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1.  Patient attitudes regarding the role of the pharmacist and interest in expanded pharmacist services 
Canadian Pharmacists Journal : CPJ  2014;147(4):239-247.
Pharmacists are consistently ranked among the most trusted professionals, and research shows high levels of satisfaction with pharmacist services. Studies have also shown that the public is generally unaware of the full range of roles and responsibilities of a pharmacist. The purpose of this study was to explore the public’s knowledge and attitudes regarding the role of the community pharmacist and to determine their likelihood of using expanded pharmacist services.
Adults across Newfoundland and Labrador were surveyed by telephone. Survey questions addressed how frequently participants visited the pharmacy, understanding of duties undertaken by pharmacists, perceptions and attitudes regarding pharmacists as health care professionals, likelihood of using expanded pharmacist services and participant demographics. Comparisons were made between responses from urban and rural participants and frequent versus nonfrequent pharmacy users, to determine if there were any differences.
The majority of participants were generally aware of what pharmacists do when filling prescriptions; those who visited the pharmacy more frequently appeared to be more informed. Participants indicated they would take advantage of the expanded services suggested, with greatest interest in receiving advice for minor ailment management and prescription refills from pharmacists. Results support the prevailing view that pharmacists are trusted health professionals who should have access to patients’ health information to provide best care.
The public is aware of aspects of the pharmacist’s role, but opportunities exist to better educate the public on the knowledge, skills and unique professional abilities of pharmacists to support uptake of expanded pharmacist services.
PMCID: PMC4212442  PMID: 25360150
2.  Elongated TCR alpha chain CDR3 favors an altered CD4 cytokine profile 
BMC Biology  2014;12:32.
CD4 T lymphocyte activation requires T cell receptor (TCR) engagement by peptide/MHC (major histocompatibility complex) (pMHC). The TCR complementarity-determining region 3 (CDR3) contains variable α and β loops critical for pMHC recognition. During any immune response, tuning of TCR usage through progressive clonal selection occurs. Th1 and Th2 cells operate at different avidities for activation and display distinct transcriptional programs, although polarization may be plastic, influenced by pathogens and cytokines. We therefore hypothesized that CDR3αβ sequence features may intrinsically influence CD4 phenotype during progression of a response.
We show that CD4 polarization involves distinct CDR3α usage: Th1 and Th17 cells favored short TCR CDR3α sequences of 12 and 11 amino acids, respectively, while Th2 cells favored elongated CDR3α loops of 14 amino acids, with lower predicted affinity. The dominant Th2- and Th1-derived TCRα sequences with14 amino acid CDR3 loops and 12 amino acid CDR3 loops, respectively, were expressed in TCR transgenics. The functional impact of these TCRα transgenes was assessed after in vivo priming with a peptide/adjuvant. The short, Th1-derived receptor transgenic T cell lines made IFNγ, but not IL-4, 5 or 13, while the elongated, Th2-derived receptor transgenic T cell lines made little or no IFNγ, but increased IL-4, 5 and 13 with progressive re-stimulations, mirrored by GATA-3 up-regulation. T cells from primed Th2 TCRα transgenics selected dominant TCR Vβ expansions, allowing us to generate TCRαβ transgenics carrying the favored, Th2-derived receptor heterodimer. Primed T cells from TCRαβ transgenics made little or no IL-17 or IFNγ, but favored IL-9 after priming with Complete Freund’s adjuvant and IL-4, 5, 9, 10 and 13 after priming with incomplete Freund’s. In tetramer-binding studies, this transgenic receptor showed low binding avidity for pMHC and polarized T cell lines show TCR avidity for Th17 > Th1 > Th2. While transgenic expression of a Th2-derived, ‘elongated’ TCR-CDR3α and the TCRαβ pair, clearly generated a program shifted away from Th1 immunity and with low binding avidity, cytokine-skewing could be over-ridden by altering peptide challenge dose.
We propose that selection from responding clones with distinctive TCRs on the basis of functional avidity can direct a preference away from Th1 effector responses, favoring Th2 cytokines.
PMCID: PMC4046507  PMID: 24886643
CD4 T cell; T cell receptor; Cytokine; Transgenic; Mouse; Th17; Th1; Th2
3.  Affinity grid-based cryo-EM of PKC binding to RACK1 on the ribosome 
Journal of structural biology  2012;181(2):10.1016/j.jsb.2012.11.006.
