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The influenza A M2 protein (AM2) is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle including viral assembly and budding. An atomic-level conformational understanding of this key player in the influenza life cycle could inform new antiviral strategies. For conformational studies of complex systems like the AM2 membrane protein, a multipronged approach using different biophysical methods and different model membranes is a powerful way to incorporate complementary data and achieve a fuller, more robust understanding of the system. However, one must be aware of how the sample composition required for a particular method impacts the data collected and how conclusions are drawn. In that spirit, we systematically compared the properties of AM2 in two different model membranes: nanodiscs and liposomes. Electron paramagnetic spectroscopy of spin-labeled AM2 showed that the conformation and dynamics were strikingly similar in both AM2-nanodiscs and AM2-liposomes consistent with similar conformations in both model membranes. Analysis of spin labeled lipids embedded in both model membranes revealed that the bilayer in AM2-liposomes was more fluid and permeable to oxygen than AM2-nanodiscs with the same lipid composition. Once the difference in the partitioning of the paramagnetic oxygen relaxation agent was taken into account, the membrane topology of AM2 appeared to be the same in both liposomes and nanodiscs. Finally, functionally relevant AM2 conformational shifts previously seen in liposomes due to the addition of cholesterol were also observed in nanodiscs.

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The influenza A M2 protein is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle. The monomeric subunits of the M2 homotetramer consist of an N-terminal ectodomain, a transmembrane domain, and a C-terminal cytoplasmic domain. The transmembrane domain forms a four-helix proton channel that promotes uncoating of virions upon host cell entry. The membrane-proximal region of the C-terminal domain forms a surface-associated amphipathic helix necessary for viral budding. The structure of the remaining ~34 residues of the distal cytoplasmic tail has yet to be fully characterized despite the functional significance of this region for influenza infectivity. Here, we extend structural and dynamic studies of the poorly characterized M2 cytoplasmic tail. We used SDSL-EPR to collect site-specific information on the mobility, solvent accessibility, and conformational properties of residues 61-70 of the full-length, cell-expressed M2 protein reconstituted into liposomes. Our analysis is consistent with the predominant population of the C-terminal tail dynamically extending away from the membranes surface into the aqueous medium. These findings provide insight into the hypothesis that the C-terminal domain serves as a sensor that regulates how M2 protein participates in critical events in the viral infection cycle.

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BACKGROUND: Primary care offers a promising setting for promoting parenting practices that shape healthy eating and physical activity behaviors of young children. This study assessed the impact of a parent-based, primary care intervention on the feeding habits, health behaviors, and body mass index (BMI) of 2⁻5 year olds with elevated or rapidly-increasing BMI. METHODS: Four private pediatric offices in West Michigan were assigned as control (n = 2) or intervention (n = 2) sites based on patient load and demographics. Treatment families were recruited at well-child visits to receive physician health-behavior counseling and four visits with a registered dietitian nutritionist (RDN) over a 6-month period. Intervention outcomes were age- and sex-specific BMI metrics, including BMI z-scores and percent of the 95th percentile (%BMIp95), the Family Nutrition and Physical Activity survey (FNPA), and the Feeding Practices and Structure Questionnaire (FPSQ). RESULTS: Of 165 enrolled families, 127 completed follow-up measures (77% retention). Mean (±SD) FNPA scores improved in treatment vs. control (4.6 ± 4.6 vs. 0.1 ± 4.2; p < 0.001), and screen time (h/day) decreased (-0.9 ± 1.8 vs. 0.3 ± 1.1; p < 0.001). Non-responsive feeding practices (i.e., reward for behavior (p = 0.006) and distrust in appetite (p < 0.015)) and structure-related feeding practices (structured meal timing (p < 0.001)) improved in treatment parents vs. controls. Reductions in child BMI measures did not differ between groups. CONCLUSIONS: Families with preschool children participating in a low-intensity, primary care intervention improved obesogenic health behaviors, parent feeding habits, and child screen time, but not child adiposity. Future research should assess the sustainability of these family lifestyle improvements, and evaluate their future impact on the health and development of the children.

