to care. Main results: 1,675 of 3,300 physicians responded (50.8%). Only 9% of physicians preferred the current employer-based financing system. Forty-nine percent favored either tax incentives or penalties to encourage the purchase of medical insurance, and 42% preferred a government-run, taxpayer-financed single-payer national health insurance program. The majority of respondents believed that all Americans should receive needed medical care regardless of ability to pay (89%); 33% believed that the uninsured currently have access to needed care. Nearly one fifth of respondents (19.3%) believed that even the insured lack access to needed care. Views about access were independently associated with support for single-payer national health insurance. Conclusions: The vast majority of physicians surveyed supported a change in the health care financing system. While a plurality support the use of financial incentives, a substantial proportion support single payer national health insurance. These findings challenge the perception that fundamental restructuring of the U.S. health care financing system receives little acceptance by physicians

Transcriptome dynamics of a broad host-range cyanophage and its hosts

Cyanobacteria are highly abundant in the oceans and are constantly exposed to lytic viruses. The T4-like cyanomyoviruses are abundant in the marine environment and have broad host-ranges relative to other cyanophages. It is currently unknown whether broad host-range phages specifically tailor their infection program for each host, or employ the same program irrespective of the host infected. Also unknown is how different hosts respond to infection by the same phage. Here we used microarray and RNA-seq analyses to investigate the interaction between the Syn9 T4-like cyanophage and three phylogenetically, ecologically and genomically distinct marine Synechococcus strains: WH7803, WH8102 and WH8109. Strikingly, Syn9 led a nearly identical infection and transcriptional program in all three hosts. Different to previous assumptions for T4-like cyanophages, three temporally regulated gene expression classes were observed. Furthermore, a novel regulatory element controlled early-gene transcription, and host-like promoters drove middle gene transcription, different to the regulatory paradigm for T4. Similar results were found for the P-TIM40 phage during infection of Prochlorococcus NATL2A. Moreover, genomic and metagenomic analyses indicate that these regulatory elements are abundant and conserved among T4-like cyanophages. In contrast to the near-identical transcriptional program employed by Syn9, host responses to infection involved host-specific genes primarily located in hypervariable genomic islands, substantiating islands as a major axis of phage-cyanobacteria interactions. Our findings suggest that the ability of broad host-range phages to infect multiple hosts is more likely dependent on the effectiveness of host defense strategies than on differential tailoring of the infection process by the phage

The positive outlook study- a randomised controlled trial evaluating the effectiveness of an online self-management program targeting psychosocial issues for men living with HIV: a study protocol

Background: The emergence of HIV as a chronic condition means that people living with HIV are required to takemore responsibility for the self-management of their condition, including making physical, emotional and socialadjustments. This paper describes the design and evaluation of Positive Outlook, an online program aiming toenhance the self-management skills of gay men living with HIV.Methods/design: This study is designed as a randomised controlled trial in which men living with HIV in Australiawill be assigned to either an intervention group or usual care control group. The intervention group willparticipate in the online group program ‘Positive Outlook’. The program is based on self-efficacy theory and uses aself-management approach to enhance skills, confidence and abilities to manage the psychosocial issues associatedwith HIV in daily life. Participants will access the program for a minimum of 90 minutes per week over seven weeks.Primary outcomes are domain specific self-efficacy, HIV related quality of life, and outcomes of health education.Secondary outcomes include: depression, anxiety and stress; general health and quality of life; adjustment to HIV;and social support. Data collection will take place at baseline, completion of the intervention (or eight weeks postrandomisation) and at 12 week follow-up.Discussion: Results of the Positive Outlook study will provide information regarding the effectiveness of onlinegroup programs improving health related outcomes for men living with HIV

Population Genetics of Duplicated Alternatively Spliced Exons of the Dscam Gene in Daphnia and Drosophila

