Identification of protein interactions of grapevine fanleaf virus RNA-dependent RNA polymerase during infection of by affinity purification and tandem mass spectrometry.

The RNA-dependent RNA polymerase (1E) is involved in replication of grapevine fanleaf virus (GFLV, , ) and causes vein clearing symptoms in . Information on protein 1E interaction with other viral and host proteins is scarce. To study protein 1E biology, three GFLV infectious clones, i.e. GHu (a symptomatic wild-type strain), GHu-1E (an asymptomatic GHu mutant) and F13 (an asymptomatic wild-type strain), were engineered with protein 1E fused to a V5 epitope tag at the C-terminus. Following -mediated delivery of GFLV clones in and protein extraction at seven dpi, when optimal 1E:V5 accumulation was detected, two viral and six plant putative interaction partners of V5-tagged protein 1E were identified for the three GFLV clones by affinity purification and tandem mass spectrometry. This study provides insights into the protein interactome of 1E during GFLV systemic infection in and lays the foundation for validation work

Long-term site fidelity of endangered smalltooth sawfish (Pristis pectinata) from different mothers

Understanding how endangered species use nursery habitats is vital for recovery planning. Research on the smalltooth sawfish (Pristis pectinata) has shown that areas of estuarine nurseries, called hotspots, are used consistently. The objectives of our study were 1) to determine whether 10 young-of-the-year smalltooth sawfish in an artificial, non-main-stem portion (i.e., a seawall canal system) of a hotspot were descended from one or different mothers and 2) to document long-term habitat use by these individuals. At least 4 mothers contributed to the group, which comprised siblings, half-siblings, and unrelated individuals. Young sawfish exhibited site fidelity to their capture location, spending 61% of their time there. Continuous residency lasted as long as 86 days, but these fish made small-scale diel

R270C polymorphism leads to loss of function of the canine P2X7 receptor

The relative function of the P2X7 receptor, an ATP-gated ion channel, varies between humans due to polymorphisms in the P2RX7 gene. This study aimed to assess the functional impact of P2X7 variation in a random sample of the canine population. Blood and genomic DNA were obtained from 69 dogs selected as representatives of a cross section of different breeds. P2X7 function was determined by flow cytometric measurements of dye uptake and patch-clamp measurements of inward currents. P2X7 expression was determined by immunoblotting and immunocytochemistry. Sequencing was used to identify P2RX7 gene polymorphisms. P2X7 was cloned from an English springer spaniel, and point mutations were introduced into this receptor by site-directed mutagenesis. The relative function of P2X7 on monocytes varied between individual dogs. The canine P2RX7 gene encoded four missense polymorphisms: F103L and P452S, found in heterozygous and homozygous dosage, and R270C and R365Q, found only in heterozygous dosage. Moreover, R270C and R365Q were associated with the cocker spaniel and Labrador retriever, respectively. F103L, R270C, and R365Q but not P452S corresponded to decreased P2X7 function in monocytes but did not explain the majority of differences in P2X7 function between dogs, indicating that other factors contribute to this variability. Heterologous expression of site-directed mutants of P2X7 in human embryonic kidney-293 cells indicated that the R270C mutant was nonfunctional, the F103L and R365Q mutants had partly reduced function, and the P452S mutant functioned normally. Taken together, these data highlight that a R270C polymorphism has major functional impact on canine P2X7

Effectiveness and cost-effectiveness of an educational intervention for practice teams to deliver problem focused therapy for insomnia: rationale and design of a pilot cluster randomised trial

Background: Sleep problems are common, affecting over a third of adults in the United Kingdom and leading to reduced productivity and impaired health-related quality of life. Many of those whose lives are affected seek medical help from primary care. Drug treatment is ineffective long term. Psychological methods for managing sleep problems, including cognitive behavioural therapy for insomnia (CBTi) have been shown to be effective and cost effective but have not been widely implemented or evaluated in a general practice setting where they are most likely to be needed and most appropriately delivered. This paper outlines the protocol for a pilot study designed to evaluate the effectiveness and cost-effectiveness of an educational intervention for general practitioners, primary care nurses and other members of the primary care team to deliver problem focused therapy to adult patients presenting with sleep problems due to lifestyle causes, pain or mild to moderate depression or anxiety. Methods and design: This will be a pilot cluster randomised controlled trial of a complex intervention. General practices will be randomised to an educational intervention for problem focused therapy which includes a consultation approach comprising careful assessment (using assessment of secondary causes, sleep diaries and severity) and use of modified CBTi for insomnia in the consultation compared with usual care (general advice on sleep hygiene and pharmacotherapy with hypnotic drugs). Clinicians randomised to the intervention will receive an educational intervention (2 × 2 hours) to implement a complex intervention of problem focused therapy. Clinicians randomised to the control group will receive reinforcement of usual care with sleep hygiene advice. Outcomes will be assessed via self-completion questionnaires and telephone interviews of patients and staff as well as clinical records for interventions and prescribing. Discussion: Previous studies in adults have shown that psychological treatments for insomnia administered by specialist nurses to groups of patients can be effective within a primary care setting. This will be a pilot study to determine whether an educational intervention aimed at primary care teams to deliver problem focused therapy for insomnia can improve sleep management and outcomes for individual adult patients presenting to general practice. The study will also test procedures and collect information in preparation for a larger definitive cluster-randomised trial. The study is funded by The Health Foundation