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We report the optimization of detergent-mediated reconstitution of an integral membrane-bound protein, full-length influenza M2 protein, by direct insertion into detergent-saturated liposomes. Detergent-mediated reconstitution is an important method for preparing proteoliposomes for studying membrane proteins, and must be optimized for each combination of protein and membrane constituents used. The purpose of the reconstitution was to prepare samples for site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) studies. Our goals in optimizing the protocol were to minimize the amount of detergent used, reduce overall proteoliposome preparation time, and confirm the removal of all detergent. The liposomes were comprised of (1-palmitoyl-2-oleyl-sn-glycero-phosphocholine (POPC) and 1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), and the detergent octylglucoside (OG) was used for reconstitution. Rigorous physical characterization was applied to optimize each step of the reconstitution process. We used dynamic light scattering (DLS) to determine the amount of OG needed to saturate the preformed liposomes. During detergent removal by absorption with Bio-Beads, we quantified the detergent concentration by means of a colorimetric assay, thereby determining the number of Bio-Bead additions needed to remove all detergent from the final proteoliposomes. We found that the overnight Bio-Bead incubation used in previously published protocols can be omitted, reducing the time needed for reconstitution. We also monitored the size distribution of the proteoliposomes with DLS, confirming that the size distribution remains essentially constant throughout the reconstitution process.

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Influenza A M2 is a membrane-associated protein with a C-terminal amphipathic helix that plays a cholesterol-dependent role in viral budding. An M2 mutant with alanine substitutions in the C-terminal amphipathic helix is deficient in viral scission. With the goal of providing atomic-level understanding of how the wild-type protein functions, we used a multipronged site-directed spin labeling electron paramagnetic resonance spectroscopy (SDSL-EPR) approach to characterize the conformational properties of the alanine mutant. We spin-labeled sites in the transmembrane (TM) domain and the C-terminal amphipathic helix (AH) of wild-type (WT) and mutant M2, and collected information on line shapes, relaxation rates, membrane topology, and distances within the homotetramer in membranes with and without cholesterol. Our results identify marked differences in the conformation and dynamics between the WT and the alanine mutant. Compared to WT, the dominant population of the mutant AH is more dynamic, shallower in the membrane, and has altered quaternary arrangement of the C-terminal domain. While the AH becomes more dynamic, the dominant population of the TM domain of the mutant is immobilized. The presence of cholesterol changes the conformation and dynamics of the WT protein, while the alanine mutant is insensitive to cholesterol. These findings provide new insight into how M2 may facilitate budding. We propose the AH-membrane interaction modulates the arrangement of the TM helices, effectively stabilizing a conformational state that enables M2 to facilitate viral budding. Antagonizing the properties of the AH that enable interdomain coupling within M2 may therefore present a novel strategy for anti-influenza drug design.

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BACKGROUND: The Family Nutrition and Physical Activity Screening Tool (FNPA) evaluates family behavioural and environmental factors associated with pediatric obesity, but it is unknown if FNPA scores differ among youth across obesity severities. Our aim was to determine the association between the FNPA and obesity severity in youth referred to weight management. METHODS: Upon initiating treatment, height, weight, and the FNPA were collected according to standard procedures. Cut-points for overweight/obesity, severe obesity (SO) class 2, and SO class 3 were calculated. FNPA scores were compared across weight status groups using analysis of covariance, and odds of SO across FNPA quartiles were evaluated with multiple logistic regression. RESULTS: Participants included 564 5-18year old who initiated treatment and completed the FNPA. After adjustment, FNPA scores differed by weight status with higher/healthier scores in youth with overweight/obesity (56.6±8.5) when compared to those with SO class 2 (55.0±7.1; p=0.015) or SO class 3 (53.6±9.0; p<0.001). Compared to those in the highest FNPA quartile, youth in the 2nd quartile had 1.8 (95% CI: 1.1, 2.9) times higher odds of SO, and those in the lowest FNPA quartile had 2.1 (95% CI: 1.3, 3.4) times higher odds of SO. Youth with SO had unhealthier subscale scores among 6 of 10 constructs, including nutritional, physical activity, sedentary, and sleep behaviours. CONCLUSIONS: Results suggest a consistent inverse relationship between the FNPA and adiposity among youth presenting for weight management. The FNPA is a useful metric for programs and clinicians targeting family behaviours and the home environment to combat obesity.