Affinity grids (AG) are specialized EM grids that bind macromolecular complexes containing tagged proteins to obtain maximum occupancy for structural analysis through single-particle EM. In this study, utilizing AG, we show that His-tagged activated PKC βII binds to the small ribosomal subunit (40S). We reconstructed a cryo-EM map which shows that PKC βII interacts with RACK1, a seven-bladed β-propeller protein present on the 40S and binds in two different regions close to blades 3 and 4 of RACK1. This study is a first step in understanding the molecular framework of PKC βII/RACK1 interaction and its role in translation.
PMCID: PMC3833090  PMID: 23228487
Affinity grid; Cryo-EM; RACK1; PKC βII; Ribosome
4.  Pharmacist and physician views on collaborative practice 
Canadian Pharmacists Journal : CPJ  2013;146(4):218-226.
Strong working relationships between pharmacists and physicians are needed to optimize patient care. Understanding attitudes and barriers to collaboration between pharmacists and physicians may help with delivery of primary health care services. The objective of this study was to capture the opinions of family physicians and community pharmacists in Newfoundland and Labrador (NL) regarding collaborative practice.
Two parallel surveys were offered to all community pharmacists and family physicians in NL. Surveys assessed the following: attitudes and experience with collaborative practice, preferred communication methods, perceived role of pharmacists, areas for more collaboration and barriers to collaborative practice. Results for both groups were analyzed separately, with comparisons between groups to compare responses with similar questions.
Survey response rates were 78.6% and 7.1% for pharmacists and physicians, respectively. Both groups overwhelmingly agreed that collaborative practice could result in improved patient outcomes and agreed that major barriers were lack of time and compensation and the need to deal with multiple pharmacists/physicians. Physicians indicated they would like more collaboration for insurance approvals and patient counselling, while pharmacists want to assist with identifying and managing patients’ drug-related problems. Both groups want more collaboration to improve patient adherence.
Both groups agree that collaborative practice can positively affect patient outcomes and would like more collaboration opportunities. However, physicians and pharmacists disagree about the areas where they would like to collaborate to deliver care. Changes to reimbursement models and infrastructure are needed to facilitate enhanced collaboration between pharmacists and physicians in the community setting.
PMCID: PMC3734911  PMID: 23940479
5.  The use of trehalose in the preparation of specimens for molecular electron microscopy 
Micron (Oxford, England : 1993)  2011;42(8):762-772.
Biological specimens have to be prepared for imaging in the electron microscope in a way that preserves their native structure. Two-dimensional (2D) protein crystals to be analyzed by electron crystallography are best preserved by sugar embedding. One of the sugars often used to embed 2D crystals is trehalose, a disaccharide used by many organisms for protection against stress conditions. Sugars such as trehalose can also be added to negative staining solutions used to prepare proteins and macromolecular complexes for structural studies by single-particle electron microscopy (EM). In this review, we describe trehalose and its characteristics that make it so well suited for preparation of EM specimens and we review specimen preparation methods with a focus on the use of trehalose.
PMCID: PMC3156378  PMID: 21752659
7.  Responses to pandemic ASO3-adjuvanted A/California/07/09 H1N1 influenza vaccine in human immunodeficiency virus-infected individuals 
BMC Immunology  2012;13:49.
Influenza infection may be more serious in human immunodeficiency virus (HIV)-infected individuals, therefore, vaccination against seasonal and pandemic strains is highly advised. Seasonal influenza vaccines have had no significant negative effects in well controlled HIV infection, but the impact of adjuvanted pandemic A/California/07/2009 H1N1 influenza hemaglutinin (HA) vaccine, which was used for the first time in the Canadian population as an authorized vaccine in autumn 2009, has not been extensively studied.
Assess vaccine-related effects on CD4+ T cell counts and humoral responses to the vaccine in individuals attending the Newfoundland and Labrador Provincial HIV clinic.
A single dose of ArepanrixTM split vaccine including 3.75 μg A/California/07/2009 H1N1 HA antigen and ASO3 adjuvant was administered to 81 HIV-infected individuals by intramuscular injection. Plasma samples from shortly before, and 1–5 months after vaccination were collected from 80/81 individuals to assess humoral anti-H1N1 HA responses using a sensitive microbead-based array assay. Data on CD4+ T cell counts, plasma viral load, antiretroviral therapy and patient age were collected from clinical records of 81 individuals.