In insects and crustaceans, the Down syndrome cell adhesion molecule (Dscam) occurs in many different isoforms. These are produced by mutually exclusive alternative splicing of dozens of tandem duplicated exons coding for parts or whole immunoglobulin (Ig) domains of the Dscam protein. This diversity plays a role in the development of the nervous system and also in the immune system. Structural analysis of the protein suggested candidate epitopes where binding to pathogens could occur. These epitopes are coded by regions of the duplicated exons and are therefore diverse within individuals. Here we apply molecular population genetics and molecular evolution analyses using Daphnia magna and several Drosophila species to investigate the potential role of natural selection in the divergence between orthologs of these duplicated exons among species, as well as between paralogous exons within species. We found no evidence for a role of positive selection in the divergence of these paralogous exons. However, the power of this test was low, and the fact that no signs of gene conversion between paralogous exons were found suggests that paralog diversity may nonetheless be maintained by selection. The analysis of orthologous exons in Drosophila and in Daphnia revealed an excess of non-synonymous polymorphisms in the epitopes putatively involved in pathogen binding. This may be a sign of balancing selection. Indeed, in Dr. melanogaster the same derived non-synonymous alleles segregate in several populations around the world. Yet other hallmarks of balancing selection were not found. Hence, we cannot rule out that the excess of non-synonymous polymorphisms is caused by segregating slightly deleterious alleles, thus potentially indicating reduced selective constraints in the putative pathogen binding epitopes of Dscam

Implementing an innovative consent form: the PREDICT experience

<p>Abstract</p> <p>Background</p> <p>In the setting of coronary angiography, generic consent forms permit highly variable communication between patients and physicians. Even with the existence of multiple risk models, clinicians have been unable to readily access them and thus provide patients with vague estimations regarding risks of the procedure.</p> <p>Methods</p> <p>We created a web-based vehicle, PREDICT, for embedding patient-specific estimates of risk from validated multivariable models into individualized consent documents at the point-of-care. Beginning August 2006, outpatients undergoing coronary angiography at the Mid America Heart Institute received individualized consent documents generated by PREDICT. In February 2007 this approach was expanded to all patients undergoing coronary angiography within the four Kansas City hospitals of the Saint Luke's Health System. Qualitative research methods were used to identify the implementation challenges and successes with incorporating PREDICT-enhanced consent documents into routine clinical care from multiple perspectives: administration, information systems, nurses, physicians, and patients.</p> <p>Results</p> <p>Most clinicians found usefulness in the tool (providing clarity and educational value for patients) and satisfaction with the altered processes of care, although a few cardiologists cited delayed patient flow and excessive patient questions. The responses from administration and patients were uniformly positive. The key barrier was related to informatics.</p> <p>Conclusion</p> <p>This preliminary experience suggests that successful change in clinical processes and organizational culture can be accomplished through multidisciplinary collaboration. A randomized trial of PREDICT consent, leveraging the accumulated knowledge from this first experience, is needed to further evaluate its impact on medical decision-making, patient compliance, and clinical outcomes.</p

Novel roles of the chemorepellent axon guidance molecule RGMa in cell migration and adhesion

The repulsive guidance molecule A (RGMa) is a contact-mediated axon guidance molecule that has significant roles in central nervous system (CNS) development. Here we have examined whether RGMa has novel roles in cell migration and cell adhesion outside the nervous system. RGMa was found to stimulate cell migration from Xenopus animal cap explants in a neogenin-dependent and BMP-independent manner. RGMa also stimulated the adhesion of Xenopus animal cap cells, and this adhesion was dependent on neogenin and independent of calcium. To begin to functionally characterize the role of specific domains in RGMa, we assessed the migratory and adhesive activities of deletion mutants. RGMa lacking the partial von Willebrand factor type D (vWF) domain preferentially perturbed cell adhesion, while mutants lacking the RGD motif affected cell migration. We also revealed that manipulating the levels of RGMa in vivo caused major migration defects during Xenopus gastrulation. We have revealed here novel roles of RGMa in cell migration and adhesion and demonstrated that perturbations to the homeostasis of RGMa expression can severely disrupt major morphogenetic events. These results have implications for understanding the role of RGMa in both health and disease