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The C-terminal amphipathic helix of the influenza A M2 protein plays a critical cholesterol-dependent role in viral budding. To provide atomic-level detail on the impact cholesterol has on the conformation of M2 protein, we spin-labeled sites right before and within the C-terminal amphipathic helix of the M2 protein. We studied the spin-labeled M2 proteins in membranes both with and without cholesterol. We used a multipronged site-directed spin-label electron paramagnetic resonance (SDSL-EPR) approach and collected data on line shapes, relaxation rates, accessibility of sites to the membrane, and distances between symmetry-related sites within the tetrameric protein. We demonstrate that the C-terminal amphipathic helix of M2 populates at least two conformations in POPC/POPG 4:1 bilayers. Furthermore, we show that the conformational state that becomes more populated in the presence of cholesterol is less dynamic, less membrane buried, and more tightly packed than the other state. Cholesterol-dependent changes in M2 could be attributed to the changes cholesterol induces in bilayer properties and/or direct binding of cholesterol to the protein. We propose a model consistent with all of our experimental data that suggests that the predominant conformation we observe in the presence of cholesterol is relevant for the understanding of viral budding.

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While crystal and NMR structures exist of the influenza A M2 protein, there is disagreement between models. Depending on the requirements of the technique employed, M2 has been studied in a range of membrane mimetics including detergent micelles and membrane bilayers differing in lipid composition. The use of different model membranes complicates the integration of results from published studies necessary for an overall understanding of the M2 protein. Here we show using site-directed spin-label EPR spectroscopy (SDSL-EPR) that the conformations of M2 peptides in membrane bilayers are clearly influenced by the lipid composition of the bilayers. Altering the bilayer thickness or the lateral pressure profile within the bilayer membrane changes the M2 conformation observed. The multiple M2 peptide conformations observed here, and in other published studies, optimistically may be considered conformations that are sampled by the protein at various stages during influenza infectivity. However, care should be taken that the heterogeneity observed in published structures is not simply an artifact of the choice of the model membrane.

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The influenza A M2 protein is a 97-residue integral membrane protein involved in viral budding and proton conductance. Although crystal and NMR structures exist of truncated constructs of the protein, there is disagreement between models and only limited structural data are available for the full-length protein. Here, the structure of the C-terminal juxtamembrane region (sites 50-60) is investigated in the full-length M2 protein using site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy in lipid bilayers. Sites 50-60 were chosen for study because this region has been shown to be critical to the role the M2 protein plays in viral budding. Continuous wave EPR spectra and power saturation data in the presence of paramagnetic membrane soluble oxygen are consistent with a membrane surface associated amphipathic helix. Comparison between data from the C-terminal juxtamembrane region in full-length M2 protein with data from a truncated M2 construct demonstrates that the line shapes and oxygen accessibilities are remarkably similar between the full-length and truncated form of the protein.

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This paper describes FitKids360, a stage 2 pediatric weight management program. FitKids360 is a physician-referred, multicomponent, low-cost healthy lifestyle program for overweight and obese youth 5-16 years of age and their families. FitKids360 provides an evidence-based approach to the treatment of pediatric overweight by targeting patients' physical activity, screen time, and dietary behaviors using a family-centered approach. The intervention begins with a two-hour orientation and assessment period followed by six weekly sessions. Assessments include lifestyle behaviors, anthropometry, and the Family Nutrition and Physical Activity (FNPA) survey, which screens for obesogenic risk factors in the home environment. Outcomes are presented from 258 patients who completed one of 33 FitKids360 classes. After completing FitKids360, patients increased moderate to vigorous physical activity by 14 minutes (P = 0.019), reduced screen time by 44 minutes (P < 0.001), and improved key dietary behaviors. Overall, FNPA scores increased by 9% (P < 0.001) and 69% of patients with "high risk" FNPA scores at baseline dropped below the "high risk" range by followup. Patients also lowered BMIs (P = 0.011) and age- and sex-adjusted BMI z-scores (P < 0.001) after completing the 7-week program. We hope this report will be useful to medical and public health professionals seeking to develop stage 2 pediatric obesity programs.