Recruiting and retaining GPs and patients in intervention studies: the DEPS-GP project as a case study

Background: Recruiting and retaining GPs for research can prove difficult, and may result in sub-optimal patient participation where GPs are required to recruit patients. Low participation rates may affect the validity of research. This paper describes a multi-faceted approach to maximise participation of GPs and their patients in intervention studies, using an Australian randomised controlled trial of a depression/suicidality management intervention as a case study. The paper aims to outline experiences that may be of interest to others considering engaging GPs and/or their patients in primary care studies. Methods: A case study approach is used to describe strategies for: (a) recruiting GPs; (b) encouraging GPs to recruit patients to complete a postal questionnaire; and (c) encouraging GPs to recruit patients as part of a practice audit. Participant retention strategies are discussed in light of reasons for withdrawal. Results: The strategies described, led to the recruitment of a higher than expected number of GPs (n = 772). Three hundred and eighty three GPs (49.6%) followed through with the intent to participate by sending out a total of 77,820 postal questionnaires, 22,251 (28.6%) of which were returned. Three hundred and three GPs (37.0%) participated in the practice audit, which aimed to recruit 20 patients per participating GP (i.e., a total of 6,060 older adults). In total, 5,143 patients (84.9%) were represented in the audit. Conclusion: Inexpensive methods were chosen to identify and recruit GPs; these relied on an existing database, minor promotion and a letter of invitation. Anecdotally, participating GPs agreed to be involved because they had an interest in the topic, believed the study would not impinge too greatly on their time, and appreciated the professional recognition afforded by the Continuing Professional Development (CPD) points associated with study participation. The study team established a strong rapport with GPs and their reception staff, offered clear instructions, and were as flexible and helpful as possible to retain GP participants. Nonetheless, we experienced attrition due to GPs' competing demands, eligibility, personnel issues and the perceived impact of the study on patients. A summary of effective and ineffective methods for recruitment and retention is provided.Michelle K Williamson, Jane Pirkis, Jon J Pfaff, Orla Tyson, Moira Sim, Ngaire Kerse, Nicola T Lautenschlager, Nigel P Stocks and Osvaldo P Almeid

Integrating Palliative Care Into the Care of Neurocritically Ill Patients: A Report From the Improving Palliative Care in the ICU Project Advisory Board and the Center to Advance Palliative Care.

OBJECTIVES: To describe unique features of neurocritical illness that are relevant to provision of high-quality palliative care; to discuss key prognostic aids and their limitations for neurocritical illnesses; to review challenges and strategies for establishing realistic goals of care for patients in the neuro-ICU; and to describe elements of best practice concerning symptom management, limitation of life support, and organ donation for the neurocritically ill. DATA SOURCES: A search of PubMed and MEDLINE was conducted from inception through January 2015 for all English-language articles using the term palliative care, supportive care, end-of-life care, withdrawal of life-sustaining therapy, limitation of life support, prognosis, or goals of care together with neurocritical care, neurointensive care, neurological, stroke, subarachnoid hemorrhage, intracerebral hemorrhage, or brain injury. DATA EXTRACTION AND SYNTHESIS: We reviewed the existing literature on delivery of palliative care in the neurointensive care unit setting, focusing on challenges and strategies for establishing realistic and appropriate goals of care, symptom management, organ donation, and other considerations related to use and limitation of life-sustaining therapies for neurocritically ill patients. Based on review of these articles and the experiences of our interdisciplinary/interprofessional expert advisory board, this report was prepared to guide critical care staff, palliative care specialists, and others who practice in this setting. CONCLUSIONS: Most neurocritically ill patients and their families face the sudden onset of devastating cognitive and functional changes that challenge clinicians to provide patient-centered palliative care within a complex and often uncertain prognostic environment. Application of palliative care principles concerning symptom relief, goal setting, and family emotional support will provide clinicians a framework to address decision making at a time of crisis that enhances patient/family autonomy and clinician professionalism

The state of the Martian climate

60°N was +2.0°C, relative to the 1981–2010 average value (Fig. 5.1). This marks a new high for the record. The average annual surface air temperature (SAT) anomaly for 2016 for land stations north of starting in 1900, and is a significant increase over the previous highest value of +1.2°C, which was observed in 2007, 2011, and 2015. Average global annual temperatures also showed record values in 2015 and 2016. Currently, the Arctic is warming at more than twice the rate of lower latitudes

Cholera Toxin Regulates a Signaling Pathway Critical for the Expansion of Neural Stem Cell Cultures from the Fetal and Adult Rodent Brains

Background: New mechanisms that regulate neural stem cell (NSC) expansion will contribute to improved assay systems and the emerging regenerative approach that targets endogenous stem cells. Expanding knowledge on the control of stem cell self renewal will also lead to new approaches for targeting the stem cell population of cancers. Methodology/Principal Findings: Here we show that Cholera toxin regulates two recently characterized NSC markers, the Tie2 receptor and the transcription factor Hes3, and promotes the expansion of NSCs in culture. Cholera toxin increases immunoreactivity for the Tie2 receptor and rapidly induces the nuclear localization of Hes3. This is followed by powerful cultured NSC expansion and induction of proliferation both in the presence and absence of mitogen. Conclusions/Significance: Our data suggest a new cell biological mechanism that regulates the self renewal and differentiation properties of stem cells, providing a new logic to manipulate NSCs in the context of regenerative disease and cancer