Overall, 36/80 responded to vaccination either by seroconversion to H1N1 HA or with a clear increase in anti-H1N1 HA antibody levels. Approximately 1/3 (28/80) had pre-existing anti-H1N1 HA antibodies and were more likely to respond to vaccination (22/28). Responders had higher baseline CD4+ T cell counts and responders without pre-existing antibodies against H1N1 HA were younger than either non-responders or responders with pre-existing antibodies. Compared to changes in their CD4+ T cell counts observed over a similar time period one year later, vaccine recipients displayed a minor, transient fall in CD4+ T cell numbers, which was greater amongst responders.
We observed low response rates to the 2009 pandemic influenza vaccine among HIV-infected individuals without pre-existing antibodies against H1N1 HA and a minor transient fall in CD4+ T cell numbers, which was accentuated in responders. A single injection of the ArepanrixTM pandemic A/California/07/2009 H1N1 HA split vaccine may be insufficient to induce protective immunity in HIV-infected individuals without pre-existing anti-H1N1 HA responses.
PMCID: PMC3482569  PMID: 22937824
HIV; influenza; pandemic; A/California/07/2009 H1N1 HA antigen; AS03 oil in water adjuvant; inflammation; CD4+ T cells; age
8.  Capturing RNA-dependent pathways for cryo-EM analysis 
Cryo-Electron Microscopy (EM) is a powerful technique to visualize biological processes at nanometer resolution. Structural studies of macromolecular assemblies are typically performed on individual complexes that are biochemically isolated from their cellular context. Here we present a molecular imaging platform to capture and view multiple components of cellular pathways within a functionally relevant framework. We utilized the bacterial protein synthesis machinery as a model system to develop our approach. By using modified Affinity Grid surfaces, we were able to recruit multiple protein assemblies bound to nascent strands of mRNA. The combined use of Affinity Capture technology and single particle electron microscopy provide the basis for visualizing RNA-dependent pathways in a remarkable new way.
PMCID: PMC3962177  PMID: 24688633
Protein synthesis; Transcription; Lipid monolayer; Affinity Capture technology
9.  Strategy for the use of Affinity Grids to prepare non-His-tagged macromolecular complexes for single-particle electron microscopy 
Journal of molecular biology  2010;400(4):675-681.
Affinity Grids are electron microscopy (EM) grids with a pre-deposited lipid monolayer containing functionalized Nickel-nitrilotriacetic acid (Ni-NTA) lipids. Affinity Grids can be used to prepare His-tagged proteins for single-particle EM from impure solutions or even directly from cell extracts. Here we introduce the concept of His-tagged adaptor molecules, which eliminate the need for the target protein or complex to be His-tagged. The use of His-tagged protein A as adaptor molecule allows Affinity Grids to be used for the preparation of virtually any protein or complex provided that a specific antibody is available or can be raised against the target protein. The principle is that the Affinity Grid is coated with a specific antibody that is recruited to the grid by His-tagged protein A. The antibody-decorated Affinity Grid can then be used to isolate the target protein directly from a cell extract. We first established this approach by preparing negatively stained specimens of both native ribosomal complexes and ribosomal complexes carrying different purification tags directly from HEK-293T cell extract. We then used the His-tagged protein A/antibody strategy to isolate RNA polymerase II (RNAP II), still bound to native DNA, from HEK-293T cell extract, allowing us to calculate a 25-Å resolution density map by single-particle cryo-EM.
PMCID: PMC2923398  PMID: 20562026
Monolayer purification; Affinity Grid; single-particle electron microscopy; ribosome; RNA polymerase II
10.  Molecular Structure and Dimeric Organization of the Notch Extracellular Domain as Revealed by Electron Microscopy 
PLoS ONE  2010;5(5):e10532.
The Notch receptor links cell fate decisions of one cell to that of the immediate cellular neighbor. In humans, malfunction of Notch signaling results in diseases and congenital disorders. Structural information is essential for gaining insight into the mechanism of the receptor as well as for potentially interfering with its function for therapeutic purposes.