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An outbreak investigation identified 15 pertussis cases among 5 infants and 10 healthcare professionals at 1 hospital's neonatal intensive care unit (NICU). The cost of the outbreak to this hospital was $97 745. Heightened awareness of pertussis in NICUs is key to preventing healthcare-associated spread and minimizing outbreak-control-related costs. Bordetella pertussis is a highly communicable bacterial pathogen that causes a prolonged cough illness and is spread by respiratory droplet transmission. Infants aged ≤6 months are most susceptible to B pertussis infection and pertussis-associated complications, including pneumonia, encephalopathy, and death, and are commonly hospitalized for treatment [ 1]. Despite a universal pertussis vaccination program, 27 550 pertussis cases were reported in the United States during 2010 [ 2]. Pertussis outbreaks in healthcare settings can be challenging and costly to control [3]. On September 13, 2011 and September 15, 2011, 3 pertussis cases, including 2 confirmed by B pertussis isolation, among preterm infants discharged ≤30 days previously from a 71-bed NICU of a general hospital (NICU A) were reported by Hospital B, a large pediatric facility, to Maricopa County Department of Public Health. This report describes the outbreak, examines outbreak-associated costs and risk factors that might have contributed to healthcare-associated transmission, and provides guidance to prevent outbreaks in healthcare settings.

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As a target of antiviral drugs, the influenza A M2 protein has been the focus of numerous structural studies and has been extensively explored as a model ion channel. In this study, we capitalize on the expanding body of high-resolution structural data available for the M2 protein to design and interpret site-directed spin-labeling electron paramagnetic resonance spectroscopy experiments on drug-induced conformational changes of the M2 protein embedded in lipid bilayers. We obtained data in the presence of adamantane drugs for two different M2 constructs (M2TM 22-46 and M2TMC 23-60). M2TM peptides were spin labeled at the N-terminal end of the transmembrane domain. M2TMC peptides were spin labeled site specifically at cysteine residues substituted for amino acids within the transmembrane domain (L36, I39, I42, and L43) and the C-terminal amphipathic helix (L46, F47, F48, C50, I51, Y52, R53, F54, F55, and E56). Addition of adamantane drugs brought about significant changes in measured electron paramagnetic resonance spectroscopy environmental parameters consistent with narrowing of the transmembrane channel pore and closer packing of the C-terminal amphipathic helices.

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The M2 protein from influenza A is a pH-activated proton channel that plays an essential role in the viral life cycle and serves as a drug target. Using spin labeling EPR spectroscopy, we studied a 38-residue M2 peptide spanning the transmembrane region and its C-terminal extension. We obtained residue-specific environmental parameters under both high- and low-pH conditions for nine consecutive C-terminal sites. The region forms a membrane surface helix at both high and low pH, although the arrangement of the monomers within the tetramer changes with pH. Both electrophysiology and EPR data point to a critical role for residue Lys 49.

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BACKGROUND: Although the value of evidence-based practice may seem obvious, the process needed to produce more effective delivery of evidence-based healthcare is not obvious. Furthermore, the continuing escalation of healthcare costs fuels the desire of providers and consumers to undertake only those treatments that have benefit. One way to effect necessary changes in healthcare organizations is through focused, interdisciplinary, collaborative projects related to evidence-based practice. OBJECTIVES: To reduce rates of ventilator-associated pneumonia and catheter-related bloodstream infection in patients in the medical intensive care unit of a large, urban tertiary referral hospital in the Southwest. METHODS: The theory of planned behavior served as the basis for providing staff members with research-based, easily controllable strategies that "fit" with the usual methods of care delivery. Implementation of the strategies and data collection were accomplished through routine rounds on patients and regular reporting of objective information. RESULTS: During a 15-month period, use of the selected strategies resulted in a 54% reduction in ventilator-associated pneumonia, a 78% reduction in catheter-related bloodstream infections, and a 18% reduction in mean length of stay in the unit. Use of a multidisciplinary, environmentally tailored approach to concerns about patients' care resulted in estimated cost savings of 1.0 million US dollars to 2.3 million US dollars. CONCLUSIONS: Early, consistent communication about the project's rationale, expected behavior, and outcomes enhanced the manageability and effectiveness of this change in an adult intensive care unit.

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A peptide designed to form a homo-oligomeric transmembrane helical bundle was reconstituted into lipid bilayers and studied by using (2)H NMR (nuclear magnetic resonance) with magic angle spinning to confirm that the helical interface corresponds to the interface intended in the design. The peptide belongs to a family of model peptides derived from a membrane-solubilized version of the water-soluble coiled-coil GCN4-P1. The variant studied here contains two asparagines thought to engage in interhelical hydrogen bonding critical to the formation of a stable trimer. For the NMR studies, three different peptides were synthesized, each with one of three consecutive leucines in the transmembrane region deuterium labeled. Prior to NMR data collection, polarized infrared spectroscopy was used to establish that the peptides were reconstituted in lipid bilayers in a transmembrane helical conformation. The (2)H NMR line shapes of the three different peptides are consistent with a trimer structure formed by the designed peptide that is stabilized by inter-helical hydrogen bonding of asparagines at positions 7 and 14.