Methodology/Principal Findings
We used the Affinity Grid approach to prepare specimens of the Notch extracellular domain (NECD) of the Drosophila Notch and human Notch1 receptors suitable for analysis by electron microscopy and three-dimensional (3D) image reconstruction. The resulting 3D density maps reveal that the NECD structure is conserved across species. We show that the NECD forms a dimer and adopts different yet defined conformations, and we identify the membrane-proximal region of the receptor and its ligand-binding site.
Our results provide direct and unambiguous evidence that the NECD forms a dimer. Our studies further show that the NECD adopts at least three distinct conformations that are likely related to different functional states of the receptor. These findings open the way to now correlate mutations in the NECD with its oligomeric state and conformation.
PMCID: PMC2866536  PMID: 20479883
11.  Structural and functional studies on the stalk of the transferrin receptor 
Transferrin (Tf) is an iron carrier protein that consists of two lobes, the N- and C-lobes, which can each bind a Fe3+ ion. Tf binds to its receptor (TfR), which mediates iron delivery to cells through an endocytotic pathway. Receptor binding facilitates iron release from the Tf C-lobe, but impedes iron release from the N-lobe. An atomic model of the Tf-TfR complex based on single particle electron microscopy (EM) indicated that receptor binding is indeed likely to hinder opening of the N-lobe, thus interfering with its iron release. The atomic model also suggested that the TfR stalks could form additional contacts with the Tf N-lobes, thus potentially further slowing down its iron release. Here, we show that the TfR stalks are unlikely to make strong interactions with the Tf N-lobes and that the stalks have no effect on iron release from the N-lobes of receptor-bound Tf.
PMCID: PMC2692427  PMID: 19258014
transferrin; transferrin receptor; receptor stalk; iron release; single particle electron microscopy
12.  The Affinity Grid: A pre-fabricated EM grid for monolayer purification 
Journal of molecular biology  2008;382(2):423-433.
We have recently developed “monolayer purification” as a rapid and convenient technique to produce specimens of His-tagged proteins or macromolecular complexes for single particle electron microscopy (EM) without prior biochemical purification. Here, we introduce the “Affinity Grid”, a pre-fabricated EM grid featuring a dried lipid monolayer that contains Ni-NTA lipids (lipids functionalized with a Nickel-nitrilotriacetic acid group). The Affinity Grid, which can be stored for several months under ambient conditions, further simplifies and extends the use of monolayer purification. After characterizing the Affinity Grid, we used it to isolate, within minutes, ribosomal complexes from E. coli cell extracts containing His-tagged rpl3, the human homolog of the E. coli 50S subunit rplC. Depending on the way the sample was applied to the Affinity Grid, ribosomal complexes with or without associated mRNA could be prepared. Vitrified Affinity Grid specimens could be used to calculate three-dimensional reconstructions of the 50S ribosomal subunit as well as the 70S ribosome and 30S ribosomal subunit from images of the same sample. In addition, we established that Affinity Grids are stable for some time in the presence of glycerol and detergents. This feature allowed us to isolate His-tagged aquaporin-9 (AQP9) from detergent-solubilized membrane fractions of Sf9 insect cells. The Affinity Grid can thus be used to prepare single particle EM specimens of soluble complexes and membrane proteins.
PMCID: PMC2564605  PMID: 18655791
monolayer purification; lipid monolayer; affinity purification; single particle; cryo-electron microscopy
13.  7 Å projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals 
Journal of structural biology  2007;160(3):313-323.
Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7 Å resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.
PMCID: PMC2149845  PMID: 17638580
electron crystallography; S-layer protein; lipid monolayers; sugar embedding
Journal of structural biology  2007;160(3):305-312.
Lipid monolayers provide a convenient vehicle for the crystallization of biological macromolecules for 3-D electron microscopy. Although numerous examples of 3-D images from 2-D protein arrays have been described from negatively stained specimens, only six structures have been done from frozen hydrated specimens. We describe here a method that makes high quality frozen-hydrated specimens of lipid monolayer arrays for cryoelectron microscopy. The method uses holey carbon films with patterned holes for monolayer recovery, blotting and plunge freezing to produce thin aqueous films which cover >90% of the available grid area. With this method, even specimens with relatively infrequent crystals can be screened using automated data collection techniques. Though developed for microscopic examination of 2-D arrays, the method may have wider application to the preparation of single particle specimens for 3-D image reconstruction.
PMCID: PMC2268103  PMID: 17561414
Electron Crystallography

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