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The M2 protein from influenza A virus is a 97-amino-acid protein with a single transmembrane helix that forms proton-selective channels essential to virus function. The hydrophobic transmembrane domain of the M2 protein (M2TM) contains a sequence motif that mediates the formation of functional tetramers in membrane environments. A variety of structural models have previously been proposed which differ in the degree of helix tilt, with proposed tilts ranging from approximately 15 degrees to 38 degrees . An important issue for understanding the structure of M2TM is the role of peptide-lipid interactions in the stabilization of the lipid bilayer bound tetramer. Here, we labeled the N terminus of M2TM with a nitroxide and studied the tetramer reconstituted into lipid bilayers of different thicknesses using EPR spectroscopy. Analyses of spectral changes provide evidence that the lipid bilayer does influence the conformation. The structural plasticity displayed by M2TM in response to membrane composition may be indicative of functional requirements for conformational change. The various structural models for M2TM proposed to date--each defined by a different set of criteria and in a different environment--might provide snapshots of the distinct conformational states sampled by the protein.

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OBJECTIVE: To describe the scope of practice and complementary role of physician assistants as physician extenders in the pediatric intensive care unit. DESIGN: Descriptive report of a 5-yr experience using a physician assistant-resident staffing model in comparison to the traditional resident-only coverage. SETTING: Six-bed pediatric intensive care unit at a tertiary care center subject to longstanding New York Hospital Code 405 restrictions on resident work hours. INTERVENTIONS: Orientation, training, credentialing, and evaluation of physician assistants. MEASUREMENTS AND MAIN RESULTS: New Accreditation Council for Graduate Medical Education regulations based on the longstanding New York Hospital Code 405 limit the number of resident hours worked per week. Our hospital employs physician assistants as physician extenders in the pediatric intensive care unit to enable regulatory compliance. Physician assistants were oriented for a period of 6 months to 1 yr to develop skill competencies, observe and learn pediatric intensive care unit practices and procedures, and complete credentialing to perform traditionally physician, nursing, and respiratory therapist functions. Physician assistants were then assigned to an independent but supervised patient care role similar to that of a resident physician. The impact of the physician assistant program was assessed by the attending physicians, and resident opinions were surveyed. CONCLUSIONS: Physician assistants play a complementary role as physician extenders in the pediatric intensive care unit, enabling compliance with New York state and Accreditation Council for Graduate Medical Education resident work hour regulations. Physician assistants perform similar tasks and activities as the pediatric intensive care unit residents and integrate well with them in enhancing bedside patient care. Over time, physician assistants provide additional direction to the residents by virtue of their familiarity with unit-specific policies and procedures and repetitive pediatric intensive care unit practice patterns. As multifunctional members of the health care team, they support nursing and respiratory therapy functions and improve the day-to-day functioning of the unit. The physician assistant serves as a key member of the pediatric intensive care unit transport team. Limitations observed include high job turnover rates among the physician assistants and confusion between their role as shift workers or professional employees.

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Although previous studies are beginning to point to the specific types of helix-helix interactions that stabilize the folds of membrane-bound helical proteins, quantitative thermodynamic data on natural membrane proteins has been very limited. Here the database is expanded substantially by adding thermodynamic data for a series of sequence variants of M2 protein from influenza A virus. The M2 protein has a single transmembrane helix that homotetramerizes to form proton-selective channels that are essential to virus function. To determine the contributions of specific residues to the folding of this protein, a series of transmembrane peptides with single-site changes near the core of the protein were studied by using sedimentation equilibrium analytical ultracentrifugation. Remarkably, a large number of the mutations increased the stability of the protein. The free energies of tetramerization of the variants can be understood in terms of current models for the structure of the protein. In general, the energetic consequences of the mutations are smaller than those observed for similar mutations in water-soluble proteins. This observation is consistent with previous studies and hence may represent a general phenomenon.

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